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Influence of mycorrhizas on plant competition for phosphorus between slash pine and grass

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Influence of mycorrhizas on plant competition for phosphorus between slash pine and grass
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Pedersen, Christian Thomas, 1958-
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English
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xii, 121 leaves : ill. ; 29 cm.

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Fungi ( jstor )
Grasses ( jstor )
Hyphae ( jstor )
Mycorrhizal fungi ( jstor )
Mycorrhizas ( jstor )
Nutrients ( jstor )
Plant competition ( jstor )
Plant roots ( jstor )
Plants ( jstor )
Soil science ( jstor )
Dissertations, Academic -- Soil and Water Science -- UF
Grasses -- Florida ( lcsh )
Mycorrhizas -- Florida ( lcsh )
Phosphorus ( lcsh )
Slash pine -- Florida ( lcsh )
Soil and Water Science thesis, Ph. D
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1995.
Bibliography:
Includes bibliographical references (leaves 101-121).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Christian Thomas Pedersen.

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INFLUENCE OF MYCORRHIZAS ON PLANT COMPETITION FOR
PHOSPHORUS BETWEEN SLASH PINE AND GRASS
















By

CHRISTIAN THOMAS PEDERSEN













A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1995



























To Karen,

for her love, support, patience

and her sense of humor that carried

us both through this adventure













ACKNOWLEDGMENTS

I thank Dr. David Sylvia for giving my wife and me a warm welcome to Florida and for his continued support throughout my studies. I also thank the members of my supervisory committee, Drs. Nicholas Comerford, James Graham, David Mitchell and Donn Shilling, for their inputs. I would like to especially thank Drs. David Mitchell, David Hubbell and Suresh Rao for their major contributions to the philosophical aspects of the Doctorate in Philosophy. Appreciation is extended to the National Science Foundation for partially funding this research (Grant No. BSR-9019788). The opportunity to use the laboratory facilities of Drs. Rao and Comerford is gratefully acknowledged. Mary McLeod, Dongping Dai, Drs. Amiel Jarstfer and Linda Lee deserve thanks for their invaluable technical guidance and patience throughout the process. Thanks also go to the many laboratory assistants that have stood by my side and who probably do not realize how much they have contributed. I appreciate Mike Allen's technical support with my Benlate field work. To the many graduate students who have passed through and the few that still remain: The interactions we had were valuable to me. Specifically, I would like to thank Dr. Pauline Grierson, Len Scinto and Dr. Philip Smethurst for their supportive conversations that put things in perspective. Many thanks also to David Farmer and Steve Trabue for the late night reality checks on the second floor of McCarty Hall. Lastly, I am grateful for my family's love and support, which, even though they were far removed from my research activities, was of great importance.

iii














TABLE OF CONTENTS



ACKNOWLEDGMENTS .................................. iii

LIST OF TABLES ..................................... v

LIST OF FIGURES .................................. viii

ABSTRACT ......................................... x

CHAPTERS

1 INTRODUCTION ................................ 1

2 MYCORRHIZAS AND PLANT COMPETITION ............... 3

Introduction ............ .......................... 3
The Autecology of the Mycorrhizal Symbiosis ................. 5
Nutrient Uptake: The Role of External Hyphae ............. 5
Other Mycorrhizal Effects ........................... 14
Synecology ... ......... ..... ....... .... ......... 19
Plant Interactions ........ ........................ 20
Environmental Conditions and Plant Competition ........... 26
Plant Succession and Community Structure ................... 30

3 LIMITATIONS IN THE USE OF BENOMYL IN
EVALUATING MYCORRHIZAL FUNCTIONING .............. 34
Introduction ................................... 34
Materials and Methods .............................. 36
Results ............. ...... .................. 41
Discussion ..................... ............... 48








iv








4 MYCORRHIZAS AFFECT PLANT COMPETITION FOR
PHOSPHORUS BETWEEN PINE AND GRASS ................ 52
Introduction ................................... 52
Materials and Methods ........................... 53
Results ..................................... 60
Discussion .................................... 69

5 CONCLUSIONS ................................... 75

APPENDICES

1 GROWTH CHAMBER COMPETITION STUDY BETWEEN
PINE AND GRASS ................................. 80
Introduction ................................... 80
Materials and Methods ........................... 80
Results ..................................... 83
Discussion .................................. 93

2 COLONIZATION OF PANICUM CHAMAELONCHE AND CORN BY
DIFFERENT ARBUSCULAR MYCORRHIZAL FUNGI ........... 95

3 PHOSPHORUS GROWTH RESPONSE CURVE FOR
NONMYCORRHIZAL PANICUM CHAMAELONCHE ............ 98

REFERENCE LIST ..................................... 101

BIOGRAPHICAL SKETCH ................................ 122




















V













LIST OF TABLES



Table Page

2-1 Summary of nutrient uptake kinetic studies ..................... 9

3-1 Test for homogeneity of slopes for the effect of Benlate* 50 DF applied
in the field on percent Panicum chamaelonche roots with arbuscules and
their activity over time ................................ 43

4-1 Pinus elliottii (pine) and Panicum chamaelonche (grass) treatment
combinations planted in the competition study. Two plants were planted per pot. The superscripts "+" and "-" signify an inoculated or noninoculated plant respectively. Pine was inoculated with Pisolithus tinctorius and the grass was inoculated with Glomus sp. (INVAM FL329,
formerly FL906) .......... ...... ................. 56

4-2 Ergosterol concentration (ug g-') of Pinus elliottii roots inoculated with
Pisolithus tinctorius (pine+) or noninoculated (pine-), and grown in combination with Pinus elliottii (pine) or Panicum chamaelonche (grass) at 0.32, 3.23 or 32.26 /M P for 18 wk. Each value represents the mean
of six replicates SE ................................ 64

4-3 Initial phosphorus uptake rate, I (pmol P cm-2 s-') and C., (jAM P), the
minimum solution concentration from which a nutrient can be absorbed, for Pinus elliottii and Panicum chamaelonche grown in a hydroponic solution containing 0.32 pM P. Each value represents the mean of three
replicates SE .................................... 68

Al-i Tests for single degree of freedom contrasts for root-length density, plant
biomass, plant P content and percent colonization. Each parameter was
analyzed separately for grass and pine ...................... 84






vi








A1-2 Mean hyphal length density (m cm3 of soil) for each competition
treatment presented separately for each compartment (one hyphal and two plant compartments). Hyphal length is made up of the sum of both AM and EM fungal hyphae. Each value represents the mean of a minimum of
six replicates SE .................................. 85

A1-3 Mean soil-phosphorus content (ug P g of soil) for each competition
treatment presented separately for each compartment (one hyphal and two plant compartments). Each value represents the mean of a minimum of six
replicates SE .................................... 90




































vii













LIST OF FIGURES


Figure age

2-1 Length of external hyphae spreading from mycorrhizal roots of Trifolium
subterraneum after (a) 28 days and (b) 47 days. Bars represent standard
error of the mean [with permission from (Jakobsen et al., 1992)] ....... 7

3-1 Assessment of arbuscular activity in Panicum chamaelonche roots from
the field site in 1991. (A) Percentage of root length with arbuscules in benomyl-treated and nontreated plots, (B) Percentage root length with metabolically active arbuscules in benomyl-treated and nontreated plots and (C) Precipitation. Each symbol represents the mean of three replicates
SE .......................................... 42

3-2 Total dry weight of mycorrhizal (M) and nonmycorrhizal (C) plants, (A)
Pinus elliottii and (B) corn in response to 0, 20, 60 or 150 kg benomyl ha1 in the greenhouse. Each symbol represents the mean of seven replicates
SE .................. .. ........ ... .... ... .... 45

3-3 Soil hyphal length (total) and activity (active) of mycorrhizal (A) Pinus
elliottii and (B) corn plants in response to 0, 20, 60 or 150 kg benomyl ha' in the greenhouse. Each symbol represents the mean of seven
replicates SE ......................................... 46

3-4 Mycorrhizal colonization of (A) slash pine and (B) corn grown in the
greenhouse in response to 0, 20, 60 or 150 kg benomyl ha1. Each symbol
represents the mean of seven replicates SE .................. 47

4-1 Pinus elliottii (A) shoot-phosphorus concentration, (B) shoot-phosphorus
content and (C) total dry weight in response to different competition treatments and grown at either 0.32, 3.23 or 32.26 /AM P for 18 wk. Each symbol represents the mean of six replicates SE. Inoculated grass was not colonized at the end of the experiment and therefore was not included
in the analysis ..................................... 62




viii








4-2 Panicum chamaelonche (A) shoot-phosphorus concentration, (B) shootphosphorus content and (C) total dry weight in response to different competition treatments and grown at either 0.32, 3.23 or 32.26 gM P for 18 wk. Each symbol represents the mean of six replicates SE.
Inoculated grass was not colonized at the end of the experiment and
therefore was not included in the analysis ................... 63

4-3 Root-length density of (A) Pinus elliottii and (B) Panicum chamaelonche
in different competition treatments and grown at 0.32, 3.23 or 32.26 pM P for 18 wk. Each symbol represents the mean of six replicates SE.
Inoculated grass was not colonized at the end of the experiment and
therefore was not included in the analysis ........... . ........ 66

4-4 Relative crowding coefficient (RCC) for (A) Pinus elliottii inoculated with
Pisolithus tinctorius grown in combination with Panicum chamaelonche and (B) noninoculated Pinus elliottii grown in combination with P.
chamaelonche at either 0.32, 3.23 or 32.26 IM P for 18 wk. Each symbol represents the mean of six replicates. Mean standard errors were smaller
than the symbols and are therefore not included ................. 67

Al-1 Plant biomass for (A) slash pine and (B) grass grown in the growth
chamber for 62 d. Each bar represents the mean of a minimum of six
replicates SE .................................... 86

A1-2 Plant-P content for (A) slash pine and (B) grass grown in the growth
chamber for 62 d. Each bar represents the mean of a minimum of six
replicates SE ................................... 87

A1-3 Mycorrhizal colonization of (A) slash pine and (B) grass grown in the
growth chamber for 62 d. Each bar represents the mean of a minimum of
six replicates SE ................................. 88

A1-4 Root-length density for (A) slash pine and (B) grass grown in the growth
chamber for 62 d. Each bar represents the mean of a minimum of six
replicates SE ................................... 91

A1-5 Uptake rate of (A) slash pine and (B) grass grown in the growth chamber
for 62 d. Uptake rate was calculated on a unit surface area basis which included both root and hyphal surface area. Each bar represents the mean
of a minimum of six replicates SE ....................... 92





ix








A2-1 Colonization of (A) Panicum chamaelonche and (B) Zea mays inoculated
with root fragments of (1) Acaulospora scrobiculata (S315), (2) Gigaspora margarita (INVAM FL215), (3) Glomus etunicatum (INVAM FL312) or (4) Glomus sp. (INVAM FL329, formerly FL906) or spores of (5) Gigaspora rosea (INVAM FL224) or (6) Scutellospora heterogama (INVAM FL225). Each bar represents the mean of four replicates
SE . . . . . . . . . . . . . . . . . . . . . . 97

A3-1 Nonmycorrhizal Panicum chamaelonche (A) plant biomass and (B) plant
phosphorus content in response to 0.001, 0.003, 0.010, 0.032, 0.100, 0.316, 0.1000, 3.162 or 10.000 mg P L-. Each symbol represents the
mean of seven replicates SE .......................... 100


































x













Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

INFLUENCE OF MYCORRHIZAS ON PLANT COMPETITION FOR
PHOSPHORUS BETWEEN SLASH PINE AND GRASS By

Christian Thomas Pedersen

December 1995


Chairperson: Dr. D. Sylvia
Major Department: Soil and Water Science

Individual plants benefit from the mycorrhizal condition primarily by improved nutrient uptake, especially of phosphorus (P), resulting in enhanced plant survival and growth in resource-limited conditions. On a broader scale, mycorrhizas have the potential to mediate plant competition and subsequently may be important at the community level.

In the southeastern United States, slash pine (Pinus elliottii Engelm. var. elliottii) is grown in plantations, where it competes for nutrients with grasses and other herbaceous vegetation. One objective of my research was to assess mycorrhizal contribution to intra- and interspecific plant competition for P in the greenhouse between pine and Panicum chamaelonche Trin., a dominant grass species at a local plantation site. The grass was inoculated or noninoculated with the arbuscular mycorrhizal (AM) fungus, Glomus sp. (INVAM FL329, formerly INVAM FL906), and pine was inoculated or


xi








noninoculated with the ectomycorrhizal (EM) fungus, Pisolithus tinctorius (isolate S 106). The effect of P (0.32, 3.23 or 32.26 1AM) on competition also was analyzed in the greenhouse because resource abundance can affect the outcome of competition and inorganic P is limiting in pine plantations. Inoculated grasses were not colonized at the end of the experiment and were excluded from data analyses. When competing with grass, inoculated pine acquired more P and had a higher total dry weight than noninoculated pine. Grass shoot-P content was reduced at the 32.26-JpM P level when grown with pine, irrespective of the pine inoculation treatment.

I evaluated the effects of benomyl over time in the field and at 0, 20, 60 and 150 kg benomyl ha1 equivalent in the greenhouse. My objective was to determine if benomyl would be suitable for controlling AM but not EM fungi as part of a larger competition experiment involving pine and weeds. No effect was observed on pine in the greenhouse. Colonized root length of benomyl-treated Zea mays L. plants in the greenhouse remained static and the response was not dose-dependent. In contrast, colonization in the control plants increased over time. Minimal reduction of grass colonization was observed in the field, where limitations to effective control were ground cover, timing in relation to mycorrhizal development and benomyl application as a spray instead of as a soil drench.













xii













CHAPTER 1
INTRODUCTION

Mycorrhizal contribution to plant nutrient uptake, especially phosphorus (P), has been extensively studied. Under nutrient-limiting conditions mycorrhizas are able to enhance plant nutrient uptake by various mechanisms, thereby ameliorating plant stress. Under these conditions plants compete for limited nutrients and mycorrhizas may modify the competitive interactions between plants. Little work has addressed the role of mycorrhizas in this area of plant synecology. The existing studies primarily deal with arbuscular mycorrhizal (AM) fungi and their contribution to plant interactions. Many of these studies investigated facilitative mycorrhizal plant associations, where mycorrhizal fungi transfer nutrients from one plant to another through common hyphal connections. Only one study addressed the function of ectomycorrhizal (EM) fungi in plant competition. No studies to date have evaluated the role of mycorrhizas in competitive interactions between AM and EM plants, which occurs frequently during succession. In the following chapter, I review the various aspects of mycorrhizal functioning in plant autoecology and synecology and I detail their role in plant competition.

Most previous studies have been performed under controlled conditions in the greenhouse without the influence of complex interactions of other environmental variables. When bringing mycorrhizal questions to the field, one of the more difficult problems is the creation of a suitable nonmycorrhizal control, since a majority of plants


1








2

are normally mycorrhizal. Fungicides can be useful in distinguishing mycorrhizal effects from other influences on plants in the field. The fungicide benomyl is selective in that it has inhibitory effects on AM but not on EM fungi. The selectivity of this fungicide would be useful in isolating the nutrient uptake mediated by AM fungi in AM and EM plant competition studies. As part of a larger competition study involving slash pine (Pinus elliottii) and weeds at a field site northwest of Gainesville, I tested benomyl in the field and in the greenhouse to determine if it would suitably control AM but not EM fungi (Chapter 3).

I addressed the direct effect of mycorrhizas on plant competition for P in greenhouse and growth chamber studies involving slash pine and Panicum chamaelonche, a dominant weed species at the field site. The main objectives of the greenhouse competition study, described in Chapter 4, were to determine if (i) mycorrhizas affect plant competition at the interspecific or intraspecific level, (ii) competition is dependent on soil nutrient concentration and (iii) competitive abilities are related to differences in P uptake kinetics. The goals of the growth chamber study, presented in the first section of the Appendix, were to assess (i) the contribution of mycorrhizal fungal hyphae to total plant P uptake and (ii) the competitive abilities of the AM and EM fungi with respect to each other.













CHAPTER 2
MYCORRHIZAS AND PLANT COMPETITION Introduction

Over the past several decades the perception of mycorrhizas has evolved from viewing them as a unique biological phenomena to understanding them as integral parts of ecosystems. Much of the literature on mycorrhizas has addressed issues pertaining to single plants. More recently, there has been a growing tendency to evaluate the synecological consequences of the mycorrhizal association. The employment of techniques such as minirhizotrons (Lussenhop and Fogel, 1993), image analysis (Smith and Dickson, 1991), root-excluding screens and radioisotope labelling, among others, is redirecting the field to a broader scale of ecology dealing with plant interactions and community structure. The challenges faced during the next decade will be even more complex, with the increasing need to study multi-organismal assemblages and their functions at the ecosystem level. The next steps towards a more holistic view of mycorrhizal function will be determined by technological advances that will allow us to gain knowledge of how microbial systems fit together into a cohesive unit. This knowledge will provide us with a better understanding of the environment and how to best manage it in a sustainable manner.

Ecosystem studies necessitate an understanding of the functional associations of organisms with each other and with their environment. For plants, one of the main


3








4

biological interactions is competition. The term competition will be used here as the interaction between two organisms requiring the same limiting resource, which results in the decreased growth, survival or reproductive capacity of one of the two organisms. Plants mainly compete for light, water and nutrients. Physiological flexibility, within genetic constraints, allows plants to adapt to changes in resource availability. Physiological flexibility is enhanced by a plant's symbiotic relationship with mycorrhizal fungi. Modification in physiology can result in alterations of nutrient absorption capacity (Marschner and Dell, 1994) and water relations (Safir et al., 1972), as well as enhance light utilization and capture (Krishna et al., 1981). Increased tolerance or resistance to other environmental stresses, such as plant diseases (Rosendahl and Rosendahl, 1990; Schonbeck, 1978), high heavy metal concentrations (Denny and Wilkins, 1987) or xenobiotics (Donnelly et al., 1993), also have been found in mycorrhizal plants. Although the vast majority of studies with mycorrhizas has been conducted with terrestrial, mycorrhizas also have been found in wetland plants and may function in nutrient uptake in vascular aquatic plants (Rickerl et al., 1994; Wigand and Stevenson, 1994).

The objective of this chapter is to review mycorrhizal effects on plant competition and community structure. However, to prepare the foundation for the synecology of the system, a review of the autecology of mycorrhizal plants is also presented.










The Autecology of the Mycorrhizal Symbiosis Nutrient Uptake: The Role of External Hyphae

During the past decade there has been a shift in mycorrhizal studies from quantification of the internal phase to assessing the external hyphal phase in soil. The external component of this symbiosis contributes to enhanced nutrient uptake of the plant primarily by extending the root's nutrient depletion zone (Sanders and Tinker, 1973). The depletion zone extends from 0.1-15 mm from the root surface, depending on the soil type and plant species (Barber, 1995). By computer modelling, Itoh and Barber (1983) determined that by doubling the length of root hairs, which have a diameter similar to some mycorrhizal hyphae, plant phosphorus (P) uptake would double. Doubling the rootP uptake rate, however, only increased P uptake by 15%. The mycorrhizal benefit is inversely proportional to the root hair length (Schweiger et al., 1992), indicating that root hairs partially offset mycorrhizal nutrient gains due to improved spatial exploitation. Various techniques have been developed to measure external hyphae (Sylvia, 1992; Dodd, 1994). As a primary tool, the use of fine-mesh screens to prevent roots from penetrating into hyphal compartments has permitted the separation of root and hyphal contribution to plant nutrient uptake (Ames et al., 1983; Schiiepp et al., 1987), as well as quantification of hyphal distribution and density.



Spatial exploitation and hyphae

As reviewed previously (Bolan, 1991; O'Keefe and Sylvia, 1991), and based on plant uptake theory (Barber, 1995; Nye and Tinker, 1977), the key parameters involved








6

in improved nutrient uptake by mycorrhizal plants include the amount of absorbing surface area, fungal growth rates, nutrient uptake kinetics and hyphal distribution. Hyphae can extend far beyond the nutrient depletion zone (primarily P) of roots. Using an exclusion screen technique, Li et al. (1991a) located hyphae of Glomus mosseae up to a maximum measured distance of 11.7 cm from roots of Trifolium repens L. after 49 d. Ectomycorrhizal rhizomorphs are likely to extend substantially further.

In a separate comparative study on arbuscular-mycorrhizal (AM) fungi and P uptake, Acaulospora laevis, Glomus sp. and Scutellospora calospora developed hyphae up to 11 cm from the roots of the host plant, Tnrifolium subterraneum L., after 47 d (Fig. 1). However, hyphal densities with increasing distance from the mycorrhizal roots were not the same for all fungi. Acaulospora laevis had a constant hyphal density up to 11 cm, while for Glomus sp. it decreased after 3 cm, and for S. calospora the highest hyphal density was observed closest to the root and declined exponentially thereafter. The hyphal P uptake rates for the three fungi (calculated average for 28-47 d) were 2.8, 0.8 and 0.6 fmol P m-' s-, respectively, with considerably higher rates for the initial 28-day period. The consequence of these differences was a substantial contrast in plant P content among the mycorrhizal treatments. The previously listed characteristics of absorbing surface area, fungal growth rates, nutrient uptake kinetics and hyphal distribution indicative of improved nutrient uptake were all favorable in the A. laevis treatment, which was also associated with the highest plant P content.

Depending on the mycorrhiza and the initial soil nutrient concentration, the contribution by hyphae to total plant nutrient uptake (Marschner and Dell, 1994;







7






30
u) A Control
25 0- Acaulospora laevis
0
20 V Scutellospora calospora

E 15

lO
J


o I
0 1 2 3 5 7 9 11 Distance From Roots (cm)









Figure 2-1. Length of external hyphae spreading from mycorrhizal roots of Trifolium subterraneum after (a) 28 days and (b) 47 days. Bars represent standard error of the mean [with permission from (Jakobsen et al., 1992)].








8

comprehensive review) has ranged between 7 to 109% for P (George et al., 1992; Li et al., 1991a; Li et al., 1991c; Pearson and Jakobsen, 1993), 16-25% for zinc (Kothari et al., 1991) and 53-62% for copper (Li et al., 1991c). In another study, in contrast to the control, mycorrhizal plants recovered 1.7 times more '5NH4+ applied 2 cm from the root compartment and 2.75 times more 15NH4 when applied at a distance of 5 cm (Johansen et al., 1993). This provides evidence for an increasing benefit with greater distance. If diffusion and mass flow of a nutrient are slower than hyphal transport, a mycorrhizal benefit could conceivably be derived even for nutrients that are not strongly adsorbed to soils. This was demonstrated when transfer of '5NO3- was increased in mycorrhizal treatments by over 400% under dry soil conditions (Tobar et al., 1994). These studies illustrate the capacity of the external phase of mycorrhizas to increase a plant's nutrient absorption. They also demonstrate that the response differs depending on the soil environment, the fungi involved and the spatial location of nutrients.



Uptake kinetics

Uptake kinetics can be quite different between mycorrhizal and nonmycorrhizal plants. Based on uptake models and mycorrhizal characteristics, however, differences in uptake kinetics appear to be of secondary importance compared to surface area and spatial distribution (O'Keefe and Sylvia, 1991). Few uptake studies comparing mycorrhizal and nonmycorrhizal plants have been performed since the review of O'Keefe and Sylvia (1991). Most studies have used root weight to standardize uptake parameters (Table 2-1). However, none have taken into account the surface area or weight









Table 2-1. Summary of nutrient uptake kinetic studies.


Fungus Host Nutrient Km I C. Reference concentration (/M) (/M) (J M)
Glomus fasciculatum Lycopersicon esculentum 1-100 KH2PO4 1.61-0.35 0.1-0.32' n.dc (Cress et al., 1986) (root segments)
none 3.9-42 0.10-0.251' n.d Glomus mosseae Glycine max 30 KH2PO4 20 58b n.d (Karunaratne et al., (whole plant) 1986) none 3.5 19b n.d. Pisolithus tinctorius Pinus caribea 20 Na2HPO4 3.89 0.30" 0.32 (Pacheco and (whole plant) Cambraia, 1992) none 16.44 0.23a 11.98 Glomus Zea mays 1.5-1070 Zn 5.3-0.38 0.08-0.47' n.d. (Sharma et al., 1992)
macrocarpon (root segments)
none 4.5-0.95 0.03-0.55" n.d.


"a mol g fresh weight hb nmol m-2 s-"
n.d. = not determined



'.0








10

contributed by mycorrhizal hyphae, which could alter the calculated kinetic parameters. Also, in uptake experiments, the use of whole plants rather than root segments would be more appropriate, since this would incorporate possible source-sink effects of the plant. The information available suggests that there may be differences in the K., I, and C, values of mycorrhizal and nonmycorrhizal plants. The term K. represents the substrate concentration at which the uptake rate is half of the maximum influx rate, I The value Ca is the minimum solution concentration from which a nutrient can be absorbed. Studies that have estimated P uptake parameters have generally found a higher I,, for mycorrhizal plants. Uptake kinetics are dependent on the solution nutrient concentration (Cress et al., 1979; Sharma et al., 1992), and, consequently, selection of relevant soil nutrient concentrations in experiments is critical. For example, both P and Zn appear to have more than one concentration dependent uptake system.

Uptake kinetics may have a major role in mycorrhizal plant survival when nutrients are limited. In nature, reduced availability of nutrients occurs due to fixation and biological immobilization. With two competing plant species, the one with the lower C, value would be at a competitive advantage, because it can reduce the nutrient concentration below the Ca value of the other organism (Tilman, 1982). In fact, Pacheco (1992) demonstrated a lower Ca for ectomycorrhizal compared to nonmycorrhizal pine, which suggests a potential advantage for mycorrhizal plants. Unavailable nutrients may be released in pulses when microbial activity is temporarily stimulated due to environmental conditions. Under these circumstances, plants with differing uptake strategies, such as emphasis on Ca in one plant versus emphasis on I,








11

in another, could survive in the same environment due to niche separation. Mycorrhizas may add flexibility to a plant's physiological strategy by allowing it to profit from a broader range of nutrient uptake mechanisms. Furthermore, mycorrhizas may add an element of efficiency to soil nutrient exploitation by roots. Campbell et al. (1991) proposed that plant species primarily acquire resources either by efficiently exploiting a given resource (precision foraging) or by extensive development of roots and occupation of a high resource site (scale foraging). Although untested, mycorrhizas may give an advantage to the plant group with a tendency to efficiently exploit a given soil volume by accessing nutrients outside of the root's nutrient depletion zone.



Exudates and secretions

Root and hyphal secretions and exudates modify nutrient availability in the soil (Darrah, 1993; Duff et al., 1994). Several different mechanisms are involved and include release of enzymes, chelating agents such as organic anions and siderophores, and changes in rhizosphere pH by CO, from respiration and H' excretion during uptake of cations.

Nitrogen and phosphorus are often present in organic forms (Stevenson, 1986), which are less available than the organic forms. Ectomycorrhizal and ericoid fungi permit plant utilization of organic N from proteins and peptides (Abuzinadah and Read, 1986; Abuzinadah and Read, 1989; Bajwa et al., 1985; Bajwa and Read, 1985), which are otherwise unavailable N sources to plants (Abuzinadah and Read, 1986). Arbuscularmycorrhizal fungi do not appear capable of utilizing complex organic-N sources (Frey








12

and Schiepp, 1993) and, thus, probably lack any significant protease release. Phosphatases are produced by plant roots (Duff et al., 1994; Tarafdar and Claassen, 1988; Cumming, 1993) and release P from bound organic forms close to the root. Phosphatase release by mycorrhizal fungal hyphae also has been demonstrated (Antibus et al., 1992; Jayachandran et al., 1992; Tarafdar and Marschner, 1994) and may increase plant uptake of P; however, mycorrhizal contribution to total P uptake by this particular mechanism has not been quantified yet. Extension of hyphae beyond the root depletion zone would permit solubilization and uptake of P from these unavailable organic-P forms. For some nonmycorrhizal plants, mobilization of the organic-P fraction can approach one third of the total P absorbed (Jungk et al., 1993). Proton release by roots, in part to compensate for NH4+ uptake, can create a substantially lower rhizosphere pH (Marschner and R6mheld, 1983) and acidification of the soil also has been documented in mycorrhizal systems (Li et al., 1991b). Although this enhances dissolution of iron (Fe), and consequently bound P, the rate of protonation is slower than by the chelating mechanism (Schwertmann, 1991).

The availability of inorganic P bound to aluminum (Al) and Fe minerals can be increased by organic anions, such as oxalate (Fox et al., 1990), by the processes of chelation, ligand exchange and dissolution of the metallophosphate complex. Ectomycorrhiza, which form fungal mats, are capable of substantially altering the chemical soil environment by increasing the oxalate anion concentration by several orders of magnitude (Griffiths et al., 1994). Soil-solution phosphate in these mats is strongly correlated with oxalate concentration. Fungi most likely vary in the quantity of organic








13

acids released, resulting in differences in mineral weathering rates, nutrient release and subsequent benefit to plants. Chelation of Al not only releases bound P, but also lowers the free ion activity and thus reduces Al toxicity to the plant (Arp and Strucel, 1989). This same mechanism may apply to other metals. Chelating agents specific to Fe are termed siderophores and are produced by plant roots (Marschner, 1986), as well as by several mycorrhizal fungi (Cress et al., 1986; Schuler and Haselwandter, 1988; Watteau and Berthelin, 1995). Watteau and Berthelin (1995) found mycorrhizal siderophores of the hydroxylate type to be more effective chelators than organic anions and less specific for Fe than for Al.

The question of accessibility of organic compounds as carbon (C) sources to mycorrhizal fungi has been debated (Harley and Smith, 1983). Recently, the ectomycorrhizal fungi Cenococcum geophilum, Laccaria bicolor, Rhizopogon vinicolor and Suillus lakei were shown to utilize C from hemicellulose, cellulose and less readily from a humic polymer mix or from Pseudotsuga menziesii needles (Durall et al., 1994). Tanesaka et al. (1993) reported that several ectomycorrhizal fungi apparently did not have the ability to degrade complex C substances such as wood. Haselwandter et al. (1990) found several ericoid and ectomycorrhizal fungi capable of lignin degradation. At present, the ability to degrade organic matter has not been documented for AM fungi. Nonetheless, they appear to be efficient at capturing P released by decomposers prior to its being immobilized again (Joner and Jakobsen, 1994), possibly due to an advantageous spatial distribution. In general, the question of mycorrhizal fungal hyphae accessing








14

nutrients in forms not available to nonmycorrhizal plants may prove to be less important than the spatial accessibility of nutrients beyond the root's nutrient depletion zone.

Fungi and plants release polysaccharides resulting in a direct mucilaginous connection to soil particles in their respective rhizospheres and hyphospheres. This matrix may enhance aggregate formation, reduce nutrient loss from leaching and reduce dehydration by increasing waterholding capacity (Chenu, 1993). Furthermore, the connections to soil particles permit direct transfer of nutrients from soil to root (Uren, 1993), which may be essential under low moisture conditions, very similar to the contact exchange described by Nye and Tinker (1977).



Other Mycorrhizal Effects

Water relations

Improved water relations in mycorrhizal plants have been documented extensively (Nelsen, 1987) and have been associated with improved plant-P status (Nelsen and Safir, 1982), though non-P responses are also reported (Aug6 et al., 1986; Bethlenfalvay et al., 1988; Davies, Jr. et al., 1992). Drought tolerance in mycorrhizal plants is enhanced by increasing plant turgor, leaf water potential, stomatal conductance and root hydraulic conductivity. In addition, Bethlenfalvay et al. (1988) have suggested that mycorrhizal fungi are able to acquire soil water at lower water potentials than roots, although Nelsen (1987) proposed that fungal hyphae gave the plant a spatial advantage by extending the water depletion zone beyond the root. Recently, Ruiz-Lozano et al. (1995) found differences in proline concentration in drought-stressed mycorrhizal plants and suggested








15

that changes in osmotic potential may contribute to their improved drought tolerance over nonmycorrhizal plants. Most studies with AM fungi do not show any major water transfer via hyphae (George et al., 1992; Nelsen and Safir, 1982), although this is not always the case (Faber et al., 1991). In contrast, ectomycorrhizal fungi appear able to directly transfer water to the plant (Boyd et al., 1985), especially through rhizomorphs (Duddridge et al., 1980).



Carbon costs

The benefits of enhanced nutrient uptake associated with mycorrhizal biomass production has energy costs associated with it that vary with the symbiosis. The plantmicrobe-soil interactions are unique to each environment and correspondingly the mycorrhizal response may vary (Sylvia et al., 1993). This may depend on the fungus, such as differing growth responses observed with 20 isolates of Pisolithus spp. on Eucalyptus grandis (Burgess et al., 1994). Responses also vary with plant species and cultivars of the same plant species (Krishna et al., 1985; Mrtensson and Rydberg, 1995; Smith et al., 1992). So, although host specificity per se has not been documented clearly, host specific responses do exist. These differences may be related to root morphology, as suggested by Baylis (1975). Negative relationships have been found between mycorrhizal dependency and root fibrousness (Hetrick et al., 1992; Pope et al., 1983) or root hair length (Crush, 1974). However, as suggested by Graham et al. (1991), other undetermined factors aside from root architecture are more likely involved. Carbon cost, measured as energy expended per unit nutrient absorbed (Tinker et al., 1994), varies








16

between different mycorrhizal associations. The observed variation in mycorrhizal growth response among closely related plants may relate to differing strategies of plant-C allocation to the symbiosis (Graham and Eissenstat, 1994), as well as to plant age (Eissenstat et al., 1993). Pearson and Jakobsen (1993) quantified the P-uptake efficiency (C utilized/P absorbed) for three different AM fungi. For each unit of P absorbed they found that Scutellospora calospora and a Glomus sp. utilized 25 and 16 times more C, respectively, than Glomus caledonium. Total-C partitioning belowground was higher in the less efficient mycorrhizal fungi, indicating that energy efficiency of the symbiosis may be one reason for differing plant growth responses to fungi.

When comparing carbon costs of mycorrhizal to nonmycorrhizal plants, 4-36% more of the total C fixed is allocated belowground due to mycorrhizas (Durall et al., 1994). To distinguish nutritional from other mycorrhizal effects on plant-C balance, mycorrhizal plants were grown at high soil-P concentrations and demonstrated a 37% higher belowground carbon allocation than nonmycorrhizal plants (Peng et al., 1993). Of this 37%, 51% was attributed to greater root biomass and 10% to construction costs of lipid-rich roots most likely associated with the mycorrhizal fungus. Enhanced photosynthesis in mycorrhizal plants can compensate to varying degrees for this increased C drain (Dosskey et al., 1990; Kucey and Paul, 1982). Plant root turnover is also associated with a high C cost, although few studies have assessed the role of mycorrhizas in controlling this process. Durall et al. (1994) determined that ectomycorrhizal roots have a lower root turnover rate than nonmycorrhizal roots. In environmental conditions where nutrient pulses occur, roots with a lower root turnover rate demonstrated a








17

competitive advantage (Campbell and Grime, 1989). This suggests that mycorrhizal plants may profit from the reduced root turnover rate by having to invest less C into nutrient absorbing structures.



Plant fitness

Although many of the previous topics dealt with improving plant growth and stress adaptation, few mycorrhizal studies have directly addressed mycorrhizal influence on plant fitness, that is the plant's ability to increase its numbers proportionately to other species (Begon et al., 1986). Enhanced efficiency of resource acquisition by mycorrhizal plants allows more energy to be allocated to growth and reproduction, which potentially increases plant fitness. The result in the next generation may be expressed in terms of improved survival, growth rate or reproduction. Mycorrhizal plants have displayed increased seed number, seed weight and P and N content (Lu and Koide, 1994), with some benefits still significantly expressed in the second generation of offspring grown in the absence of mycorrhizal fungi (Koide, T. and Lu, 1992). Increased P status of seed has been associated with subsequently higher P content and plant biomass (Bolland and Paynter, 1992). For some plant species, the presence of mycorrhizas enhanced seedling emergence rate (Hartnett et al., 1994). High P concentration in seed has resulted in increased number of emerging seedlings and a higher rate of emergence (Thomson and Bolger, 1993), factors which also have been identified as important predictors of competitive success in secondary succession (Stockey and Hunt, 1994).








18

Xenobiotics

In many ecosystems plants and mycorrhizal fungi are exposed to a wide variety of toxic compounds (xenobiotics and in some instances naturally occurring toxic compounds). Mycorrhizal fungi may effectively mediate and alter the interaction between plant and xenobiotic compounds. Various papers have assessed or reviewed pesticide effects on mycorrhizal fungi (Dehn et al., 1990; Trappe et al., 1984). Mycorrhizal fungi may function in the translocation of herbicides. In one study with apple and three herbicides (dichlobenil, paraquat and simazine), root dry weight of noninoculated plants exposed to herbicides was reduced by 46% in contrast to a 63 % decrease in mycorrhizal plants (Hamel et al., 1994). Although no effect on hyphal length was found at the highest simazine concentration applied, 75 % of the mycorrhizal plants died compared to none in the control treatment. The authors attributed this to facilitated herbicide flow to the host plant mediated by the mycorrhizal fungus. Uptake and translocation of the herbicide atrazine was also found in mycorrhizal corn, which is atrazine-tolerant (Nelson and Khan, 1992). Although the quantity absorbed was small compared to direct root uptake, the question of how this may affect an atrazine-sensitive plant remains unanswered. Certain mycorrhizal fungi also have demonstrated the capacity to degrade herbicides such as atrazine and to a lesser extent 2,4-dichlorophenoxyacetic acid (Donnelly et al., 1993). This provokes the question as to whether mycorrhizal fungi offer some protection against xenobiotics. In corn and sorghum certain herbicide safening effects by AM fungi have been found against the herbicides imazaquin, imazethapyr and pendimethalin (Siqueira et al., 1991).








19

Metal cations and soil acidity

High metal cation concentrations can be toxic to plants. The high solubility of Al, due to the acidic nature of Oxisols and Ultisols, is a growth-limiting factor for plants in many tropical countries. Natural selection of mycorrhizal ecotypes leads to varying genotypic sensitivity to soil acidity (Robson and Abbott, 1989), as well as to high metal concentrations (Gildon and Tinker, 1983; Griffioen et al., 1994). Several studies have found mycorrhizas capable of alleviating toxic effects to plants caused by Al, cadmium, Cu and Zn (Bradley et al., 1982; Colpaert and Van Assche, 1993; Denny and Wilkins, 1987; Dueck et al., 1986; Koslowsky and Boerner, 1989). Two mechanisms currently explain this response. Firstly, electronegative sites on the hyphal cell walls bind the positively charged heavy metal cations (Denny and Wilkins, 1987; Galli et al., 1994). The observation that under acidic soil conditions heavy metal uptake is increased (Killham and Firestone, 1983) partly confirms this. It is possible that protonation of negatively charged sites in the plant or fungal walls results in less binding and greater uptake of the metal cation. The second path is the immobilization of the cations by complexation in vacuoles with polyphosphates (Martin et al., 1994) or associated metallothionein-like peptides (Turnau et al., 1994).



Synecology

Co-evolution of mycorrhizal fungi and plants has been suggested (Allen, 1991; Harley and Smith, 1983). Since selection for more fit species occurs continuously, and a larger proportion of plants show mycorrhizal dependency than not, it follows that there








20

must be some measure of improved fitness derived from mycorrhiza; otherwise the symbiosis would have been selected against. The alternative is that mycorrhizal fungi are parasites with maximum adaptability to plant resistance strategies. This, however, is unlikely considering the exchange of nutrients between the two organisms, which is characteristic of a true mutualism.

The effects of the mutualism on plant growth and survival influence interactions beyond the single plant level (Brundrett, 1991; Francis and Read, 1994). Plants rarely grow alone, except in extreme or anthropogenic environments, and consequently end up competing for similar resources, especially inorganic nutrients, water and light. Under conditions limiting growth, mycorrhizal plants have distinct competitive advantages. Thus, from a holistic and functional perspective, mycorrhizal research reaches its full value when applied to natural or managed ecosystems where interactions occur. Current issues pertain to the involvement of mycorrhizas in plant community development, stabilization and diversity, as well as to questions of environmental sustainability and the economics of agricultural production systems. A relevant question, then, is to what extent is the force of this symbiosis manifested in plant communities?



Plant Interactions

During competition, plants utilize several different strategies for optimal resource capture with many of them overlapping those found in the mycorrhizal symbiosis. The choice of strategy depends primarily on a site's resources and the amount of disturbance (Grime, 1979; Tilman, 1982). Literature summarized in the first part of this chapter has








21

shown that mycorrhizas can enhance resource capture. Environmental factors strongly influence the mycorrhizal benefit derived by a plant and consequently also its competitive ability.



Resource competition

Competition occurs when a resource is inadequate to meet the needs of the competitors. Nutrient availability fluctuates with the chemical environment and moisture content of the soil. Soil heterogeneity frequently compounds the intensity of competition in some areas, since resources are not evenly distributed. Phosphorus has been the focus of mycorrhizal research, because it is required by plants in proportionately large quantities, and yet, in the soil it is easily immobilized chemically and biologically. Consequently, the use of P also dominates mycorrhizal studies involving competition.

When mycorrhizal plants compete under nutrient-limiting conditions, niche differentiation may be of considerable importance. Plants competing intraspecifically will have similar nutrient requirements and acquisition strategies which may vary depending on plant age. Conversely, in interspecific interactions, some competition may be alleviated by niche differentiation. For example, a potential growth response associated with spatial niche separation by roots of two grass species only became evident by experimentally increasing soil depth (Van Auken et al., 1994). The varying plant responses to different mycorrhizal species in the literature suggest the involvement of a combination of the earlier reviewed mechanisms, including hyphal spatial distribution and access to less available nutrients. However, if the mycorrhizal contribution to nutrient








22

uptake is primarily related to spatial niche differences between roots and hyphae, then larger soil volumes would be preferable in experiments; otherwise root nutrient depletion zones quickly overlap and the potential mycorrhizal benefit is not realized (O'Keefe and Sylvia, 1991). As an intermediate approach between pot and field competition studies, artificial micro- or mesocosms have been used (Campbell et al., 1991; Grime et al., 1987), which, among other things, allow for the exploration of large soil volumes by external hyphae, the creation of resource gradients or patches and the longer-term monitoring of plant growth and reproduction in a regulated environment. Further consideration should be given to the incorporation of an unsterilized soil control into experiments. The inclusion of plant pathogens, soil arthropods and microbes which affect resource abundance and mycorrhizal plant growth (Newsham et al., 1994), as well as subsequent plant interactions, would provide a more realistic extrapolation of experimental results to natural phenomena.

A number of mycorrhizal plant competition studies have demonstrated that AM fungi affect competition to varying degrees (Brown et al., 1992; Francis and Read, 1994; Fitter, 1977; Hetrick et al., 1989; Hartnett et al., 1993; Newman et al., 1992). Plant competition between two host plants involving a single species of AM fungus account for the majority of the data. Apparently only one plant competition study dealt with different groups or species of mycorrhizal fungi and it is also the only EM plant competition study (Perry et al., 1989). There is evidence that competitive success is related to mycorrhizal dependency (Hartnett et al., 1993; Hetrick et al., 1989). Mycorrhizal dependency is very variable and depends on the particular environment and host plant. Hartnett et al. (1993)








23
and BMith and Hayman (1984) determined that, in a given soil volume, mycorrhizal benefit for a plant decreases with increasing density of its competitors. Higher plant density is paralleled by an increase in root and hyphal density in the soil and proportionately greater overlap of nutrient depletion zones. In intraspecific competition of inoculated plants of high mycorrhizal dependency, density-related competition was observed, but this did not occur when mycorrhizal fungi were absent. Inoculated plants with low mycorrhizal dependency lacked this response, indicating their ability to more efficiently extract nutrients from the soil than the nonmycorrhizal plants with high mycorrhizal dependency.

Plants of the same species but different plant age also have been compared for competitive interactions. Eissenstat and Newman (1990) evaluated the possible advantages of mycorrhizas to seedling establishment in the presence of an older plant of the same species. The results indicated that there is not a facilitative but rather a competitive relationship between the two plants, similar to that observed in the absence of mycorrhizal fungi. In another study, Franson et al. (1994) found that competition intensity between an established and a seedling soybean plant was not altered by increasing the stress on the younger plant.

Plant competitive interactions between mycorrhizal host and nonhost plants have been investigated in a limited number of studies. It is worth noting that some have documented a reduction in biomass of nonhost plants when such plants were grown under mycorrhizal conditions (Allen et al., 1989; Ocampo, 1986). Francis and Read (1994) found evidence for a chemical factor, which was extracted from soil of mycorrhizal








24

plants, that inhibited root growth of nonhost plants. This suggests that mycorrhizas may have effects beyond those currently known.

In summary, mycorrhizas can enhance a plant's competitive ability, and the effect is generally associated with increased nutrient uptake. The greatest benefit of mycorrhizas appears to lie in their ability to buffer the plant from adverse environmental conditions that reduce resource availability.



Mycorrhiza-mediated reduction of competition

With most plants possessing similar nutritional requirements, competition is a key factor in their interactions. The existence of hyphal connections between plants is well known. Various studies, especially those using root-excluding screens, have unequivocally demonstrated that nutrient transfer between root zones of a donor and receiver plant can be mediated by mycorrhizal hyphae (Newman, 1988; Newman et al., 1992). Although it is possible for hyphae from the receiver mycorrhiza to scavenge nutrients from the rhizosphere of the donor plant, most likely the majority of transfer is by direct hyphal connections between plants. For example, radio-labelled C from an ectomycorrhizal donor plant has been found solely in ectomycorrhizal plants and not in neighbor AM neighbor plants; by using autoradiography, no visual evidence existed of a direct interspecific C transfer between intermingling roots of donor and receiver plants (Read et al., 1985). In another study, 46% of the total C transferred directly from plant to plant was via mycorrhizal connections, 15 % of uptake was indirectly mediated by mycorrhiza, and 39% was translocated by other processes (Martins, 1993). These








25
fractions could be verified further by comparing nutrient transfer from a mycorrhizal donor plant to either a myc- mutant (a mutant plant not able to form mycorrhiza) or a normal mycorrhizal receiver plant. The quantity obtained by the receiver is variable, and appears to depend on the nutrient involved. Generally, P is not transferred at fast rates (Newman and Eason, 1993) or in quantities that significantly affect growth (Ikram et al., 1994). The transfer of N by mycorrhizas has been documented (Newman, 1988), with most studies utilizing a legume, because of its importance in intercropping systems, as the donor plant. The quantity of N transferred from the root zone of donor plant to the receiver plant varies (Bethlenfalvay et al., 1991; Frey and Schiiepp, 1993). By increasing competitive pressures for N in intercropping systems, mycorrhizal fungi at certain times may enhance nitrogen fixation (Barea et al., 1989), although this is not always the case (Reeves, 1992). Both of these studies and others (Hamel and Smith, 1991; Ikram et al., 1994) have found minimal amounts to no N transferred. The quantitative significance of mycorrhizal transfer of nutrients to total uptake by the receiver plant still remains unclear.

The phenomenon of increased survival of certain plant species in mycorrhizal microcosm studies (Grime et al., 1987) deserves further attention, especially, since no direct cause was found. Source-sink gradients, such as those created by shading or lownutrient status of one plant, have been suggested as the force behind nutrient transfer. For interplant C transfer, shading of the receiver plant increased C translocation to that plant (Read et al., 1985). However, shading does not always produce this effect (Franson et al., 1994; Hirrel and Gerdemann, 1979). In contrast, clipping of leaves to simulate








26

herbivory and to produce a C sink resulted in C transfer away from the clipped plant (Waters and Borowicz, 1994). In settings where young seedlings compete for nutrients with established plants, the seedlings become more quickly colonized by the preexisting mycorrhizal network; however, no further benefit to the seedlings was detected (Franson et al., 1994; Eissenstat and Newman, 1990). In Grime's (1987) study, '4C-labelling of one dominant plant resulted in substantially more C being transferred to subdominants when plants were mycorrhizal compared to nonmycorrhizal. Although competition does occur in these systems, several plants colonized by the same mycorrhizal type will be closely tied together by the hyphal network and may benefit from C transfer among plants.



Environmental Conditions and Plant Competition Non-resource edaphic factors

Several soil characteristics may indirectly influence the assorted mycorrhizal mechanisms that enhance a plant's competitive ability. Soil acidity is an important factor influencing soil nutrient availability. Acidic soils are a natural result of soil weathering, and, as stated earlier, Al toxicity is one of the main associated problems. Mycorrhizas may enable a plant to survive unfavorable conditions caused by toxic concentrations of metal cations, including Al (Koslowsky and Boerner, 1989). Although mycorrhizas may facilitate growth of plants under acidic soil conditions, I am not aware of any studies that systematically address the effect this may have on plant competition.








27

Soil chemical processes associated with organic matter turnover and mycorrhizas may also play a yet unstudied role in plant interactions. As organic matter is degraded by microbes, various compounds, including phenolic materials, are released to the soil. Phenolics have been implicated in various allelopathic interactions (Rice, 1984). Researchers have demonstrated both inhibition and stimulation of mycorrhizal fungi by phenolic compounds (Baar et al., 1994; Boufalis and Pellissier, 1994; Siqueira et al., 1991). Different microbial responses to phenolics have been attributed to variability in degradation capacity of the microbes, phenolic concentration, soil characteristics and availability of inorganic soil nutrients (Blum and Shafer, 1988). Similarly, mycorrhizal fungi vary in their capacity to chemically alter different forms of phenolic compounds (Giltrap, 1982; Ramstedt and Soderhall, 1983; Tam and Griffiths, 1993). Garbaye (1994) hypothesized that phenolic compounds may be degraded by bacteria closely associated with mycorrhizal fungi, thereby also enhancing the establishment of mycorrhizal fungi. Although no clear link has been found between mycorrhizal sensitivity to phenolic compounds and plant competitive ability, the results of a few studies suggest a possible connection (Leake et al., 1989; Wacker and Safir, 1990). Because mycorrhizal fungi occur in competitive environments, such as forests, with the potential of allelopathy (Horsley, 1987; Pellissier, 1994), it is important to determine what growth-limiting factors, as well as their magnitudes, actually occur. Although it is difficult to distinguish resource competition from interference competition, several researchers have been successful in differentiating these two phenomena (Nilsson, 1994; Shilling et al., 1992; Thus, 1994; Wardle et al., 1994).








28

Associated soil biota

Fitter and Garbaye (1994) have summarized the current information about belowground interactions of mycorrhizas and rhizosphere microbes. Unfortunately, few studies have addressed how these interactions affect plant populations or communities. Mycorrhizas influence the rhizosphere environment by modifying plant exudation and rhizodeposition (Leyval and Berthelin, 1993), and subsequently affect microbial composition and metabolic activity in varying degrees. Inversely, certain fluorescent pseudomonads and spore-forming bacilli, similar to growth-promoting rhizobacteria, may significantly regulate the mycorrhizal benefit to the plant (Garbaye, 1994; Schreiner and Koide, 1993); however, mechanisms of action are still largely unknown. These bacteria have demonstrated some fungal, but not plant, host specificity. With appropriately matched mycorrhizal fungi and bacteria it is conceivable that a plant would possess a competitive advantage over other plants without selected associations. Rabatin and Stinner (1991) reviewed the effects of microfauna, many of which are fungivores, on mycorrhiza. As an example, Boerner and Harris (1988) conducted a competition study between mycorrhizal Panicum virgatum and the nonhost Brassica napa, where the addition of Collembola reduced the competitive ability of the grass, resulting in a reduction of biomass compared to the mycorrhizal P. virgatum without competition.

Studies of plant disease control by mycorrhizas interacting with plant pathogens have yielded variable results (Linderman, 1994; Duchesne, 1994). Various mechanisms have been reported that are unique to the environment, host and microbes involved. Based on field studies utilizing the fungicide benomyl, Carey et al. (1992) suggested that,








29

aside from direct physiological benefits to the plant, mycorrhizal contributions to plant health in the field may be a common but subtle phenomenon, because it is buried within complex interactions.

To make the situation more complex, few studies have included interactions between mycorrhizas and other plant endophytes (Clay, 1992). The fungal endophyte Acremonium sp., for example, has reduced colonization and reproduction by Glomus sp. (Chu-Chou et al., 1992; Guo et al., 1992). Reduction of insect herbivory has been attributed to secondary metabolite production by fungal endophytes (Clay, 1991). Another study found that mycorrhizas may reduce feeding inhibition of an insect herbivore induced by Acremonium sp. (Barker, 1987). Additionally, nonmycorrhizal endophytes are capable of altering competitive relationships between plants (Clay et al., 1993) and plant drought resistance (White, 1992) in ways similar to mycorrhiza. The data suggest that endophytes are involved in various effects observed in plant studies and consequently they deserve further consideration.



Herbivory

Herbivores generally have either an inhibitory or neutral effect on mycorrhizas (Barbosa et al., 1991; Gehring and Whitham, 1994). Herbivory results in increased plantC allocation to the replacement of aboveground parts instead of to maintenance of the mycorrhizal symbiosis (Jones and Last, 1991). There are also a few studies on the inverse effect of mycorrhizas on herbivores (Gange et al., 1994; Rabin and Pacovsky, 1985). Generally, mycorrhizas had an inhibitory effect on the herbivorous insects. Gange








30

and West (1994) found that compared to fungicide-treated plants, mycorrhizal plants had lower soluble neutral sugars, starch, total N, and amino acids (alanine and tyrosine/valine) and a higher concentration of the anti-feedant chemicals, aucubin and catalpol. In their study, chewing insects were negatively impacted when feeding on mycorrhizal plants; however, sucking insects developed better on mycorrhizal plants. The authors hypothesized that a higher C/N ratio in the mycorrhizal plants allowed more C to be allocated to plant defense mechanisms, such as secondary plant metabolite production. Localization of the secondary metabolites may partly account for the differential response between insect types. Viewed in terms of plant competition, plants able to efficiently modify their C balance to simultaneously reduce insect pests and still maintain the mycorrhizal association may have a competitive advantage in the long run.



Plant Succession and Community Structure

Limited information is available on the ecological relevance of mycorrhizas in plant competition. Plant competition can be viewed in terms of single plant interactions, but its importance lies at the population and community levels. The interactions occurring at the ecosystem level are obviously complex and many have been set aside for the sake of simplicity. As has been suggested by various authors (Brundrett, 1991; Francis and Read, 1994; Newman, 1988) mycorrhizas are likely involved in plant community structuring, but the magnitude of their effect is unknown. Increasing the competitive ability of individuals within a population enhances their ability to capture resources and improves their fitness. One of the major components determining early succession is








31

plant competition for limited nutrients (Wilson and Shure, 1993). Under nutrient limitations, resource acquisition enhanced by mycorrhizas occurs at the expense of other plants, which results in the highly competitive plants becoming more abundant and dominant in the community. Continuous growth of a plant in the same soil eventually will select a microbial community well adapted to that environment. Over time the adapted microbial community can become disadvantageous for growth of that plant species, but not for others, and, in this manner, may contribute to plant succession (Bever, 1994; Van der Putten et al., 1993). In these studies it was suggested that this negative feedback on growth may be related to pathogen buildup. Mycorrhizal fungi were not considered, because of the assumption that mycorrhizal effects are usually beneficial. However, if there is a selection for less efficient mycorrhizal fungi occurring over time, then this may similarly contribute to succession by decreasing a plant's C-use efficiency and its competitive ability. In monocultural settings, a shift of mycorrhizal fungal species composition over time was identified by Johnson et al. (1992a; 1992b) and Wacker et al. (1990). In both cases there was an associated decline in plant growth, indicating that mycorrhizal fungi should not be discarded a priori as a contributing factor to growth declines.

Succession of ectomycorrhizal fungi from "early" to "late" stage fungi occurs in undisturbed forest systems (Deacon and Fleming, 1992). Differing fungal resource requirements, as well as changes in other soil microbial components, have been postulated to cause the succession (Garbaye, 1994). Recent research indicates that this succession may be tied closely to factors found in the soil organic matter. Removal of








32

litter and humus in Pinus sylvestris stands increased mycorrhizal fungal species richness and reverted the species composition to the early successional types (Devries et al., 1995). In other systems, the increased buildup of organic matter also has been associated with higher concentrations of phenolic compounds (Kuiters and Sarink, 1986; Leake et al., 1989), which have demonstrated allelochemical effects. Perhaps resistance to and the ability to degrade phenolic compounds determines which fungal species are capable of growing at a certain stage of succession. Leake et al. (1989) demonstrated that ericoid mycorrhizas were capable of enhancing ericoid plant growth and survival, possibly by a detoxification mechanism. Whereas AM fungi are found more commonly in mineral soils, ectomycorrhizal fungi are often associated with environments high in organic matter and are physiologically adapted to utilizing complex substrates (Francis and Read, 1994). Also, ectomycorrhizal mantles surrounding root tips are capable of protecting these from potentially toxic compounds. As a consequence, tolerance to adverse environmental conditions allows the plant to focus more of its energy on resource acquisition strategies without substantial tradeoffs of energy for other mechanisms, thereby making it a better competitor.

Plant competition, as affected by mycorrhizal fungi, could be relevant in plant community structuring and succession. As such, mycorrhizal benefits to single plants may prove functionally significant at the ecosystem level. In addition, positive interactions in communities are often neglected (Bertness and Callaway, 1994) and should also be considered in the discussion of plant interactions mediated by mycorrhizas (Amaranthus and Perry, 1994). Mycorrhizas can moderate plant competition (Perry et al., 1989) and







33

provide resilience to disturbance (Amaranthus and Perry, 1994). Mycorrhizal connections between dying and living plants also limit soil nutrient loss by leaching and immobilization (Eason and Newman, 1990). The network of hyphal bridges connecting neighboring plants can affect coexistence by increasing species richness and diversity (Gange et al., 1993; Grime et al., 1987). The current literature indicates that this is perhaps more likely due to transfer of C than of inorganic nutrients. Furthermore, a higher plant species diversity has been associated with increased ecosystem stability in a stressed environment (Tilman and Downing, 1994). Obviously, with the multitude of effects and interactions mediated by mycorrhiza, a quantification of the net mycorrhizal influence in ecosystems is a formidable challenge. Still, with the current emphasis on environmentally sound management of ecosystems, it is important to include them in considerations of appropriate technologies in managed ecosystems.













CHAPTER 3
LIMITATIONS IN THE USE OF BENOMYL IN EVALUATING MYCORRHIZAL FUNCTIONING

Introduction

A limitation to mycorrhizal field research is the difficulty of obtaining an appropriate nonmycorrhizal control, since plants in nature are normally colonized. Soil fumigation has been used to control mycorrhizal fungi; however, the broad biocidal effects limit the usefulness of this technique. Fungicides are more specific and alter fewer biological soil processes. Paul et al. (1989) summarized the ideal properties of a fungicide used to chemically exclude an organism from an experiment. The fungicide properties should include: (i) moderate persistence to reduce mechanical disturbance from the application process, (ii) an appropriate activity spectrum that targets selected organisms only and (iii) no direct physiological effects on the plant.

The systemic fungicide benomyl, a benzimidazole, has been used frequently to reduce arbuscular mycorrhizal (AM) activity in experimental treatments (Jalali and Domsch, 1975; Kough et al., 1987; Fitter and Nichols, 1988; Hartnett et al., 1994; Newsham et al., 1995; West et al., 1993). Benomyl's lack of direct effects on plants and somewhat selective effects against AM fungi (Zygomycetes) currently make it a better choice compared to other fungicides (Paul et al., 1989; Sukarno et al., 1993). Nonetheless, the amount of mycorrhizal control achieved with benomyl has varied. Reduction of colonization or biomass of mycorrhizal plants has been observed in several 34








35

cases (Evans and Miller, 1988; Fitter and Nichols, 1988; Trappe et al., 1984), but these results are not always achieved (Koide et al., 1988; Fitter, 1986; Trappe et al., 1984). Much of this variability is likely attributable to the experimental conditions such as soil type, method and timing of fungicide application and potentially more complex interactions occurring within the soil microbial community. For example, benomyl can inhibit nematodes (Elamayem et al., 1978) and different fungi that do not form mycorrhizas (Edgington et al., 1971), thereby indirectly altering mycorrhizal effects.

Several studies have addressed the effects of arbuscular mycorrhizas on plant interactions (Fitter, 1977; Hall, 1978; Newman et al., 1992), and some have utilized benomyl (Hartnett et al., 1993; Hetrick et al., 1989; Newsham et al., 1995) or other fungicides (Gange et al., 1993) to create control treatments. Only one study addressed the influence of EM fungi on plant competition (Perry et al., 1989). Very few studies have taken place under field conditions, and apparently none have addressed the role of mycorrhizas in the interactions between AM and EM plants. Benomyl's putative selective effect against AM fungi and neutral effects on EM fungi (Trappe et al., 1984) could be valuable in sorting out the individual benefits of these two types of mycorrhizal symbioses to different host plants competing for the same nutrients.

As part of a larger plant competition study between AM and EM plants, the usefulness of benomyl as a tool to selectively control mycorrhizas was tested. The main objectives were to: (i) compare the efficacy of benomyl in controlling mycorrhizas in the greenhouse to that in the field, (ii) differentiate effects of benomyl on external hyphae








36

from those on the internal mycorrhizal phase and (iii) determine if the intensity and longevity of the fungicide's effect was dose-dependent.



Materials and Methods

Field Study

The site was located 21 km northwest of Gainesville, Florida and was part of a larger plant competition study involving slash pine (Pinus elliottii Engelm. var. elliottii)) and weeds. Slash pine had been planted in April 1990 in beds approximately 26 cm in height and about 2 m in width with rows spaced approximately 213 cm apart. Soil was a Pomona fine sand (a sandy, siliceous, hyperthermic Ultic Haplaquod). The surface 10 cm of soil had 7 ltg P g-' extractable in 2 mM CaCI, and a soil solution with pH 3.9. Approximately 3.3% weight was lost upon ignition. The dominant weeds were Panicum chamaelonche Trin., P. aciculare Dec.ex Poir. in Lam., Andropogon spp., Paspalum spp. Rubus sp. and Serenoa repens. In December 1991, less than 1 spore of mycorrhizal fungi g' of field soil was detected; the populations consisted of a mix of Glomus sp., Gigaspora sp. and Scutellospora sp. In the greenhouse, pot cultures of P. chamaelonche originating from the field and grown in field soil yielded two AM isolates, Gigaspora rosea (INVAM FL224) and Scutellospora heterogama (INVAM FL225) which were submitted to and identified by J. Morton at INVAM.

Two areas (each 18.4 m by 11 m) containing slash pine and weeds were selected randomly for this study. The control plot received no fungicide sprays. Benlate* 50 DF (E.I. du Pont de Nemours & Co., Inc., Wilmington, DE) was applied to the second area








37
with a CO,-pressurized backpack sprayer by covering the area once and then making a second application perpendicular to the first. The first spray (2 April 1991) was applied at the rate of 5 kg benomyl ha-' using the equivalent of approximately 150 ml of water m2. Subsequent sprays (30 May, 11 July and 19 Sept. 1991) were applied at a rate of 20 kg benomyl hai'.

Panicum chamaelonche was chosen as the indicator plant of AM fungal activity because it was a dominant weed species at the site. Samples were taken on 2 April, 4 April, 30 May, 10 June, 2 July, 22 July, 13 August, 10 October 1991. At each sampling, three plants were selected randomly and removed from each plot. The roots were washed and cut into lengths of 1 to 2 cm. To determine fungicide effects on colonization and metabolic activity, 1- to 2-g subsamples of roots were stained at room temperature for 8 h in a solution containing 0.2 M Tris HCI (pH 7.4), 1 mg ml-' of iodonitrotetrazolium violet (INT) and 3 mg ml-' of NADH (Sylvia, 1988). This was followed by clearing the roots in a boiling, saturated solution of chloral hydrate for 10 min and subsequent counterstaining overnight in 0.5% aniline blue in lactoglycerol. The chloral hydrate treatment proved unnecessary and was eliminated in samplings collected after May. The roots were destained in lactoglycerol and a minimum of 25 1-cm-long root segments per plant were laid out parallel to each other on slides. The percentage of root segments with arbuscules and the percentage of total arbuscules that were active (those staining with INT) were estimated using bright-field microscopy at 400x magnification. The effect of benomyl on mycorrhizal development was evaluated using the relationship of time and either arbuscule abundance or activity. The slopes of linear regression of benomyl-treated








38

versus nontreated plants were compared using the General Linear Model procedure of SAS (SAS Institute, Inc., 1989).



Greenhouse Study

Both of the following experiments had completely randomized factorial designs (two mycorrhizal treatments x four benomyl levels) with seven replications each. To maintain uniform daylength of approximately 12 h, extra light (800 /Lmol m2 s-' from 17:00 to 20:00 hr) was provided. Plants in all experiments were fertilized semiweekly with 3.2 MM NH4NO3, 7.5 yM Ca(NO3),, 7.7 ttM KCI, 1.0 MM MgSO4, 20 nM NaFeEDTA, 5.0 nM CuSO4-4H20, 240 nM H3BO4, 20 nM MnC124H20, 5 nM Na2MoO4-2H20 and 20 nM ZnSO47H20. The nutrient solution for corn or pine contained, respectively, 3.2 nM H3PO4 or 0.32 nM H3PO4. All data were analyzed by analysis of variance using the General Linear Model procedure (SAS Institute, Inc., 1989). Both experiments were repeated once under similar environmental conditions.



Benomyl effects on pine

Slash pine seeds were disinfested for 2 min in a 5.25% sodium hypochlorite solution with 0.2 ml Liqui-Nox surfactant (Alconox, Inc., New York, NY) and then rinsed thoroughly with tap water. Plants were raised from seed for 12 d in a growth chamber [29 CO/23 Co (day/night), with a 15-h light period and irradiance of 1000 pmol m- s1-] in a vermiculite/sand (1:1) mix. They were then transplanted into sand in 50-ml pots (5 cm2 of surface area) grown in the greenhouse for 6 wk where they received water








39

only. Pisolithus tinctorius (Pers.) Coker & Couch (isolate S106) was grown with no shaking in a modified Melin-Norkrans liquid medium (Marx, 1969) containing glucose instead of sucrose. Just prior to use, fungal mats were washed with tap water, added to a food processor with water and chopped (Rousseau and Reid, 1990). Eight-week-old pines were inoculated with the fungus by immersing the washed roots in the suspension and then grown in the greenhouse in 500 ml of sand in DeepotsT (28 cm2 of surface area; McConkey, Co., Sumner, WA). Six weeks after inoculation, 10 ml of a suspension of Benlate* 50 WP in deionized water was applied once at 0, 20, 60 or 150 kg benomyl ha-' equivalent (based on pot surface area). Plants were grown from January to March 1993 under a mean photosynthetic photon flux density of 535 Amol m-2 s"' and 17/30.C (min./max.) temperature regime.

Groups of plants were harvested before, and then 2 and 4 wk after benomyl application. Prior to harvesting the plants, a soil core (15.5-mm diam. by 15-cm deep) was removed from each pot. Hyphal length and activity were evaluated by a slightly modified procedure of Sylvia (1988). A thoroughly mixed, 10-g, wet-mass subsample of soil was added to 500 ml of water and chopped in a Waring blender at the high setting for 20 seconds. The resulting suspension was allowed to settle for 20 seconds before a 25-ml portion was removed and filtered through a 0.45-ptm-pore size membrane (GN-6 Metricel"; Gelman, Ann Arbor, MI). The hyphae on the membrane were stained for

6 h with INT solution, destained with tap water, counterstained for 30 min with 0.1% trypan blue in lactoglycerol and destained again with tap water. Using a gridline-intercept








40

method, total and active hyphal lengths were determined microscopically at 400x from 20 randomly selected fields on the filter.

Pine needles were removed from seedlings and dried overnight at 65C, and P content was determined colorimetrically (Murphy and Riley, 1962). Ergosterol (a sterol found in fungal, but not plant, membranes) content in the root was used to provide a relative estimate of total fungal biomass present (Martin et al., 1990; Salmanowicz et al., 1989). Fresh roots were washed, ground in liquid nitrogen and thoroughly mixed. A 0.1to 0.3-g subsample was extracted overnight at room temperature with 5 ml of 100% ethanol. This sample was filtered through a 0.45-/Am syringe filter and then assayed for free ergosterol by high-pressure liquid chromatography (Waters 715 Ultra WISP, Gilson 115 UV detector). Separation was achieved on a C-18 column (Supelcosil" LC-18; Supelco, Inc., Bellefonte, PA) at 400C using a methanol-water mobile phase (92:8) flowing at 2 ml min-' with detection at 282 nm.



Benomyl effects on corn

The effect of benomyl on colonization by the AM fungus Glomus sp. (INVAM FL329, formerly FL906) was studied in a separate experiment. Germinated corn (Zea mays L. cv. Silver Queen) seed was planted in sand in Deepots" with 5 g of soil inoculum (83 spores g-') placed 2 to 3 cm below the seedling. Control plants received a 5-ml suspension of inoculum filtrate obtained by mixing 60 g of soil from a pot culture with 1.2 L of water and then filtering this through a 10-a/m membrane filter. Benlate 50 WP was applied 19 d after planting to the soil surface at rates of 0, 20, 60 and 150 kg








41

benomyl ha1 equivalent. Plants were grown from March to May 1993 under a mean photosynthetic photon flux density of 608 pmol m2 s- and 18/350C (min./max.) temperature regime.

The plants were sampled before, and then 2, 4 and 6 wk after benomyl application. The harvest procedures were the same as for pine, with the exception of estimation of root colonization. Washed root segments (1 to 2 cm) were cleared with 10% KOH for 30 min, rinsed several times with tap water, acidified for 30 min in 5% HCl and stained overnight in 0.05% aniline blue in lactoglycerol. Colonization was determined using a gridline-intersect method (Giovannetti and Mosse, 1980). Although fungi other than AM existed in this particular system, the differentiation of saprophytic from characteristic AM fungal hyphae was based on gross morphological differences. Arbuscular mycorrhizal fungi generally had a somewhat larger hyphal diameter (4 compared to <2 1Am), stained darker with aniline blue, were not dematiacious, lacked septation or clamp connections and demonstrated a less angular growth pattern compared to other fungi present. Prior to statistical analysis, percentage colonization was transformed using the arcsine, square root transformation.



Results

Field Study

Initial AM colonization of P. chamaelonche in the field was high, indicating that root growth and mycorrhizal development commenced earlier than the first fungicide









42

S* CONTROL A
S100
2 M BENOMYL


80



0 60
0
I l I I I I I I I I

tt B

U


< 80






40



C
E 60




40


20
r-d
0









4o 0 0 V O


Sampling Date


Figure 3-1. Assessment of arbuscular activity in Panicum chamaelonche roots from the field site in 1991. (A) Percentage of root length with arbuscules in benomyl-treated and nontreated plots, (B) Percentage root length with metabolically active arbuscules in benomyl-treated and nontreated plots and (C) Precipitation. Each symbol represents the mean of three replicates + SE.








43




Table 3-1. Test for homogeneity of slopes for the effect of Benlate* 50 DF applied in the field on percent Panicum chamaelonche roots with arbuscules and their activity over time.


Slope over time
Roots with arbuscules Roots with active arbuscules
(%) (%)
Control -0.104" -0.164 Benlate -0.012 -0.010 indicates slope value is significantly different from 0 at P < 0.01








44

application on 2 April (Fig. 3-1A). Over the entire growing season, both the proportion of roots with arbuscules and the activity for benomyl-treated plants did not change significantly, whereas samples from the control plots had significantly negative slopes with time for both arbuscule abundance and activity (Table 3-1). Early in the season ground cover was sparse and the spray was applied directly to the soil. This was paralleled by a short-term decrease in the proportion of roots with arbuscules (Fig. 3-1A) as well as metabolic activity (Fig. 3-1B). As ground cover increased through the growing season, more of the spray was intercepted by foliage leaving less to penetrate through to the soil. Concomitant with this, the differences between treated and nontreated plots disappeared. In late summer, as the plants started to senesce, roots of benomyl-treated plants had more arbuscules and arbuscule activity than nontreated plants. In a concurrent study, no effect of benomyl on shoot P status was observed at samplings taken in June and August. There was no apparent relationship between precipitation, application of benomyl and mycorrhizal response (Fig.3-1C).



Greenhouse Study

Benomyl effects on pine

There were no significant effects of benomyl on inoculated or noninoculated pine biomass (Fig. 3-2A). Phosphorus content of the needles increased over time for all treatments from a mean of 320 mg to 450 mg per plant, but this was not related to the benomyl treatments (data not shown). Similarly, benomyl had no effect on the length or







45


0.8
A 0 kg benomyl ha-1
0.7 20 kg benomyl ha' EE0B 60 kg benomyl ha1 0.6 150 kg benomyl ha"
0.5 0.4

01 0.3 0.2
0.1


1.4 B

1.2

I- 1.0

0.8 0.6 0.4

0.2

0.0
C M C M C M C M
0 2 4 6 Week After Benomyl Application

Figure 3-2. Total dry weight of mycorrhizal (M) and nonmycorrhizal (C) plants, (A) Pinus elliottii and (B) corn in response to 0, 20, 60 or 150 kg benomyl ha1 in the greenhouse. Each symbol represents the mean of seven replicates SE.







46

550 0 kg benomyl ha"' A
500 20 kg benomyl ha-' 450 1 60 kg benomyl ha' 400 150 kg benomyl ha-1
350
300
0 250
200
O
150
0D 100 E 50
8 B IM 80 B ) 70..J
60 50
40
30




TOTAL ACTIVE TOTAL ACTIVE TOTAL ACTIVE TOTAL ACTIVE
0 2 4 6 Week After Benomyl Application

Figure 3-3. Soil hyphal length (total) and activity (active) of mycorrhizal (A) Pinus elliottii and (B) corn plants in response to 0, 20, 60 or 150 kg benomyl ha-' in the greenhouse. Each symbol represents the mean of seven replicates SE.







47

6 A
A
S 5
0
4 o 4

I 3
E






E 3.5 B 1 0 kg benomyl ha0 3.0 I 20 kg benomyl ha- E 60 kg benomyl ha. 2.5 -ISS 150 kg benomyl ha-'
2.0

1.5
1.0
- 0.5

0.0
0 2 4 6

Week After Benomyl Application




Figure 3-4. Mycorrhizal colonization of (A) slash pine and (B) corn grown in the greenhouse in response to 0, 20, 60 or 150 kg benomyl ha-'. Each symbol represents the mean of seven replicates + SE.








48

viability of external hyphae of the ectomycorrhizal fungus (Fig. 3-3A). There was a difference in EM colonization, as measured by ergosterol concentration, at 4 wk between the 60 and 150 kg benomyl ha' treatments (Fig. 3-4A); however, this was not repeatable.



Benomyl effects on corn

Benomyl at all concentrations arrested further root colonization by the AM fungus, whereas colonization in the treatment receiving no benomyl continued to increase over the 6-wk period (Fig. 3-4B). There was no dose-related response in colonization. Noninoculated plants remained noncolonized. Total biomass of mycorrhizal and nonmycorrhizal plants was reduced by benomyl by approximately 12% (Fig. 3-2B); however, this was unrelated to the fungicide concentration applied. The length of external hyphae of AM fungi or their viability was not affected significantly or consistently by the different rates of benomyl (Fig. 3-3B). The P concentration of corn leaves decreased steadily throughout the experiment from 3.64 to 0.62 mg P g-' without any evidence of a benomyl effect (data not shown).



Discussion

Corn was used as a substitute for P. chamaelonche due to lack of native plant material. Benomyl arrested mycorrhizal development of corn in the greenhouse experiment. This is consistent with the mode of action of benomyl, which entails inhibition of nuclear division by binding to tubulin (Davidse, 1986). There was no dose-








49
dependent response by the mycorrhizal grass in the greenhouse. The range of concentrations was based on previous values published in the literature (Trappe et al., 1984). The sand used in the greenhouse minimized possible adsorption phenomena that normally occur in field soils. Consequently, all the concentrations tested were above the threshold required to obtain a maximum inhibition of mycorrhizal development. Not only was all of the fungicide readily available, but it was also well above the manufacturer's recommended application rate, which together presumably caused a decrease in plant biomass unrelated to the plant's mycorrhizal status. In agreement with previous literature (Trappe et al., 1984), the effect of benomyl on mycorrhizal pine was neutral although sometimes an increase in growth has been observed (De la Bastide and Kendrick, 1990; Pawuk and Barnett, 1981).

Arbuscules were counted in the field, since they are a distinguishing characteristic of the mycorrhizal fungus, and, more importantly, they represent the site where active exchange of nutrients between the symbionts occurs. The initial decrease in arbuscule activity in the field following benomyl application has been documented in a greenhouse study as well (Sukarno et al., 1993). Since the response to 5 kg benomyl ha' was minor compared to total colonization, the benomyl application rate was increased. At the last sampling as the plants started to senesce, the increase in arbuscule number and activity in roots of plants treated with benomyl may be due to a reduction in the impact of nonmycorrhizal fungi on plant growth and subsequent mycorrhizal functioning. Low colonization and arbuscule numbers in the greenhouse study made it difficult to obtain a reliable measure of arbuscule abundance to compare to the results in the field. The lack








50

of AM response to benomyl in the field during most of the growing season may be attributed to the increased interception of the fungicide by ground cover. Although benomyl can enter through leaves, systemic translocation is not as efficient generally as direct application to the target site (Hassall, 1990), in this case, the roots.

Larsen et al. (1994) determined that benomyl applied directly to the leaves of cucumber had little effect on mycorrhizal efficiency, yet when benomyl was applied to the soil, complete inhibition of P uptake by hyphae occurred within 5 d. Although no fungicide effect on fungal alkaline phosphatase activity was found inside the root, the rapid response, nonetheless, suggests some direct influence on uptake or transport mechanisms. Kough et al. (1987) and Thingstrup and Rosendahl (1994) have observed suppressive effects of benomyl on internal fungal enzyme activity in mycorrhizal plants. Although benomyl demonstrated no significant effect on external hyphal length or viability in this study, an inhibitory response has been found in another system (Sukarno et al., 1993).

Benomyl can be an effective tool for inhibiting AM activity in the field; however, researchers need to be aware of the limitations of this approach. The timing of root colonization and initial nutrient contribution to mycorrhizal dependent seedlings can be critical to their survival (Hartnett et al., 1994; Hetrick et al., 1989; Plenchette and Perrin, 1992). Fungicide applications in the field should be timed according to the plant's optimal benefit from mycorrhizas, which, correspondingly, would provide the full impact of the fungicide treatment on mycorrhizal functioning (Gange et al., 1993; Newsham et al., 1995). Furthermore, the frequency of application is determined by fungicide








51

persistence in the soil, which is variable (Ware, 1992) due to degradation and sorption in different soil environments. In sandy soils where sorption is low, somewhat comparable to the sand used in the greenhouse study, persistence may be longer, assuming leaching does not occur, so that an application every 5 to 6 wk may suffice. Soils with higher levels of organic matter or clay may require more frequent applications or higher concentrations.

The method of application is also critical. Although benomyl is considered a systemic fungicide, translocation from leaves to the active site in the roots appears to be minimal. A soil drench is the optimal method of application (Fitter and Nichols, 1988; Hassall, 1990; Perrin and Plenchette, 1993). Appropriate preparations should be made to accommodate the increasing ground cover as treatments are applied later in the growing season. Tall ground cover may be compensated for by applying a large volume of water to wash the active ingredient to the soil. Benomyl concentrations applied experimentally have ranged from 0.5 to 300 kg benomyl ha-' (Trappe et al., 1984). Treatments of as little as 3 kg ha' biweekly in a short turf grass setting have been adequate to reduce AM colonization by 80% (Rhodes and Larsen, 1981). As a consequence, the combination of concentration, volume of water used and frequency of application balanced with the environmental conditions should provide the desired reduction of mycorrhizal activity.













CHAPTER 4
MYCORRHIZAS AFFECT PLANT COMPETITION FOR PHOSPHORUS
BETWEEN PINUS ELLIOTTII and PANICUM CHAMAELONCHE Introduction

Soil fertility largely determines the amount of plant biomass an environment can support (Donald, 1951). In environments with low nutrients, plants are stressed directly by the lack of adequate nutrients, and they survive primarily by stress tolerance mechanisms (Grime, 1979). An environment with more nutrients has the potential to produce more plant biomass, which increases plant growth and also raises the chances that root nutrient depletion zones of two plants will overlap. As a consequence, plant competition for nutrients becomes one of the factors governing plant growth and survival. Environmentally induced stress on a plant, therefore, can be considered a gradient extending from direct physical stress on an individual plant to stress produced biologically by plant interactions (Berkowitz et al., 1995; Grime, 1979).

Autecological studies have extensively documented that mycorrhizas can increase plant tolerance to environmental stresses and contribute to a plant's survival and growth (Sylvia and Williams, 1992). The various mycorrhizal contributions that enhance individual plant health similarly benefit a plant when competing with neighboring plants. Much less research has quantitatively addressed the influence of mycorrhizas on the synecology of plants. Previous studies have demonstrated that mycorrhizas can enhance a plant's competitive ability (Allen and Allen, 1984; Fitter, 1977; Hall, 1978; Hartnett 52








53
et al., 1993; Hetrick et al., 1989). The majority of these studies relate to competition between arbuscular-mycorrhizal (AM) plants. To my knowledge, only one study has addressed ectomycorrhizal (EM) effects on plant competition (Perry et al., 1989). Plant competition between EM and AM plants has not been explored specifically.

The goal of this research was to assess the effect of mycorrhizas on the competitive ability of slash pine (Pinus elliottii Engelm. var. elliottii), which commonly is grown for pulpwood in the southeastern United States. Grasses, among other plants, compete extensively in new slash pine plantations since weed control is practiced infrequently. The specific objectives of the study were to determine (i) if mycorrhizas alter the competitive ability of pine when growing with grass and (ii) how this relationship is modified by phosphorus (P) concentration.



Materials and Methods

Greenhouse Competition Study

All experiments were conducted in acid-washed sand. Acid-washing was accomplished by treating the sand with 25% HCI for 24 h, then draining the acid and rinsing the sand until the pH increased to that of the deionized water being used. Eighty percent of the sand was in the particle size range of 0.160 to 1 mm, and the majority of the remaining portion was larger than 1 mm.

Slash pine seeds were disinfested for 2 min in a 5.25% sodium hypochlorite solution with 0.2 ml Liqui-Nox surfactant (Alconox, Inc., New York, N.Y.) and then rinsed thoroughly with tap water. Seedlings were raised from seed for 2 wk in a growth








54

chamber [29/23 Co (day/night), with a 15-h light period and irradiance of 1000 jmol mn' s-'] in sand and then transplanted to 50-ml pots (5 cm' of surface area) and grown in sand in the greenhouse for 8 wk where they received water only. To inoculate pine, washed roots were dipped in a slurry of rinsed and chopped Pisolithus tinctorius (Pers.) Coker & Couch (isolate S106) grown in a liquid suspension culture containing modified MelinNorkrans liquid medium (Marx, 1969) using glucose instead of sucrose. Roots of control plants were dipped in tap water. After a further 6 wk of growth in 500 ml of sand in Deepots" (28 cm' of surface area; McConkey, Co., Sumner, WA), pine roots were gently rinsed free of adhering sand particles and planted in the appropriate competition treatments as described below.

Grass plants of a dominant competing weed species in the field (Panicum chamaelonche Trin.) were obtained from cultures maintained in sand in the greenhouse. Plants were started from seed and vegetatively propagated in 150-ml pots (7 cm2 of surface area). Two months in advance of the experiment, grass plants were inoculated with pot culture inoculum of Glomus sp. (INVAM FL329, formerly FL906) previously cultured on sorghum in pasteurized soil. Roots of plants were washed and the plants transplanted into sand in DeepotsT containing 5 g of soil inoculum (83 spores g-1) located 2-3 cm below the sand surface. Control plants were transplanted into sand without inoculum and received a 5-ml suspension of inoculum filtrate obtained by mixing 60 g of soil from a pot culture with 1.2 L of water and then filtering the mixture through a 10-gm membrane filter. Just prior to the experiment the grass roots were washed as described for the method of pine roots.








55

Pine and grass plants were sorted separately into three size classes at the start of the experiment. Noninoculated pine had no visual indication of colonization, whereas inoculated pine was heavily colonized. Inoculated grasses had a mean root colonization of 30% at the start of the experiment. There were no significant differences in biomass between inoculated and noninoculated plants at the beginning of the experiment. Intraspecific and interspecific paired combinations of plant species (Table 4-1), inoculated or not, were made by selecting plants from the same size class. Plants were planted together in 500 ml of sand. There were six replications per treatment. Plants were grown in the greenhouse with mean temperatures of 21/340C (min./max) and a mean photosynthetic photon flux density of 1240 Mmol m-2 s- from January to June 1994. A repeat of the experiment was run from May to October 1994 with seven replications. The greenhouse temperature regime was 24/360 (min./max.) with a mean photosynthetic photon flux density of 1490 14mol m-2 s '. The plants were fertilized semiweekly with a solution containing: 660 AM NH4NO3, 660 1M (NH4)2SO4, 616 pM KCI, 80g m MgSO4, 54 1M NaFeEDTA, 600 pM CaCl2, 0.25 pM CuSO4, 14 pM H3BO3, 40 pM NaMoO4, 2.75 pM MnCI and 1.25 IpM ZnSO4. Phosphorus was supplied at either 0.32, 3.23 or 32.26 pM H3PO4. In the repeat of this experiment the 0.32 pM H3PO, treatment was replaced with 323.58 pM H3PO4, since plant growth was very slow at the lowest P concentration. Soil solution pH was measured from several pots during the experiment by thoroughly watering pots with deionized water and collecting the leachate.

Plants were removed from the pots after 129 d, and the roots of individual plants were separated carefully from each other. The exception was the intraspecific grass








56




Table 4-1. Pinus elliottii (pine) and Panicum chamaelonche (grass) treatment combinations planted in the competition study. Two plants were planted per pot. The superscripts "+" and "-" signify an inoculated or noninoculated plant respectively. Pine was inoculated with Pisolithus tinctorius and the grass was inoculated with Glomus sp. (INVAM FL329, formerly FL906).


Plant Competition Treatments Intraspecific Interspecific



pine+ x pine+ pine' x grass+ pine x pine- pine+ x grassgrass+ x grass+ pine- x grass+ grass- x grass- pine- x grass-








57



combination where the roots were treated as one unit and then half the value allotted to each plant. Root wet and dry mass were determined. An estimate of root length was obtained using calculations of specific root length (cm root g' of root fresh weight) for pine and grass from a previous experiment and expressed here as root-length density (cm root cm' of soil). For grass, root colonization was determined using a gridline-intersect method for the AM treatments (Giovannetti and Mosse, 1980) after clearing the roots for 30 min in 10% KOH and staining in 0.05% aniline blue overnight. For pine, root ergosterol concentration was used as an estimate of EM fungal biomass (Martin et al., 1990; Salmanowicz et al., 1989). Fresh pine roots were washed, ground in liquid nitrogen and thoroughly mixed. A 0.1- to 0.3-g subsample was extracted overnight at room temperature with 5 ml of 100% ethanol. This sample was filtered through a 0.45 pm-syringe filter and then assayed for free ergosterol by high-pressure liquid chromatography (Waters 715 Ultra WISP, Gilson 115 UV detector). Separation was carried out using a C-18 column (SupelcosilT LC-18; Supelco Inc., Bellefonte, PA) at 40C with a methanol-water mobile phase (92:8) flowing at 2 ml min-', with detection at 282 nm.

Shoots were analyzed separately from roots. Shoot wet mass was determined and dry mass was measured after drying overnight at 65*C. The shoots were ground and then ashed at 5000C for a minimum of 4 h. Phosphorus analysis of the shoot tissue was performed using the method of Murphy and Riley (1962).








58

To compare the competitive abilities of the two plant species, the relative crowding coefficient (RCC; Harper, 1977) was calculated. To avoid subjective pairing of plants between treatments all possible combinations were used to calculate the RCC. The following is a sample calculation of the RCC for grass total dry weight when growing with pine:

grass (interspecific)

pine (interspecific)
RCC (shoot P, mg P) =
grass (intraspecific)

pine (intraspecific)


Data for grass and pine were analyzed separately. To determine if plant competition was affected by the plant species, data for each plant species were subjected to analysis of variance and statistically planned contrasts using the General Linear Model (SAS Institute, Inc., 1989). Data for colonization were transformed to arcsine square roots prior to analysis (Steel and Torrie, 1980). The least-squares means statement within SAS was used to compare means.



Determination of P Uptake Kinetics for Pine and Grass

Pine and grass plants were inoculated with their respective mycorrhizal fungi or noninoculated. Pine plants were grown in DeepotsT for a further 24 wk after inoculation with P. tinctorius. Grasses were inoculated 3 wk prior to transferal to 1-L Erlenmeyer flasks by applying to each plant root system a minimum of 20 spores of a mixed culture of Gigaspora rosea (INVAM FL224) and Scutellospora heterogama (INVAM FL225),








59

cultured on P. chamaelonche in field soil in the greenhouse. The AM fungal species were isolated from P. chamaelonche growing in a Spodosol at a field site 21 km northwest of Gainesville. The grasses were replanted together in a 15-L pot of sand. Noninoculated plants were treated in the same manner, except that no spores were added to the roots.

Three grasses and pines, inoculated or noninoculated, were selected, and their roots were gently rinsed free of adhering sand. Each plant was transferred to a single I-L Erlenmeyer flask covered with aluminum foil. Plants were grown in a growth chamber [29/23 C' (day/night), with a 15-h light period and irradiance of 1000 1pmol m-' s-'] in a continuously aerated nutrient solution with the following nutrient composition: 660 AM NH4NO3, 616 pM KCI, 800 AM MgSO4, 54 ~M NaFeEDTA, 600 /M Ca(NO3)2,4H20, 0.75 AM CuSO4, 52 gM H3BO3, 120 AM NaMoO4, 8.25 yM MnCI and 3.75 jM ZnSO4. Phosphorus was supplied at 3.23 !M H3PO4. The solution was changed semiweekly. At the start of the experiment a minimum 4-wk acclimatization period was given allowing external hyphae to regrow from the colonized roots.

To quantify uptake kinetics, root systems were rinsed with deionized water and placed in additional deionized water for 1 h. One liter of fresh nutrient solution, identical to the one used previously, was added to 1-L acid-washed Erlenmeyer flasks. At the start of the experiment, plant roots were gently patted dry with paper towels, placed in the nutrient solution and weighed. At regular intervals, 23 ml of solution for P analysis were removed and immediately filtered through 0.45-Am syringe filters. The solution was replaced with sufficient deionized water to bring the system back to its original starting weight. Twenty milliliters of sample removed for P analysis were evaporated to dryness.








60

Twenty milliliters of concentrated HCI were added to the sample and also evaporated to dryness. Phosphorus was determined colorimetrically by a slightly modified procedure of Murphy and Riley (1962). Reagent, quantitatively diluted with deionized water, was added directly to the samples. Since some solutions were at the detection limit, less reagent was added to later samples in order to concentrate them. The resulting Pdepletion curve was fit using a curve-fitting procedure (SigmaPlot; Jandell Scientific, San Rafael, California), and uptake calculations were made based upon this idealized curve. Total surface area of roots and hyphae were determined using image analysis software (Mocha; Jandell Scientific, San Rafael, California) or gridline-intersect methods (Giovannetti and Mosse, 1980). The maximum uptake rate, I., was calculated based on the root surface area and the quantity of P absorbed from the nutrient solution during the first 45 min. The minimum solution concentration from which a nutrient can be absorbed, C., was considered the asymptotic value where the solution P concentration no longer decreased.

Results

Greenhouse Competition Study

No colonization was found in the inoculated grass plants at the end of the experiment. Therefore these treatments were excluded from further analyses. Also, the ubiquitous EM fungus, Thelephora terrestris (Ehrh.) Fr., was found growing on the noninoculated pine (pine-) treatments, but not in the inoculated pine (pine+) treatments. The mean soil solution pH was 4.0.








61

Pine shoot-P concentration increased in all treatments with increasing level of applied P (Fig. 4-1A). A higher shoot-P concentration was observed in pine+ compared to the pine- (P < 0.001). In the interspecific competition treatments where pine was grown with grass, pine+ had an elevated shoot-P content compared to pine- (Fig. 4-1B). The difference became more apparent with increasing P level. In the treatments where pine was grown with pine, pine- and pine+ acquired similar quantities of P at all levels of applied P. Total dry weight of pine was not affected by the level of applied P (Fig. 4-1C). Overall pine+ had a higher total dry weight than pine- (P = 0.07) and more so when grown with grass (P < 0.01). Pines grown with other pines had a lower dry weight than when grown with grass (P 0.01). Colonization was also higher in the pine+ treatments inoculated with P. tinctorius than in the pine- treatments that became colonized with T. terrestris (Table 4-2).

Similar trends were observed in the repeat of the experiment, although differences were smaller and not always significant. Pine+ grown with grass had a 70% higher shoot-P level compared to pine- (P < 0.05), but only at the 32.26-IPM P level. The total pine+ biomass was 31% larger than pine- at 32.26-1pM P (P < 0.05), and only when grown with grass.

For grass shoot-P concentration there was a significant interaction between the level of applied P and the competition treatment (P < 0.05). At the 32.26-gM P level, the shoot-P concentration of grass when grown with pine+ was lower than when grown with pine- (Fig. 4-2A). The grass intraspecific competition treatment at this P level was higher than both pine treatments. The shoot-P content at the 32.26-aRM P level was also







62
r 2500 62
2 A 0 Pine+ x Pine+
2 2000 Pine- x Pine
3 1500 Pine+ x Grass" o V Pine- x Grass0 1000

o 500


8000 B

6000

S4000
0

4 2000
0
O 0
S7 C






o
6




3


0.32 3.22 32.26 P Applied (pM P), Log Scale


Figure 4-1. Pinus elliottii (A) shoot-phosphorus concentration, (B) shoot-phosphorus content and (C) total dry weight in response to different competition treatments and grown at either 0.32, 3.23 or 32.26 IM P for 18 wk. Each symbol represents the mean of six replicates SE. Inoculated grass was not colonized at the end of the experiment and therefore was not included in the analysis.







~63
'm 3000 .. A Grass" x Grass" a 2500 S2500 A Pine+ x Grassa 2000 C V Pine x Grass S1500
0.
L 1000
0
o 500
0 I
0. 3000
E 2500 '" 2000 c 1500
0
0 1000
500
0
0
1.5


p 1.0


o 0.5

o
I- 0.0
0.32 3.22 32.26 P Applied (pM P), Log Scale



Figure 4-2. Panicum chamaelonche (A) shoot-phosphorus concentration, (B) shootphosphorus content and (C) total dry weight in response to different competition treatments and grown at either 0.32, 3.23 or 32.26 jM P for 18 wk. Each symbol represents the mean of six replicates SE. Inoculated grass was not colonized at the end of the experiment and therefore was not included in the analysis.








64




Table 4-2. Ergosterol concentration (pg g-) of Pinus elliottii roots inoculated with Pisolithus tinctorius (pine+) or noninoculated (pine-), and grown in combination with Pinus elliottii (pine) or Panicum chamaelonche (grass) at either 0.32, 3.23 or 32.26 pM P for 18 wk. Each value represents the mean of six replicates SE.

Competition Phosphorus added (pM P)
Treatment

0.32 3.23 32.26 Pine+ x pine+ 181 20 282 34 297 33 Pine- x pine- 129 13 104 11 150 11 Pine+ x grass- 192 8 297 46 260 26 Pine- x grass- 137 21 139 12 140 17








65

lower in the treatments where grass competed with pine (Fig. 4-2B). Total dry weight of grass was not significantly different at any level of applied P or for any competition treatment (Fig. 4-2C). In the repeat of the experiment there were no differences in shootP content between the different grass competition treatments, except at the 322.58-IPM P concentration where grass grown with pine had a 39% higher shoot-P content (P 0.02) than when grown with another grass. Grass total dry weight at that P concentration was higher in the interspecific treatment with pine than in the intraspecific treatment with grass (P < 0.01).

Pine+ had a higher root length than pine- over all treatments (P 0.001), even though there were no differences in biomass between inoculated and noninoculated plants at the beginning of the experiment (Fig. 4-3A). Pine root length did not change with the level of P applied. In the repeat of the experiment, response of pine root length did not differ between the competition treatments or between the 0.32- and 3.22-ApM P levels (data not shown). When grass was grown with grass, there was an increase in grass root length at the 32.26-pM P level (Fig. 4-3B) which was paralleled by an increase in shootP content. At the 32.26-pM P level, grass growing with grass had a higher root length than grass in the interspecific treatments. When grass was grown with pine+, there was an increase in grass root length from the 0.32- to the 3.22-zpM P level, whereas grass root length for pine- was not different between P levels. In the repeat of the experiment, root length increased between the 0.32- and 3.22-pM P levels for grass grown with grass only (data not shown).







66

E0 A Pine x Pine+ S20 Pine' xPine U) A Pine+ x Grass" o 15 V Pine x Grass"

E

0
0



20
?o B

S15




Grass" x Grass" 0 5 A Pine' x Grass" o, v Pine- x Grass'
0 I I
0.32 3.22 32.26 P Applied (pM P), Log Scale





Figure 4-3. Root-length density of (A) Pinus elliottii and (B) Panicum chamaelonche in different competition treatments and grown at 0.32, 3.23 or 32.26 pIM P for 18 wk. Each symbol represents the mean of six replicates SE. Inoculated grass was not colonized at the end of the experiment and therefore was not included in the analysis.







3 67


A Pine'
SGrassCO



o B U Pine" S3 Grass"


2






0.32 3.22 32.26 P Applied (pM P), Log Scale



Figure 4-4. Relative crowding coefficient (RCC) for total dry weight of (A) Pinus elliottii inoculated with Pisolithus tinctorius grown in combination with Panicum chamaelonche and (B) noninoculated Pinus elliottii grown in combination with P. chamaelonche at either 0.32, 3.23 or 32.26 IM P for 18 wk. Each symbol represents the mean of six replicates SE. Mean standard errors were smaller than the symbols and are therefore not included.








68




Table 4-3. Maximum uptake rate, I., (pmol P cm-2 S-') and C. (UM P), the minimum solution concentration from which a nutrient can be absorbed, for Pinus elliottii and Panicum chamaelonche grown in a hydroponic solution containing 0.32 IAM P. Each value represents the mean of three replicates SE.


Plant species I, C.


Pinus elliottii 0.116 0.027 0.080 0.018 Panicum chamaelonche 0.075 0.016 0.028 0.014








69

At each level of P, pine+ had a higher RCC than grass (Fig. 4-4A). In contrast, pine- was more competitive than grass only above the 0.32-,pM P level (Fig. 4-4B). In the repeat of the experiment, grass RCC between 32.26 and 322.58 pM P rose by 279 and 144% when competing with pine- and pine+ respectively. At this same P level the RCC for pine- and pine+ dropped by 75 and 27% respectively.



Determination of P Uptake Kinetics for Pine and Grass

Again, no colonization was observed in the grass+ plants, even though I attempted to use indigenous fungi, so the treatment was excluded from analysis. On a root surface area basis, I., was not different between pine+ and pine-; however, I, based on total surface area, which included mycorrhizal hyphae, was much lower in the pine+. Consequently only the values for the nonmycorrhizal pine and grass are shown. A higher I value was observed for pine, whereas grass had a lower C, value (Table 4-3).



Discussion

Inoculation of slash pine with P. tinctorius enhanced P acquisition of pine when grown with nonmycorrhizal grass. This response is dependent on at least two conditions of the experimental design, namely soil volume and nutrient availability. In large soil volumes mycorrhizal fungi are able to enhance plant nutrient uptake by accessing areas beyond the root's nutrient depletion zone. This mechanism is much less important in smaller volumes of soil, such as in this experiment, since roots and fungal hyphae proliferate throughout the pot, essentially making the entire volume a single nutrient








70

depletion zone. Since an acid-washed sand was used, soluble inorganic nutrients were the only source of nutrients available to both the roots and mycorrhizal fungi. This made the potential ability to utilize nutrients in different forms inconsequential. As a result, differences in P acquisition most likely were related to a combination of differences in absorbing surface area and uptake rates. Although plant density also may affect the outcome of plant competition (Hartnett et al., 1993; Taylor and Aarssen, 1989) this was not tested.

Previous researchers comparing uptake by mycorrhizal and nonmycorrhizal plants have observed a higher uptake rate for mycorrhizal plants (Cress et al., 1979; Karunaratne et al., 1986; Pacheco and Cambraia, 1992). However, these estimates generally are reported on a root weight or root length basis only. If the estimates included hyphal surface area the uptake rates would be greatly reduced for the mycorrhizal plants. In the current study, pine+ and pine- had similar uptake rates if based on root surface area alone. The lower uptake rate of pine+ compared to pine-, based on total surface area, strongly suggests that hyphal nutrient depletion zones were overlapping. If inadequate mixing of the nutrient solution occurs, the rate-limiting step for uptake would be the replenishment of P absorbed inside the dense mass of hyphae. The P uptake kinetics of nonmycorrhizal pine and grass determined hydroponically in a 0.32-tM P solution were different, but the variability was relatively high due to the low number of replications. The higher I. demonstrated by pine would permit pine to sequester more P than grass, which would give the pine a competitive advantage over grass under the regular fertilization schedule followed here. The lower C for grass








71

would be more advantageous where low nutrient concentrations persist over longer time periods, such as in the field but not in this greenhouse study where the time between fertilization was relatively brief. These observations suggest that differences in P uptake kinetics partially may be responsible for the outcome of competition.

Pine and grass root growth rates vary with P concentration, resulting in different absorbing surface areas. The poor relationship I observed between root length and shootP content indicates that root length is not the only factor contributing to the pine's competitive interaction with grass. Nonetheless, the increase in root length in the pine' compared to the pine- treatments suggests that mycorrhizal fungi increased pine root length and thereby enhanced nutrient uptake. As a result the competitive ability of pine was increased compared to grass.

The exact nature of the relationship between intensity of competition and resource abundance is still under debate, but it depends on the environmental conditions and plant species involved (Di Tommaso and Aarssen, 1991; Grace, 1995). Tilman (1982) stated that competition increases with decreasing resource availability. An alternate viewpoint is espoused by Grime (1979) who maintained that competition intensity increases with increasing habitat fertility. By definition competition is expressed as effects on plant biomass, survival or reproduction. In this study plants did not demonstrate any dry weight response to P application, which suggests that P was not the only nutrient limiting plant growth. At the 0.32-j/M P level plant growth was marginal as a result of inadequate P in the system. Although dry weight was not altered by the different levels of applied P, P uptake by both pine and grass was affected. Since nutrient uptake is part of the








72

mechanism leading to differences in plant biomass, it is very likely that this would influence plant competition. Differences between P status of grass and pine indicate that P capture by pine reduced the amount of P taken up by grass, specifically at the 32.26/AM P level. Pine, based on its higher RCC, appeared to be more competitive than grass when the two competed with each other. When grown with grass the enhanced P uptake of pine+ corresponded with a larger total dry weight compared to pine-, indicating that inoculation with P. tinctorius did alter the competitive ability of pine.

Although not validated in a repeated experiment, grass at 322.58 JM P had a 7.5 or 4.6 times larger RCC than pine+ or pine-, respectively. A change in competitive dominance between the species Rumnex acetosella and Poa pratensis with changes in soil fertility also has been documented (Fowler, 1982). Dual-phasic, P uptake kinetics dependent on solution-P concentration have been found in fungi (Jennings, 1995), plants (Barber, 1972) and in mycorrhizal roots (Cress et al., 1979). If a dual-phasic uptake system exists for each of the plant species, then a higher affinity enzyme system in one of the species could provide a possible explanation for plant dominance based on uptake ability.

When pine was grown with pine, competition was equally intense if the plants were inoculated with P. tinctorius or colonized with T. terrestris. Since the pine- plants were not uniformly colonized with T. terrestris, I was not able to determine if the EM inoculation treatments substantially altered the intensity of intraspecific competition of pine with and without mycorrhizas. However, in an EM competition study by Perry et








73

al. (1989), biomass of plants in intraspecific competition (12 trees potr) was altered by different EM fungi, indicating that competition intensity varies with the fungal species.

The somewhat reduced response in the repeat of this experiment may be related to temperature differences between the two experiments. The first one ended in June and the second ended in October, which resulted in not only a 2C higher maximum temperature in the second study, but a longer daily exposure to higher temperatures as well. The mean colonization of pine+ in the repeat was 46% lower compared to the first run of the experiment. A decrease of mycorrhizal effectiveness has been observed at temperatures of 34 to 35C for certain Pisolithus tinctorius isolates (Marx et al., 1970) grown on pine, as well as for Glomus spp. (Fabig et al., 1989) grown on several grass hosts. The lack of colonization in the grass plants may be related to the high soil acidity (pH 4.0) and the lack of buffering capacity of the sand. Activity of Glomus spp. is optimal above pH 5.3 (Abbott and Robson, 1985; Wang et al., 1985).

The conclusion of this study is that P. tinctorius can increase P acquisition of pine when grown with grass, which consequently could lead to an increase in competitive ability. The controlled conditions used in this experiment allow the isolation of specific variables that affect a plant's competitive ability. The actual proportion of a plant's total competitive ability contributed by the mycorrhizal component can only be determined under field conditions where soil chemical, physical and biological parameters modify plant interactions and the mycorrhizal response. Yet, our ability to isolate the different components of a plant's competitive ability and determine the relative importance of each








74

component is limited precisely by the interwoven nature of the plant and soil complex. When this is accomplished, we will be closer to determining the magnitude of mycorrhizal effects on the ecology or economy of an ecosystem.













CHAPTER 5

CONCLUSION



Simultaneously evaluating arbuscular mycorrhizal (AM) and ectomycorrhizal

(EM) effects on plant competition for nutrients involves discerning the complex interactions between four different organisms. To do this under controlled experimental conditions requires an understanding of the growth requirements of each species. One of the main problems in my studies was the difficulty in obtaining colonization in the AM grass treatments. This was likely related to the artificial environmental conditions created for the experiments. The main factors distinguishing the field soil from the acid-washed sand were the presence of greater buffering capacity and organic matter in the soil, and differing microbial composition and nutrient regimes. Since Panicum chamaelonche was highly colonized in the field compared to the greenhouse, it is likely that modification of one or several of these factors would increase colonization.

Distinguishing the effects of one mycorrhizal type from another can be accomplished by the use of fungicides. In the research presented here the use of benomyl as a tool to control the AM fungal component was tested. The following conclusions can be drawn from this research.





75








76

1. Benomyl can successfully inhibit development of an AM fungus under controlled

conditions in the greenhouse with no side effects on the EM fungus, Pisolithus

tinctorius.

2. Early in the season with low ground cover in the field, benomyl caused a slight

reduction in arbuscule activity. Later as ground cover increased, systemic translocation of benomyl from shoot to roots of grasses apparently was insufficient to reduce mycorrhizal colonization, even at high benomyl

concentrations.

3. Soil drench of benomyl would be a more effective method to place the fungicide

directly at the target site, namely the roots.

4. Sufficient water should be used to permit penetration of benomyl into the soil as

ground cover increases.


Previous studies have demonstrated that mycorrhizas can enhance a plant's competitive ability. The role of mycorrhizas in competition between EM and AM plants and the effects of different P levels have not been explored specifically. The greenhouse study I conducted to address these interactions yielded the following results.


1. Both the reduction in P acquisition of grass when grown with pine compared to

another grass at the 32.26-14M P level amd the higher relative crowding coefficient for total dry weight indicate that pine is more competitive than grass

under the conditions tested.








77

2. Inoculation of slash pine with P. tinctorius enhanced both P uptake and total dry

weight and hence the competitive ability of pine when competing with

nonmycorrhizal grass.

3. When grown in intraspecific competition, no difference was observed in the

competitive ability of pine colonized with P. tinctorius or Thelephora terrestris. 4. The different P levels added did not affect grass or pine biomass which suggests

that P was not the only limiting factor to growth.

5. A higher Im value for pine and the lower Cra for grass suggest that differing P

uptake kinetics can contribute to competitive interactions.


Several additional factors would have to be elucidated to draw conclusions from these results about pine and grass interactions in the field. In a separate field competition study involving slash pine and weeds (primarily grasses) pine growth was substantially decreased in contrast to the greenhouse where pine exhibited a higher competitive ability than grass. In the Spodosol at the field site, organic forms of P are the major source of P, which is released during periodic pulses of nutrient cycling triggered by increases in soil moisture. This contrasts with the inorganic P used in the greenhouse study, which was applied at frequent and regular intervals and thus maintained a relatively consistent P concentration in the system. Also, the buffering capacity of the field soil was absent in the greenhouse, and this would modify plant-soil-microbe interactions by altering nutrient availability and flux. As a consequence differing pine and grass P uptake kinetics expressed in the greenhouse would not necessarily provide the same competitive








78

advantages in the field. The use of a field soil in subsequent studies would incorporate, at least in part, these effects.

In the hydroponic study on P-uptake an attempt was made to measure the effects of mycorrhizas on P uptake kinetics. The results involving mycorrhizas were inconclusive since mycorrhizal plants with a higher total absorbing surface area demonstrated a lower I, value than the nonmycorrhizal plants. Based on visual observations this is due to extensive hyphal development in the mycorrhizal treatments most likely resulting in overlapping depletion zones of roots and hyphae. Allowing hyphae to regrow for a period of 2 wk instead of 4 wk prior to P uptake measurement probably would have avoided the problem. Although not quantified in the hydroponic study, mycorrhizal fungi may have different IL and C, values from the host plant. If the fungus has a higher I, or a lower Ca than the host plant, as well as the competing plant, this would confer a competitive advantage to the host plant.

In the field, root-length density measured down to a depth of 87 cm was several fold higher for grass than for pine. The 500-mL soil volume used in the greenhouse experiment created a root-bound condition which did not fully permit this difference to be expressed. Much of the contribution of mycorrhizas is due to their accessing nutrients beyond the root's nutrient depletion zone; however, since hyphae and roots were able to access nutrients in most of the soil volume in pots, this probably did not contribute a competitive advantage in my study. The spatial advantage mycorrhizal hyphae would provide to a host plant by their presence directly at the site of nutrient mineralization, such as in and around organic matter, also was not expressed. Although external








79

mycorrhizal hyphae contribute to nutrient uptake and thus plant competitive ability in the field due to their spatial distribution, part of this component could be reduced by hyperparasitism by parasitic fungi and the effects of fungal feeding by Arthropods. Incorporation of a larger nonsterilized, soil volume in future competition studies would allow more components of a plant's competitive ability to function.

The simplification of the soil environment achieved by using an acid-washed sand allowed the isolation of specific mycorrhizal effects that influence plant competition. Ideally, the next step in this process would be to address the competition between mycorrhizal plants in field soil or directly in the field while acknowledging the limitations imposed by the complexity and heterogeneity of field soil conditions.













APPENDIX 1
GROWTH CHAMBER COMPETITION STUDY BETWEEN PINUS ELLIOTTII AND PANICUM CHAMAELONCHE

Introduction

A competition study involving pine (Pinus elliottii Engelm. var. elliottii)) and grass (Panicum chamaelonche Trin.) was set up to determine: (i) the contribution of mycorrhizal fungal hyphae to total plant P uptake and (ii) the competitive abilities of arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) fungi with respect to each other. This experiment was conducted twice. No colonization was obtained in the inoculated grass treatments the first time this experiment was run, and therefore no competition resulted between plants.



Materials and Methods

The study was initiated as part of a larger field investigation into competition for nutrients between pine and grass at a field site 21 km northwest of Gainesville, Florida. Pine and grass were grown in acid-washed sand and inoculated as in the Material and Methods of Chapter 4. Grasses were inoculated 14 wk prior to the start of the experiment by applying to each plant root system a minimum of 20 spores of a mixed culture of Gigaspora rosea (INVAM FL224) and Scutellospora heterogama (INVAM FL225) as in the Materials and Methods section of Chapter 4. At the start of the experiment, grass had a mean root colonization of 13%. Pine roots were visually inspected to prevent inclusion 80








81

of noninoculated pine colonized by Thelephora terrestris. At the start of the experiment plant roots were washed free of all sand and divided into three size classes. Different combinations of plant species, inoculated or noninoculated, were made by selecting pairs of plants from the same size class to create the competition treatments listed in Table 4-1 of Chapter 4. There was a minimum of six replications per treatment.

Growth boxes were constructed with two plant compartments (416 g of dry sand each) on opposite sides of a hyphal compartment (225 g of dry sand). The plant compartments were separated from the hyphal compartment by root-excluding nylon screens (Tetko, Inc., Depew, N.Y.) with a mesh size of either 15 ptm for the grass or 40 plm for the pine. The internal dimensions of each plant compartment were 4 x 9 x 11.5 cm (width x length x depth) and 2.5 x 9 x 11.5 cm for the hyphal compartment. Plant fresh weights were measured at the start of the experiment. Eight pine and grass plants, inoculated or noninoculated, were used to determine plant water content and initial P status. After planting, water was added to reach 10% of the soil gravimetric water content and the boxes were then weighed. Deionized water was added to maintain this weight during the experiment. Plant compartments were fertilized separately from the hyphal compartments. In weeks 1, 2, 3 and 6, plant compartments were fertilized three times weekly with 1.4 tsmoles P as NaH2PO4 along with 10 ml of nutrient solution containing 2.8 mM NH4NO3, 2.8 mM Ca(NO3)2, 2.6 mM KCI, 3.4 mM MgSO4, 230 /LM NaFeEDTA, 3.2 IAM CuSO4, 221 IAM H3BO3, 510 pM NaMoO4, 35.1 pM MnCI and 15.9 1M ZnSO4. During weeks 4 and 5 they received 15.1 moles P at each fertilization. Starting in the fourth week, hyphal compartments received 8.4 upmoles P. To prevent








82

massflow of nutrients from the hyphal to the plant compartment, water used to bring the boxes to their original weight was added only to the plant compartment. Plants were grown in a growth chamber with mean temperatures of 23/29"C (dark/light cycle, respectively) and a mean photosynthetic photon flux density of 1000 /mol m-2 s-I at plant height. Plant shading was minimized by the distance between plants. Boxes were randomized each time plants were watered in the growth chamber.

Plants were harvested after 62 d. The surface sand layer containing a crusted algal mat approximately 5-mm thick was removed and treated separately. Sand was removed from roots first by shaking and then by rinsing the roots with water. All root pieces were collected. A sample of subsurface sand was retained for further analysis. Plant tissue-P status, colonization and root and hyphal surface areas were measured as in the Materials and Methods of Chapter 4. Root and hyphal surface areas were measured separately for the plant and hyphal compartments. For absorbing surface area calculations involving intraspecifically competing mycorrhizal plants, half of the total hyphal surface area from the hyphal compartment was added to the surface area of the plant on each side. Plant P-uptake rate was calculated as the change in plant P content during the time of the experiment based on combined root and hyphal surface area. The mean total hyphal density for each compartment and treatment was calculated.

Sand for P analysis was thoroughly mixed prior to analysis. A 4.5- to 5.5-g sample in 20-ml, borosilicate vials was treated overnight with 5 ml of concentrated HCI. This was evaporated and 10 ml of 0.1 N HCI were added. Phosphorus was analyzed after 24 h following the procedure of Murphy and Riley (1960). Surface and subsurface soil








83

samples from the plant and hyphal compartments were treated separately by the same procedure.

Data for grass and pine were analyzed separately. To determine if plant competition was affected by the plant species, data for each plant species were tested by single degree of freedom contrasts using the General Linear Model procedure of SAS (SAS Institute, Inc., 1989). For the contrasts, target plants were analyzed based on mycorrhizal status, type of competition (intraspecific /interspecific) or mycorrhizal status of the competing neighbor plant. Data for colonization were arcsine, square roottransformed prior to analysis (Steel and Torrie, 1980). The least-squares means statement within SAS was used to compare means.



Results

Pine+ exhibited greater plant biomass compared to the pine- treatments (Fig. Al1A, Table Al-1). There was no difference between intraspecific and interspecific competition in the pine- treatments irrespective of the mycorrhizal status of grass. No difference was observed for grass response to intra- or interspecific competition either inoculated or noninoculated (Fig. Al-1B).

Similar to pine biomass, pine' had a higher plant-P content than pine- (Fig. Al2A, Table Al-1). Grass' had a lower P content than grass- when competing with both pine+ and pine- (Fig. A1-2B). No such difference was found in the intraspecific treatments where grass competed with grass.









Table Al-1. Tests for single degree of freedom contrasts for root-length density, plant biomass, plant
P content and percent colonization. Each parameter was analyzed separately for grass and pine.


Root-Length Density Plant Plant P Colonization Uptake Rate (m cm3 of soil) Biomass Content (%) (fmol P cm2 s-1)
(g) (mg P g')
Contrasts for grass:
Grass+ / grass- .06 n.s. .02 .001 .05 (Grass+ / grass-) over all pine .06 n.s. .03 .02 n.s. (Pine+ / pine-) over all grass .04 n.s. n.s. n.s. n.s. Intra- / interspecific n.s. n.s. n.s. .05 n.s.


Contrasts for pine:
Pine+ / pine- <.001 .001 .01 .006 .03 (Pine+ / pine-) over all grass <.001 <.001 .06 .01 n.s. (Grass+ / grass-) over all pine n.s. n.s. n.s. n.s. n.s.
Intra- / interspecific n.s. n.s. .07 n.s. n.s.













Table A1-2. Mean hyphal length density (m cm-3 of soil) for each competition treatment presented separately for each compartment (one hyphal and two plant compartments). Hyphal length is made up of the sum of both AM and EM hyphae. Each value represents the mean of a minimum of six replicates SE.


Compartment Plant Compartment A Hyphal Compartment Plant Compartment B
A B
Pine+ Pine+ 131.26 13.58 113.12 15.87 131.26 15.58 Pine- Pine- 94.82 8.69 80.73 12.36 94.82 8.69 Pine' Grass+ 82.40 10.55 63.69 8.01 32.48 6.39 Pine+ Grass- 100.43 :7.76 64.32 6.44 21.16 3.69 Pine- Grass+ 75.02 14.88 48.13 11.61 14.47 4.20 Pine- Grass- 162.27 28.67 65.40 7.71 22.40 6.04 Grass' Grass+ 7.42 1.08 3.75 + 0.64 7.42 1.08 Grass- Grass 6.73 0.82 2.38 0.31 6.73 0.82








86


3 A



2








E

II 2










0


e, e, be.
x$ xl- xl-



Figure Al-i. Plant biomass for (A) slash pine and (B) grass grown in the growth chamber for 62 d. Each bar represents the mean of a minimum of six replicates SE.







87

5000
A
4000

3000

C 2000

1000


0 o
C 4000 3500 B 3000
2500 2000
1500
1000
500
0






Figure A1-2. Plant-P content for (A) slash pine and (B) grass grown in the growth chamber for 62 d. Each bar represents the mean of a minimum of six replicates SE.







88

800

0 700 o 600 M 500

300 -Io 200
.5
u 100






6

0
4
o 2

0




x o X+ x + ,+




Figure A1-3. Mycorrhizal colonization of (A) slash pine and (B) grass grown in the growth chamber for 62 d. Each bar represents the mean of a minimum of six replicates + SE.




Full Text
INFLUENCE OF MYCORRHIZAS ON PLANT COMPETITION FOR
PHOSPHORUS BETWEEN SLASH PINE AND GRASS
By
CHRISTIAN THOMAS PEDERSEN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1995


103
Baylis, G.T. 1975. The magnolioid mycorrhiza and mycotrophy in root systems derived
from it. p. 373-389. In F.E. Sanders, B. Mosse and P.B. Tinker (ed.) Endomycorrhizas.
Academic Press, New York, N.Y.
Begon M., J.L. Harper, and C.R. Townsend. 1986. Ecology: Individuals, Populations
and Communities. Sinauer, Sunderland, MA.
Berkowitz A.R., C.D. Canham, and V.R. Kelly. 1995. Competition vs. facilitation of
tree seedling growth and survival in early successional communities. Ecology
76:1156-1168.
Bertness M.D. and R. Callaway. 1994. Positive interactions in communities. Trends
Ecol. Evol. 9:191-193.
Bethlenfalvay G.J., M.S. Brown, R.N. Ames, and R.S. Thomas. 1988. Effects of
drought on host and endophyte development in mycorrhizal soybeans in relation to water
use and phosphate uptake. Physiol. Plantar. 72:565-571.
Bethlenfalvay G.J., M.G. Reyes-Solis, S.B. Camel, and R. Ferrera-Cerrato. 1991.
Nutrient transfer between the root zones of soybean and maize plants connected by a
common mycorrhizal mycelium. Physiol. Plantar. 82:423-432.
Bever J.D. 1994. Feedback between plants and their soil communities in an old field
community. Ecology 75:1965-1977.
Blum U. and S.R. Shafer. 1988. Microbial populations and phenolic acids in soil. Soil
Biol. Biochem. 20:793-800.
Boln N.S. 1991. A critical review on the role of mycorrhizal fungi in the uptake of
phosphorus by plants. Plant Soil 134:189-207.
Bolland M.D.A. and B.H. Paynter. 1992. Increasing phosphorus concentration in seed
of annual pasture legume species increases herbage and seed yields. Plant Soil
125:197-205.
Boufalis A. and F. Pellissier. 1994. Allelopathic effects of phenolic mixtures on
respiration of two spruce mycorrhizal fungi. J. Chem. Ecol. 20:2283-2289.
Boyd, R.T., R.T. Furbank, and D.J. Read. 1985. Ectomycorrhiza and the water relations
of trees, p. 689-693. In V. Gianinazzi-Pearson and S. Gianinazzi (ed.) Physiological and
Genetical Aspects of Mycorrhizae. Proc. of the 1st European Symposium on Mycorrhizae,
Dijon, 1-5 July, 1985. Institu National de Recherche Agronomique, Dijon, France.


117
Rousseau J.V.D. and C.P.P. Reid. 1990. Effects of phosphorus and ectomycorrhizas on
the carbon balance of loblolly pine seedlings. For. Sci. 36:101-112.
Ruiz-Lozano J.M., R. Azcn, and M. Gomez. 1995. Effects of arbuscular-mycorrhizal
Glomus species on drought tolerance: Physiological and nutritional plant responses. Appl.
Environ. Microbiol. 61:456-460.
Safir G.R., J.S. Boyer, and J.W. Gerdemann. 1972. Nutrient status and mycorrhizal
enhancement of water transport in soybean. Plant Physiol. 49:700-703.
Salmanowicz B., J.-E. Nylund, and H. Wallander. 1989. High performance liquid
chromatography assay of ergosterol: a technique to estimate fungal biomass in roots
with ectomycorrhiza. Agrie. Ecosyst. Environ. 28:437-440.
Sanders F.E. and P.B. Tinker. 1973. Phosphate flow into mycorrhizal roots. Pestic. Sci.
4:385-395.
SAS Institute Inc. 1989. SAS software, release 6.08. 6th ed. SAS Institute, Inc. Cary,
N.C.
Schnbeck F. 1978. Effect of the endotrophic mycorrhizae on disease resistance of higher
plants. Pflanzenkrankheiten und Pflanzenschutz 85:191-6.
Schreiner R.P. and R.T. Koide. 1993. Streptomycin reduces plant response to
mycorrhizal infection. Soil Biol. Biochem. 25:1131-1133.
Schuler R. and K. Haselwandter. 1988. Hydroxamate siderophore production by ericoid
mycorrhizal fungi. J. Plant Nutr. 11:907-913.
Schepp H., D.D. Miller, and M. Bodmer. 1987. A new technique for monitoring
hyphal growth of vesicular-arbuscular mycorrhizal fungi through soil. Trans. Br. Mycol.
Soc. 89:429-435.
Schweiger, P., A.D. Robson, N.J. Barrow, and L.K. Abbott. 1992. Root hair length
determines beneficial effect of a Glomus sp. on shoot growth of some pasture species.
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Schwertmann U. 1991. Solubility and dissolution of iron oxides. Plant Soil 130:1-25.
Sharma A.K., P.C. Srivastava, B.N. Johri, and V.S. Rathore. 1992. Kinetics of zinc
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Frtil. Soils 13:206-210.


10
contributed by mycorrhizal hyphae, which could alter the calculated kinetic parameters.
Also, in uptake experiments, the use of whole plants rather than root segments would be
more appropriate, since this would incorporate possible source-sink effects of the
plant. The information available suggests that there may be differences in the Km, I,^
and Cmin values of mycorrhizal and nonmycorrhizal plants. The term Km represents the
substrate concentration at which the uptake rate is half of the maximum influx rate, 1^.
The value C,,^ is the minimum solution concentration from which a nutrient can be
absorbed. Studies that have estimated P uptake parameters have generally found a higher
Imax for mycorrhizal plants. Uptake kinetics are dependent on the solution nutrient
concentration (Cress et al., 1979; Sharma et al., 1992), and, consequently, selection of
relevant soil nutrient concentrations in experiments is critical. For example, both P and
Zn appear to have more than one concentration dependent uptake system.
Uptake kinetics may have a major role in mycorrhizal plant survival when
nutrients are limited. In nature, reduced availability of nutrients occurs due to fixation
and biological immobilization. With two competing plant species, the one with the lower
Cmin value would be at a competitive advantage, because it can reduce the nutrient
concentration below the C,^ value of the other organism (Tilman, 1982). In fact,
Pacheco (1992) demonstrated a lower for ectomycorrhizal compared to
nonmycorrhizal pine, which suggests a potential advantage for mycorrhizal plants.
Unavailable nutrients may be released in pulses when microbial activity is temporarily
stimulated due to environmental conditions. Under these circumstances, plants with
differing uptake strategies, such as emphasis on C,^ in one plant versus emphasis on 1^


5
The Autecologv of the Mvcorrhizal Symbiosis
Nutrient Uptake: The Role of External Hvphae
During the past decade there has been a shift in mycorrhizal studies from
quantification of the internal phase to assessing the external hyphal phase in soil. The
external component of this symbiosis contributes to enhanced nutrient uptake of the plant
primarily by extending the roots nutrient depletion zone (Sanders and Tinker, 1973).
The depletion zone extends from 0.1-15 mm from the root surface, depending on the soil
type and plant species (Barber, 1995). By computer modelling, Itoh and Barber (1983)
determined that by doubling the length of root hairs, which have a diameter similar to
some mycorrhizal hyphae, plant phosphorus (P) uptake would double. Doubling the root-
P uptake rate, however, only increased P uptake by 15%. The mycorrhizal benefit is
inversely proportional to the root hair length (Schweiger et al., 1992), indicating that
root hairs partially offset mycorrhizal nutrient gains due to improved spatial exploitation.
Various techniques have been developed to measure external hyphae (Sylvia, 1992;
Dodd, 1994). As a primary tool, the use of fine-mesh screens to prevent roots from
penetrating into hyphal compartments has permitted the separation of root and hyphal
contribution to plant nutrient uptake (Ames et al., 1983; Schiiepp et al., 1987), as well
as quantification of hyphal distribution and density.
Spatial exploitation and hvphae
As reviewed previously (Boln, 1991; OKeefe and Sylvia, 1991), and based on
plant uptake theory (Barber, 1995; Nye and Tinker, 1977), the key parameters involved


72
mechanism leading to differences in plant biomass, it is very likely that this would
influence plant competition. Differences between P status of grass and pine indicate that
P capture by pine reduced the amount of P taken up by grass, specifically at the 32.26-
pM P level. Pine, based on its higher RCC, appeared to be more competitive than grass
when the two competed with each other. When grown with grass the enhanced P uptake
of pine+ corresponded with a larger total dry weight compared to pine', indicating that
inoculation with P. tinctorius did alter the competitive ability of pine.
Although not validated in a repeated experiment, grass at 322.58 M P had a 7.5
or 4.6 times larger RCC than pine+ or pine', respectively. A change in competitive
dominance between the species Rumex acetosella and Poa pratensis with changes in soil
fertility also has been documented (Fowler, 1982). Dual-phasic, P uptake kinetics
dependent on solution-P concentration have been found in fungi (Jennings, 1995), plants
(Barber, 1972) and in mycorrhizal roots (Cress et al., 1979). If a dual-phasic uptake
system exists for each of the plant species, then a higher affinity enzyme system in one
of the species could provide a possible explanation for plant dominance based on uptake
ability.
When pine was grown with pine, competition was equally intense if the plants
were inoculated with P. tinctorius or colonized with T. terrestris. Since the pine' plants
were not uniformly colonized with T. terrestris, I was not able to determine if the EM
inoculation treatments substantially altered the intensity of intraspecific competition of
pine with and without mycorrhizas. However, in an EM competition study by Perry et


21
shown that mycorrhizas can enhance resource capture. Environmental factors strongly
influence the mycorrhizal benefit derived by a plant and consequently also its competitive
ability.
Resource competition
Competition occurs when a resource is inadequate to meet the needs of the
competitors. Nutrient availability fluctuates with the chemical environment and moisture
content of the soil. Soil heterogeneity frequently compounds the intensity of competition
in some areas, since resources are not evenly distributed. Phosphorus has been the focus
of mycorrhizal research, because it is required by plants in proportionately large
quantities, and yet, in the soil it is easily immobilized chemically and biologically.
Consequently, the use of P also dominates mycorrhizal studies involving competition.
When mycorrhizal plants compete under nutrient-limiting conditions, niche
differentiation may be of considerable importance. Plants competing intraspecifically will
have similar nutrient requirements and acquisition strategies which may vary depending
on plant age. Conversely, in interspecific interactions, some competition may be
alleviated by niche differentiation. For example, a potential growth response associated
with spatial niche separation by roots of two grass species only became evident by
experimentally increasing soil depth (Van Auken et al., 1994). The varying plant
responses to different mycorrhizal species in the literature suggest the involvement of a
combination of the earlier reviewed mechanisms, including hyphal spatial distribution and
access to less available nutrients. However, if the mycorrhizal contribution to nutrient


15
that changes in osmotic potential may contribute to their improved drought tolerance over
nonmycorrhizal plants. Most studies with AM fungi do not show any major water
transfer via hyphae (George et al., 1992; Nelsen and Safir, 1982), although this is not
always the case (Faber et al., 1991). In contrast, ectomycorrhizal fungi appear able to
directly transfer water to the plant (Boyd et al., 1985), especially through rhizomorphs
(Duddridge etal., 1980).
Carbon costs
The benefits of enhanced nutrient uptake associated with mycorrhizal biomass
production has energy costs associated with it that vary with the symbiosis. The plant-
microbe-soil interactions are unique to each environment and correspondingly the
mycorrhizal response may vary (Sylvia et al., 1993). This may depend on the fungus,
such as differing growth responses observed with 20 isolates of Pisolithus spp. on
Eucalyptus granis (Burgess et al., 1994). Responses also vary with plant species and
cultivars of the same plant species (Krishna et al., 1985; Mrtensson and Rydberg, 1995;
Smith et al., 1992). So, although host specificity perse has not been documented clearly,
host specific responses do exist. These differences may be related to root morphology,
as suggested by Baylis (1975). Negative relationships have been found between
mycorrhizal dependency and root fibrousness (Hetrick et al., 1992; Pope et al., 1983)
or root hair length (Crush, 1974). However, as suggested by Graham et al. (1991), other
undetermined factors aside from root architecture are more likely involved. Carbon cost,
measured as energy expended per unit nutrient absorbed (Tinker et al., 1994), varies


27
Soil chemical processes associated with organic matter turnover and mycorrhizas
may also play a yet unstudied role in plant interactions. As organic matter is degraded
by microbes, various compounds, including phenolic materials, are released to the soil.
Phenolics have been implicated in various allelopathic interactions (Rice, 1984).
Researchers have demonstrated both inhibition and stimulation of mycorrhizal fungi by
phenolic compounds (Baar et al., 1994; Boufalis and Pellissier, 1994; Siqueira et al.,
1991). Different microbial responses to phenolics have been attributed to variability in
degradation capacity of the microbes, phenolic concentration, soil characteristics and
availability of inorganic soil nutrients (Blum and Shafer, 1988). Similarly, mycorrhizal
fungi vary in their capacity to chemically alter different forms of phenolic compounds
(Giltrap, 1982; Ramstedtand Soderhall, 1983; Tam and Griffiths, 1993). Garbaye (1994)
hypothesized that phenolic compounds may be degraded by bacteria closely associated
with mycorrhizal fungi, thereby also enhancing the establishment of mycorrhizal fungi.
Although no clear link has been found between mycorrhizal sensitivity to phenolic
compounds and plant competitive ability, the results of a few studies suggest a possible
connection (Leake et al., 1989; Wacker and Safir, 1990). Because mycorrhizal fungi
occur in competitive environments, such as forests, with the potential of allelopathy
(Horsley, 1987; Pellissier, 1994), it is important to determine what growth-limiting
factors, as well as their magnitudes, actually occur. Although it is difficult to distinguish
resource competition from interference competition, several researchers have been
successful in differentiating these two phenomena (Nilsson, 1994; Shilling et al., 1992;
Thus, 1994; Wardleetal., 1994).


8
comprehensive review) has ranged between 7 to 109% for P (George et al., 1992; Li et
ah, 1991a; Li et ah, 1991c; Pearson and Jakobsen, 1993), 16-25% for zinc (Kothari et
ah, 1991) and 53-62% for copper (Li et ah, 1991c). In another study, in contrast to the
control, mycorrhizal plants recovered 1.7 times more 15NH4+ applied 2 cm from the root
compartment and 2.75 times more 15NH4+ when applied at a distance of 5 cm (Johansen
et ah, 1993). This provides evidence for an increasing benefit with greater distance. If
diffusion and mass flow of a nutrient are slower than hyphal transport, a mycorrhizal
benefit could conceivably be derived even for nutrients that are not strongly adsorbed to
soils. This was demonstrated when transfer of 15N03' was increased in mycorrhizal
treatments by over 400% under dry soil conditions (Tobar et ah, 1994). These studies
illustrate the capacity of the external phase of mycorrhizas to increase a plants nutrient
absorption. They also demonstrate that the response differs depending on the soil
environment, the fungi involved and the spatial location of nutrients.
Uptake kinetics
Uptake kinetics can be quite different between mycorrhizal and nonmycorrhizal
plants. Based on uptake models and mycorrhizal characteristics, however, differences in
uptake kinetics appear to be of secondary importance compared to surface area and
spatial distribution (OKeefe and Sylvia, 1991). Few uptake studies comparing
mycorrhizal and nonmycorrhizal plants have been performed since the review of OKeefe
and Sylvia (1991). Most studies have used root weight to standardize uptake parameters
(Table 2-1). However, none have taken into account the surface area or weight


96
the Materials and Methods of Chapter 4. This experiment was not repeated. Colonization
of corn by AM fungi was lower than that for P. chamaelonche, possibly due to the way
the two plant species were started. Both plant species in the treatments with two
Gigaspora spp. and A. scrobiculata were more highly colonized than plants in the other
treatments, suggesting that these AM fungi may be better suited to the particular
combination of soil and greenhouse environment.


35
cases (Evans and Miller, 1988; Fitter and Nichols, 1988; Trappe et al., 1984), but these
results are not always achieved (Koide et al., 1988; Fitter, 1986; Trappe et al., 1984).
Much of this variability is likely attributable to the experimental conditions such as soil
type, method and timing of fungicide application and potentially more complex
interactions occurring within the soil microbial community. For example, benomyl can
inhibit nematodes (Elamayem et al., 1978) and different fungi that do not form
mycorrhizas (Edgington et al., 1971), thereby indirectly altering mycorrhizal effects.
Several studies have addressed the effects of arbuscular mycorrhizas on plant
interactions (Fitter, 1977; Hall, 1978; Newman et al., 1992), and some have utilized
benomyl (Hartnett et al., 1993; Hetrick et al., 1989; Newsham et al., 1995) or other
fungicides (Gange et al., 1993) to create control treatments. Only one study addressed
the influence of EM fungi on plant competition (Perry et al., 1989). Very few studies
have taken place under field conditions, and apparently none have addressed the role of
mycorrhizas in the interactions between AM and EM plants. Benomyls putative selective
effect against AM fungi and neutral effects on EM fungi (Trappe et al., 1984) could be
valuable in sorting out the individual benefits of these two types of mycorrhizal
symbioses to different host plants competing for the same nutrients.
As part of a larger plant competition study between AM and EM plants, the
usefulness of benomyl as a tool to selectively control mycorrhizas was tested. The main
objectives were to: (i) compare the efficacy of benomyl in controlling mycorrhizas in the
greenhouse to that in the field, (ii) differentiate effects of benomyl on external hyphae


64
Table 4-2. Ergosterol concentration (fig g'1) of Pinus elliottii roots inoculated with
Pisolithus tinctorius (pine+) or noninoculated (pine), and grown in combination with
Pinus elliottii (pine) or Panicum chamaelonche (grass) at either 0.32, 3.23 or 32.26 /xM
P for 18 wk. Each value represents the mean of six replicates SE.
Competition
Treatment
Phosphorus added (fiM P)
0.32
3.23
32.26
Pine+ x pine+
181 20
282 34
297 33
Pine' x pine'
129 13
104 11
150 11
Pine+ x grass'
192 8
297 46
260 26
Pine' x grass'
137 21
139 12
140 17


2
are normally mycorrhizal. Fungicides can be useful in distinguishing mycorrhizal effects
from other influences on plants in the field. The fungicide benomyl is selective in that
it has inhibitory effects on AM but not on EM fungi. The selectivity of this fungicide
would be useful in isolating the nutrient uptake mediated by AM fungi in AM and EM
plant competition studies. As part of a larger competition study involving slash pine
(Pinus elliottii) and weeds at a field site northwest of Gainesville, I tested benomyl in the
field and in the greenhouse to determine if it would suitably control AM but not EM
fungi (Chapter 3).
I addressed the direct effect of mycorrhizas on plant competition for P in
greenhouse and growth chamber studies involving slash pine and Panicum chamaelonche,
a dominant weed species at the field site. The main objectives of the greenhouse
competition study, described in Chapter 4, were to determine if (i) mycorrhizas affect
plant competition at the interspecific or intraspecific level, (ii) competition is dependent
on soil nutrient concentration and (iii) competitive abilities are related to differences in
P uptake kinetics. The goals of the growth chamber study, presented in the first section
of the Appendix, were to assess (i) the contribution of mycorrhizal fungal hyphae to total
plant P uptake and (ii) the competitive abilities of the AM and EM fungi with respect to
each other.


116
Plenchette C. and R. Perrin. 1992. Glasshouse evaluation of fungicide effects on
mycorrhizal development on leek and wheat. Mycorrhiza 1:59-62.
Pope P.E., W.R. Chaney, J.D. Rhodes, and S.H. Woodhead. 1983. The mycorrhizal
dependency of four hardwood tree species. Can. J. Bot. 61:412-417.
Rabatin, S.C. and B.R. Stinner. 1991. Vesicular-arbuscular mycorrhizae, plant, and
invertebrate interactions in soil. p. 141-168. In P. Barbosa, V.A. Krischik and C.G.
Jones (ed.) Microbial Mediation of Plant-Herbivore Interactions. John Wiley & Sons,
New York, N.Y.
Rabin L.B. and R.S. Pacovsky. 1985. Reduced larva growth of two Lepiodoptera
(Noctuidae) on excised leaves of soybean infected with a mycorrhizal fungus. J. Econ.
Entomol. 78:1358-1363.
Ramstedt M. and K. Soderhall. 1983. Protease, phenoloxidase and pectinase activities
in mycorrhizal fungi. Trans. Br. Mycol. Soc. 81:157-161.
Read, D.J., R. Francis, and R.D. Finlay. 1985. Mycorrhizal mycelia and nutrient
cycling in plant communities, p. 193-217. In A.H. Fitter, D. Atkinson, D.J. Read and
M.B. Usher (ed.) Ecological Interactions in Soil: Plants, Microbes and Animals.
Blackwell Scientific Publications, Oxford, United Kingdom.
Reeves M. 1992. The role of VAM fungi in nitrogen dynamics in maize-bean intercrops.
Plant Soil 144:85-92.
Rhodes L.H. and P.O. Larsen. 1981. Effect of fungicides on mycorrhizal development
of creeping grass. Plant Dis. 65:145-147.
Rice E. 1984. Allelopathy. Academic Press, Orlando, FL.
Rickerl D.H., F.O. Sancho, and S. Ananth. 1994. Vesicular-arbuscular endomycorrhizal
colonization of wetland plants. J. Environ. Qual. 23:913-916.
Robson, A.D. and L.K. Abbott. 1989. The effect of soil acidity on microbial activity
in soils, p. 140-165. In A.D. Robson (ed.) Soil Acidity and Plant Growth. Academic
Press, Melbourne, Australia.
Rosendahl C.N. and S. Rosendahl. 1990. The role of vesicular-arbuscular mycorrhiza
in controlling damping-off and growth reduction in cucumber caused by Pythium
ultimum. Symbiosis 9:363-366.


51
persistence in the soil, which is variable (Ware, 1992) due to degradation and sorption
in different soil environments. In sandy soils where sorption is low, somewhat
comparable to the sand used in the greenhouse study, persistence may be longer,
assuming leaching does not occur, so that an application every 5 to 6 wk may suffice.
Soils with higher levels of organic matter or clay may require more frequent applications
or higher concentrations.
The method of application is also critical. Although benomyl is considered a
systemic fungicide, translocation from leaves to the active site in the roots appears to be
minimal. A soil drench is the optimal method of application (Fitter and Nichols, 1988;
Hassall, 1990; Perrin and Plenchette, 1993). Appropriate preparations should be made
to accommodate the increasing ground cover as treatments are applied later in the
growing season. Tall ground cover may be compensated for by applying a large volume
of water to wash the active ingredient to the soil. Benomyl concentrations applied
experimentally have ranged from 0.5 to 300 kg benomyl ha1 (Trappe et al., 1984).
Treatments of as little as 3 kg ha'1 biweekly in a short turf grass setting have been
adequate to reduce AM colonization by 80% (Rhodes and Larsen, 1981). As a
consequence, the combination of concentration, volume of water used and frequency of
application balanced with the environmental conditions should provide the desired
reduction of mycorrhizal activity.


TABLE OF CONTENTS
ACKNOWLEDGMENTS iii
LIST OF TABLES v
LIST OF FIGURES viii
ABSTRACT x
CHAPTERS
1 INTRODUCTION 1
2 MYCORRHIZAS AND PLANT COMPETITION 3
Introduction 3
The Autecology of the Mycorrhizal Symbiosis 5
Nutrient Uptake: The Role of External Hyphae 5
Other Mycorrhizal Effects 14
Synecology 19
Plant Interactions 20
Environmental Conditions and Plant Competition 26
Plant Succession and Community Structure 30
3 LIMITATIONS IN THE USE OF BENOMYL IN
EVALUATING MYCORRHIZAL FUNCTIONING 34
Introduction 34
Materials and Methods 36
Results 41
Discussion 48
IV


APPENDIX 1
GROWTH CHAMBER COMPETITION STUDY BETWEEN PINUS ELLIOTTII
AND PANICUM CHAMAELONCHE
Introduction
A competition study involving pine (Pinus elliottii Engelm. var. elliottii)) and
grass {Panicum chamaelonche Trin.) was set up to determine: (i) the contribution of
mycorrhizal fungal hyphae to total plant P uptake and (ii) the competitive abilities of
arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) fungi with respect to each other.
This experiment was conducted twice. No colonization was obtained in the inoculated
grass treatments the first time this experiment was run, and therefore no competition
resulted between plants.
Materials and Methods
The study was initiated as part of a larger field investigation into competition for
nutrients between pine and grass at a field site 21 km northwest of Gainesville, Florida.
Pine and grass were grown in acid-washed sand and inoculated as in the Material and
Methods of Chapter 4. Grasses were inoculated 14 wk prior to the start of the experiment
by applying to each plant root system a minimum of 20 spores of a mixed culture of
Gigaspora rosea (INVAM FL224) and Scutellospora heterogama (INVAM FL225) as in
the Materials and Methods section of Chapter 4. At the start of the experiment, grass had
a mean root colonization of 13%. Pine roots were visually inspected to prevent inclusion
80


A1-2 Mean hyphal length density (m cm'3 of soil) for each competition
treatment presented separately for each compartment (one hyphal and two
plant compartments). Hyphal length is made up of the sum of both AM
and EM fungal hyphae. Each value represents the mean of a minimum of
six replicates SE 85
A1-3 Mean soil-phosphorus content (fig P g'1 of soil) for each competition
treatment presented separately for each compartment (one hyphal and two
plant compartments). Each value represents the mean of a minimum of six
replicates SE 90
Vll


25
fractions could be verified further by comparing nutrient transfer from a mycorrhizal
donor plant to either a myc' mutant (a mutant plant not able to form mycorrhiza) or a
normal mycorrhizal receiver plant. The quantity obtained by the receiver is variable, and
appears to depend on the nutrient involved. Generally, P is not transferred at fast rates
(Newman and Eason, 1993) or in quantities that significantly affect growth (Ikram et al.,
1994). The transfer of N by mycorrhizas has been documented (Newman, 1988), with
most studies utilizing a legume, because of its importance in intercropping systems, as
the donor plant. The quantity of N transferred from the root zone of donor plant to the
receiver plant varies (Bethlenfalvay et al., 1991; Frey and Schiiepp, 1993). By increasing
competitive pressures for N in intercropping systems, mycorrhizal fungi at certain times
may enhance nitrogen fixation (Barea et al., 1989), although this is not always the case
(Reeves, 1992). Both of these studies and others (Hamel and Smith, 1991; Ikram et al.,
1994) have found minimal amounts to no N transferred. The quantitative significance of
mycorrhizal transfer of nutrients to total uptake by the receiver plant still remains
unclear.
The phenomenon of increased survival of certain plant species in mycorrhizal
microcosm studies (Grime et al., 1987) deserves further attention, especially, since no
direct cause was found. Source-sink gradients, such as those created by shading or low-
nutrient status of one plant, have been suggested as the force behind nutrient transfer.
For interplant C transfer, shading of the receiver plant increased C translocation to that
plant (Read et al., 1985). However, shading does not always produce this effect (Franson
et al., 1994; Hirrel and Gerdemann, 1979). In contrast, clipping of leaves to simulate


74
component is limited precisely by the interwoven nature of the plant and soil complex.
When this is accomplished, we will be closer to determining the magnitude of
mycorrhizal effects on the ecology or economy of an ecosystem.


113
Leyval C. and J. Berthelin. 1993. Rhizodeposition and net release of soluble organic
compounds by pine and beech seedlings inoculated with rhizobacteria and
ectomycorrhizal fungi. Biol. Frtil. Soils 15:259-267.
Li X.-L., E. George, and H. Marschner. 1991a. Extension of the phosphorus depletion
zone in VA-mycorrhizal white clover in a calcareous soil. Plant Soil 136:41-48.
Li X.-L., E. George, and H. Marschner. 1991b. Phosphorus depletion and pH decrease
at the root-soil and hyphae-soil interfaces of VA mycorrhizal white clover fertilized with
ammonium. New Phytol. 119:397-404.
Li X.-L., H. Marschner, and E. George. 1991c. Acquisition of phosphorus and copper
by VA-mycorrhizal hyphae and root-to-shoot transport in white clover. Plant Soil
136:49-57.
Linderman, R.G. 1994. Role of VAM fungi in biocontrol, p. 1-26. In F.L. Pfleger and
R.G. Linderman (ed.) Mycorrhizae and Plant Health. APS Press, St. Paul, MN.
Lu X. and R.T. Koide. 1994. The effects of mycorrhizal infection on components of
plant growth and reproduction. New Phytol. 128:211-218.
Lussenhop J. and R. Fogel. 1993. Observing soil biota in situ. Geoderma 56:25-36.
Marschner H. 1986. Mineral Nutrition of Higher Plants. Academic Press, New York,
N.Y.
Marschner H. and B. Dell. 1994. Nutrient uptake in mycorrhizal symbiosis. Plant Soil
159:89-102.
Marschner H. and V. Romheld. 1983. In vitro measurement of root-induced pH changes
at the soil-root interface: Effect of plant species and nitrogen sources. Z.
Pflanzenphysiol. 111:241-251.
Martin F., C. Delaruelle, and J.L. Hilbert. 1990. An improved ergosterol assay to
estimate fungal biomass in ectomycorrhizas. Mycol. Res. 94:1059-1064.
Martin F., P. Rubini, R. Cote, and I. Kottke. 1994. Aluminium polyphosphate
complexes in the mycorrhizal basidiomycete Laceara bicolor: A 27A1 nuclear magnetic
resonance study. Planta 194:241-246.
Martins M.A. 1993. The role of the external mycelium of arbuscular mycorrhizal fungi
in the carbon transfer process between plants. Mycol. Res. 97:807-810.


70
depletion zone. Since an acid-washed sand was used, soluble inorganic nutrients were the
only source of nutrients available to both the roots and mycorrhizal fungi. This made the
potential ability to utilize nutrients in different forms inconsequential. As a result,
differences in P acquisition most likely were related to a combination of differences in
absorbing surface area and uptake rates. Although plant density also may affect the
outcome of plant competition (Hartnett et al., 1993; Taylor and Aarssen, 1989) this was
not tested.
Previous researchers comparing uptake by mycorrhizal and nonmycorrhizal plants
have observed a higher uptake rate for mycorrhizal plants (Cress et al., 1979;
Karunaratne et al., 1986; Pacheco and Cambraia, 1992). However, these estimates
generally are reported on a root weight or root length basis only. If the estimates
included hyphal surface area the uptake rates would be greatly reduced for the
mycorrhizal plants. In the current study, pine+ and pine' had similar uptake rates if based
on root surface area alone. The lower uptake rate of pine+ compared to pine', based on
total surface area, strongly suggests that hyphal nutrient depletion zones were
overlapping. If inadequate mixing of the nutrient solution occurs, the rate-limiting step
for uptake would be the replenishment of P absorbed inside the dense mass of hyphae.
The P uptake kinetics of nonmycorrhizal pine and grass determined hydroponically in a
0.32-piM P solution were different, but the variability was relatively high due to the low
number of replications. The higher 1^ demonstrated by pine would permit pine to
sequester more P than grass, which would give the pine a competitive advantage over
grass under the regular fertilization schedule followed here. The lower Cmin for grass


69
At each level of P, pine+ had a higher RCC than grass (Fig. 4-4A). In contrast,
pine' was more competitive than grass only above the 0.32-/*M P level (Fig. 4-4B). In
the repeat of the experiment, grass RCC between 32.26 and 322.58 /M P rose by 279
and 144% when competing with pine' and pine+ respectively. At this same P level the
RCC for pine' and pine+ dropped by 75 and 27% respectively.
Determination of P Uptake Kinetics for Pine and Grass
Again, no colonization was observed in the grass+ plants, even though I attempted
to use indigenous fungi, so the treatment was excluded from analysis. On a root surface
area basis, 1^ was not different between pine+ and pine'; however, Imax based on total
surface area, which included mycorrhizal hyphae, was much lower in the pine+.
Consequently only the values for the nonmycorrhizal pine and grass are shown. A higher
Imax value was observed for pine, whereas grass had a lower C^ value (Table 4-3).
Discussion
Inoculation of slash pine with P. tinctorius enhanced P acquisition of pine when
grown with nonmycorrhizal grass. This response is dependent on at least two conditions
of the experimental design, namely soil volume and nutrient availability. In large soil
volumes mycorrhizal fungi are able to enhance plant nutrient uptake by accessing areas
beyond the roots nutrient depletion zone. This mechanism is much less important in
smaller volumes of soil, such as in this experiment, since roots and fungal hyphae
proliferate throughout the pot, essentially making the entire volume a single nutrient


4 MYCORRHIZAS AFFECT PLANT COMPETITION FOR
PHOSPHORUS BETWEEN PINE AND GRASS 52
Introduction 52
Materials and Methods 53
Results 60
Discussion 69
5 CONCLUSIONS 75
APPENDICES
1 GROWTH CHAMBER COMPETITION STUDY BETWEEN
PINE AND GRASS 80
Introduction 80
Materials and Methods 80
Results 83
Discussion 93
2 COLONIZATION OF PANICUM CHAMAELONCHE AND CORN BY
DIFFERENT ARBUSCULAR MYCORRHIZAL FUNGI 95
3 PHOSPHORUS GROWTH RESPONSE CURVE FOR
NONMYCORRHIZAL PANICUM CHAMAELONCHE 98
REFERENCE LIST 101
BIOGRAPHICAL SKETCH 122
v


88
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o
-*->
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o
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700
600
500
400
300
200
100
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8
7
6
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4
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2
1
0
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* #
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4*
+ + X + X *f
^ ^
* o'*'
r ,& <* + +
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Figure Al-3. Mycorrhizal colonization of (A) slash pine and (B) grass grown in the
growth chamber for 62 d. Each bar represents the mean of a minimum of six replicates
SE.


APPENDIX 2
COLONIZATION OF PANICUM CHAMAELONCHE AND CORN BY DIFFERENT
ARBUSCULAR MYCORRHIZAL FUNGI
The objective of this experiment was to determine the ability of different
arbuscular mycorrhizal (AM) fungi to form mycorrhizas with either corn (Zea mays L.
cv. Silver Queen) or Panicum chamaelonche Trin. under the conditions in the
greenhouse.
Panicum chamaelonche plants were obtained from cultures maintained in sand in
the greenhouse. Plants were started from seed collected from the field and vegetatively
propagated in 150-ml pots (7 cm2 of surface area). Corn plants were grown in 150-ml
pots for 2 wk prior to inoculation. At the start of the experiment plant roots were washed
free of sand. Four plants of each species were inoculated with either a minimum of 0.5
g of onion root fragments colonized by Acaulospora scrobiculata (S315), Gigaspora
margarita (INVAM FL215), Glomus etunicatum (INVAM FL312), or Glomus sp.
(INVAM FL329, formerly FL906), or a minimum of 20 spores of Gigaspora rosea
(INVAM FL224) or Scutellospora heterogama (INVAM FL225). The latter two had been
isolated from pot cultures of P. chamaelonche originating from the field and grown in
field soil in the greenhouse. There were 4 replicates for each treatment. Plants had the
same environmental conditions as in the Materials and Methods of Chapter 4 and were
fertilized with the same nutrient solution at the 32.26 iM NaH2P04 level. After 9 wk,
plants were harvested and colonization was determined using the same technique used in
95


Root Length Colonized (m) Ergosterol (mg g-^ of root dwt.)
47
6
5
4
3
2
1
0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
1
B
i
V//A 0 kg benomyl ha'1
20 kg benomyl ha'1
l l 60 kg benomyl ha'1
150 kg benomyl ha'1
ii
I
(
n
I
i
0 2 4 6
Week After Benomyl Application
Figure 3-4. Mycorrhizal colonization of (A) slash pine and (B) corn grown in the
greenhouse in response to 0, 20, 60 or 150 kg benomyl ha1. Each symbol represents the
mean of seven replicates SE.


46
TOTAL ACTIVE TOTAL ACTIVE TOTAL ACTIVE TOTAL ACTIVE
0 2 4 6
Week After Benomyl Application
Figure 3-3. Soil hyphal length (total) and activity (active) of mycorrhizal (A) Pinus
elliottii and (B) corn plants in response to 0, 20, 60 or 150 kg benomyl ha'1 in the
greenhouse. Each symbol represents the mean of seven replicates SE.


118
Shilling D.G., J.A. Dusky, and M.A. Mossier, 1992. Allelopathic potential of celery
residues on lettuce. J. Amer. Soc. Hort. Sci. 117:308-312.
Siqueira J.O., M.G. Nair, R. Hammerschmidt, and G.R. Safir. 1991. Significance of
phenolic compounds in plant-soil-microbial systems. Crit. Rev. Plant Sci. 10:63-121.
Siqueira J.O., G.R. Safir, and M.G. Nair. 1991. VA-mycorrhizae and mycorrhiza
stimulating isoflavonoid compounds reduce plant herbicide injury. Plant Soil
134:233-242.
Smith S.E. and S. Dickson. 1991. Quantification of active vesicular-arbuscular
mycorrhizal infection using image analysis and other techniques. Aust. J. Plant Physiol.
18:637-648.
Smith S.E., A.D. Robson, and L.K. Abbott. 1992. The involvement of mycorrhizas in
assessment of genetically dependent efficiency of nutrient uptake and use. Plant Soil
146:169-179.
Steel R. and J. Torrie. 1980. Principles and Procedures of Statistics: A Biometrical
Approach. McGraw-Hill, New York, N.Y.
Stevenson F.J. 1986. Cycles of Soil. John Wiley & Sons, Inc. New York, N.Y.
Stockey A. and R. Hunt. 1994. Predicting secondary succession in wetland mesocosms
on the basis of autecological information on seeds and seedlings. J. Appl. Ecol.
31:543-559.
Sukarno N., S.E. Smith, and E.S. Scott. 1993. The effect of fungicides on
vesicular-arbuscular mycorrhizal symbiosis. 1. The effects on vesicular- arbuscular
mycorrhizal fungi and plant growth. New Phytol. 125:139-147.
Sylvia D.M. 1988. Activity of external hyphae of vesicular-arbuscular mycorrhizal fungi.
Soil Biol. Biochem. 20:39-43.
Sylvia, D.M. 1992. Quantification of external hyphae of vesicular-arbuscular mycorrhizal
fungi, p. 53-66. In J.R. Norris, D.J. Read and A.K. Varma (ed.) Methods in
Microbiology: Techniques for the Study of Mycorrhiza. Academic Press, New York,
N.Y.
Sylvia, D.M. and S.E. Williams. 1992. Vesicular-arbuscular mycorrhizae and
environmental stress, p. 101-124. In R.G. Linderman and G.J. Bethlenfalvay (ed.)
Mycorrhizae in Sustainable Agriculture. ASA Special Publication no 54, Madison, WI.


BIOGRAPHICAL SKETCH
I was born on April 29, 1958 in Queens, New York. I grew up bilingually
learning both Swiss-German as well as English. At the age of 13 my family moved to
Switzerland, where I attended the public school system. I completed a four-year degree
from the College of Business Administration and Economics in Zurich. In 1980, I
returned to the United States where I completed a B.S. in horticulture from The
Pennsylvania State University in 1983 specializing in vegetable and fruit production.
After various jobs from agricultural consulting to landscaping, I took on a position as
assistant winemaker at Bucks Country Vineyards in Pennsylvania. Surrounded by
fermenting musts and grape diseases I took an interest in microbiology. Not to forsake
plants in the process, I decided to return for graduate studies in soil microbiology.
Specifically, my work dealt with tissue-cultured asparagus and mycorrhizas. I completed
my M.S. in the Department of Botany and Plant Pathology at the Michigan State
University in 1990. From there, to round out my background, I continued towards a
Ph.D. in the Soil and Water Science Department at the University of Florida.
122


14
nutrients in forms not available to nonmycorrhizal plants may prove to be less important
than the spatial accessibility of nutrients beyond the roots nutrient depletion zone.
Fungi and plants release polysaccharides resulting in a direct mucilaginous
connection to soil particles in their respective rhizospheres and hyphospheres. This
matrix may enhance aggregate formation, reduce nutrient loss from leaching and reduce
dehydration by increasing waterholding capacity (Chenu, 1993). Furthermore, the
connections to soil particles permit direct transfer of nutrients from soil to root (Uren,
1993), which may be essential under low moisture conditions, very similar to the contact
exchange described by Nye and Tinker (1977).
Other Mvcorrhizal Effects
Water relations
Improved water relations in mycorrhizal plants have been documented extensively
(Nelsen, 1987) and have been associated with improved plant-P status (Nelsen and Safir,
1982), though non-P responses are also reported (Aug et al., 1986; Bethlenfalvay et al.,
1988; Davies, Jr. et al., 1992). Drought tolerance in mycorrhizal plants is enhanced by
increasing plant turgor, leaf water potential, stomatal conductance and root hydraulic
conductivity. In addition, Bethlenfalvay et al. (1988) have suggested that mycorrhizal
fungi are able to acquire soil water at lower water potentials than roots, although Nelsen
(1987) proposed that fungal hyphae gave the plant a spatial advantage by extending the
water depletion zone beyond the root. Recently, Ruiz-Lozano et al. (1995) found
differences in proline concentration in drought-stressed mycorrhizal plants and suggested


61
Pine shoot-P concentration increased in all treatments with increasing level of
applied P (Fig. 4-1 A). A higher shoot-P concentration was observed in pine+ compared
to the pine' (P < 0.001). In the interspecific competition treatments where pine was
grown with grass, pine+ had an elevated shoot-P content compared to pine' (Fig. 4-1B).
The difference became more apparent with increasing P level. In the treatments where
pine was grown with pine, pine' and pine+ acquired similar quantities of P at all levels
of applied P. Total dry weight of pine was not affected by the level of applied P (Fig.
4-1C). Overall pine+ had a higher total dry weight than pine' (P = 0.07) and more so
when grown with grass (P < 0.01). Pines grown with other pines had a lower dry
weight than when grown with grass (P < 0.01). Colonization was also higher in the
pine+ treatments inoculated with P. tinctorius than in the pine' treatments that became
colonized with T. terrestris (Table 4-2).
Similar trends were observed in the repeat of the experiment, although differences
were smaller and not always significant. Pine+ grown with grass had a 70% higher
shoot-P level compared to pine' (P < 0.05), but only at the 32.26-/M P level. The total
pine+ biomass was 31% larger than pine' at 32.26-/xM P (P < 0.05), and only when
grown with grass.
For grass shoot-P concentration there was a significant interaction between the
level of applied P and the competition treatment (P < 0.05). At the 32.26-/M P level,
the shoot-P concentration of grass when grown with pine+ was lower than when grown
with pine' (Fig. 4-2A). The grass intraspecific competition treatment at this P level was
higher than both pine treatments. The shoot-P content at the 32.26-/xM P level was also


53
et al., 1993; Hetrick et al., 1989). The majority of these studies relate to competition
between arbuscular-mycorrhizal (AM) plants. To my knowledge, only one study has
addressed ectomycorrhizal (EM) effects on plant competition (Perry et al., 1989). Plant
competition between EM and AM plants has not been explored specifically.
The goal of this research was to assess the effect of mycorrhizas on the
competitive ability of slash pine (Pinus elliottii Engelm. var. elliottii), which commonly
is grown for pulpwood in the southeastern United States. Grasses, among other plants,
compete extensively in new slash pine plantations since weed control is practiced
infrequently. The specific objectives of the study were to determine (i) if mycorrhizas
alter the competitive ability of pine when growing with grass and (ii) how this
relationship is modified by phosphorus (P) concentration.
Materials and Methods
Greenhouse Competition Study
All experiments were conducted in acid-washed sand. Acid-washing was
accomplished by treating the sand with 25% HC1 for 24 h, then draining the acid and
rinsing the sand until the pH increased to that of the deionized water being used. Eighty
percent of the sand was in the particle size range of 0.160 to 1 mm, and the majority of
the remaining portion was larger than 1 mm.
Slash pine seeds were disinfested for 2 min in a 5.25% sodium hypochlorite
solution with 0.2 ml Liqui-Nox surfactant (Alconox, Inc., New York, N.Y.) and then
rinsed thoroughly with tap water. Seedlings were raised from seed for 2 wk in a growth


107
Durall D.M., A.W. Todd, and J.M. Trappe. 1994. Decomposition of 14C-labelled
substrates by ectomycorrhizal fungi in association with Douglas fir. New Phytol.
127:725-729.
Eason W.R. and E.I. Newman. 1990. Rapid cycling of nitrogen and phosphorus from
dying roots of Lolium perenne. Oecologia 82:432-436.
Edgington L.V., K.L. Khew, and G.L. Barron. 1971. Fungitoxic spectrum of
benzimidazole compounds. Phytopath. 61:42-44.
Eissenstat D.M., J.H. Graham, J.P. Syvertsen, and D.L. Drouillard. 1993. Carbon
economy of sour orange in relation to mycorrhizal colonization and phosphorus status.
Annals of Botany 71:1-10.
Eissenstat D.M. and E.I. Newman. 1990. Seedling establishment near large plants:
effects of vesicular-arbuscular mycorrhizas on the intensity of plant competition. Funct.
Ecol. 4:95-99.
Elamayem M.M.A., M.R.A. Shehata, G.A. Tantawy, I.K. Ibrahim, and M.A. Schuman.
1978. Effect of CGA 12223 and benomyl on Meloidogyne javanica and Rhizoctonia
solani. Phytopath. Z. 92:289-293.
Evans D.G. and M.H. Miller. 1988. Vesicular-arbuscular mycorrhizas and the
soil-distrubance-induced reduction of nutrient absorption in maize I. Causal relations.
New Phytol. 110:67-74.
Faber B.A., R.J. Zasoski, D.N. Munns, and K. Shackel. 1991. A method for measuring
hyphal nutrient and water uptake in mycorrhizal plants. Can. J. Bot. 69:87-94.
Fabig B., A.M. Moawad, and W. Achtnich. 1989. Effect of VA mycorrhiza on dry
weight and phosphorus content in shoots of cereal crops fertilized with rock phosphate
at different soil pH and temperature levels. Z. Pflanzenernaehr. Bodenkd. 152:255-260.
Fitter A.H. 1977. Influence of mycorrhizal infection on competition for phosphorus and
potassium by two grasses. New Phytol. 79:119-125.
Fitter A.H. 1986. Effect of benomyl on leaf phosphorus concentration in alpine
grasslands: A test of mycorrhizal benefit. New Phytol. 103:767-776.
Fitter A.H. and J. Garbaye. 1994. Interactions between mycorrhizal fungi and other soil
organisms. Plant Soil 159:123-132.
Fitter A.H. and R. Nichols. 1988. The use of benomyl to control infection by
vesicular-arbuscular mycorrhizal fungi. New Phytol. 110:201-206.


92
Figure Al-5. Uptake rate of (A) slash pine and (B) grass grown in the growth chamber
for 62 d. Uptake rate was calculated on a unit surface area basis which included both
root and hyphal surface area. Each bar represents the mean of a minimum of six
replicates SE.


39
only. Pisolithus tinctorius (Pers.) Coker & Couch (isolate S106) was grown with no
shaking in a modified Melin-Norkrans liquid medium (Marx, 1969) containing glucose
instead of sucrose. Just prior to use, fungal mats were washed with tap water, added to
a food processor with water and chopped (Rousseau and Reid, 1990). Eight-week-old
pines were inoculated with the fungus by immersing the washed roots in the suspension
and then grown in the greenhouse in 500 ml of sand in Deepots (28 cm2 of surface
area; McConkey, Co., Sumner, WA). Six weeks after inoculation, 10 ml of a suspension
of Benlate 50 WP in deionized water was applied once at 0, 20, 60 or 150 kg benomyl
ha'1 equivalent (based on pot surface area). Plants were grown from January to March
1993 under a mean photosynthetic photon flux density of 535 panol m'2 s'1 and 17/30C
(min./max.) temperature regime.
Groups of plants were harvested before, and then 2 and 4 wk after benomyl
application. Prior to harvesting the plants, a soil core (15.5-mm diam. by 15-cm deep)
was removed from each pot. Hyphal length and activity were evaluated by a slightly
modified procedure of Sylvia (1988). A thoroughly mixed, 10-g, wet-mass subsample of
soil was added to 500 ml of water and chopped in a Waring blender at the high setting
for 20 seconds. The resulting suspension was allowed to settle for 20 seconds before a
25-ml portion was removed and filtered through a 0.45-pim-pore size membrane (GN-6
Metricel; Gelman, Ann Arbor, MI). The hyphae on the membrane were stained for
6 h with INT solution, destained with tap water, counterstained for 30 min with 0.1 %
trypan blue in lactoglycerol and destained again with tap water. Using a gridline-intercept


45
O)
i
o
S
o
i-
X///X O kg benomyl ha1
fcS&l 20 kg benomyl ha'1
I f-hH 60 kg benomyl ha'1
IN>NM 150 kg benomyl ha
-i
Week After Benomyl Application
Figure 3-2. Total dry weight of mycorrhizal (M) and nonmycorrhizal (C) plants, (A)
Pinus elliottii and (B) corn in response to 0, 20, 60 or 150 kg benomyl ha-1 in the
greenhouse. Each symbol represents the mean of seven replicates SE.


CHAPTER 3
LIMITATIONS IN THE USE OF BENOMYL IN EVALUATING
MYCORRHIZAL FUNCTIONING
Introduction
A limitation to mycorrhizal field research is the difficulty of obtaining an
appropriate nonmycorrhizal control, since plants in nature are normally colonized. Soil
fumigation has been used to control mycorrhizal fungi; however, the broad biocidal
effects limit the usefulness of this technique. Fungicides are more specific and alter fewer
biological soil processes. Paul et al. (1989) summarized the ideal properties of a
fungicide used to chemically exclude an organism from an experiment. The fungicide
properties should include: (i) moderate persistence to reduce mechanical disturbance from
the application process, (ii) an appropriate activity spectrum that targets selected
organisms only and (iii) no direct physiological effects on the plant.
The systemic fungicide benomyl, a benzimidazole, has been used frequently to
reduce arbuscular mycorrhizal (AM) activity in experimental treatments (Jalali and
Domsch, 1975; Kough et al., 1987; Fitter and Nichols, 1988; Hartnett et al., 1994;
Newsham et al., 1995; West et al., 1993). Benomyls lack of direct effects on plants and
somewhat selective effects against AM fungi (Zygomycetes) currently make it a better
choice compared to other fungicides (Paul et al., 1989; Sukarno et al., 1993).
Nonetheless, the amount of mycorrhizal control achieved with benomyl has varied.
Reduction of colonization or biomass of mycorrhizal plants has been observed in several
34


Table Al-3. Mean soil P content (/xg P g'1 of soil) for each competition treatment presented separately for each compartment (one
hyphal and two plant compartments). Values represent the mean of a minimum of six replicates SE.
Compartment
A B
Plant Compartment A
Hyphal Compartment
Plant Compartment B
Pine+
Pine+
1758 99
2543 224
1758 99
Pine'
Pine'
2210 122
2971 61
2210 122
Pine+
Grass+
2009 68
2321 155
1865 + 44
Pine+
Grass'
2091 238
2622 204
1722 203
Pine'
Grass+
2172 228
3233 201
2077 249
Pine"
Grass'
2426 174
2529 194
1580 169
Grass+
Grass+
2142 261
2732 255
2142 261
Grass'
Grass'
1827 150
2477 223
1827 150
s


Table A1-2. Mean hyphal length density (m cm'3 of soil) for each competition treatment presented
separately for each compartment (one hyphal and two plant compartments). Hyphal length is made up
of the sum of both AM and EM hyphae. Each value represents the mean of a minimum of six replicates
SE.
Compartment
A B
Plant Compartment A
Hyphal Compartment
Plant Compartment B
Pine+
Pine+
131.26 13.58
113.12 15.87
131.26 15.58
Pine'
Pine'
94.82 8.69
80.73 12.36
94.82 + 8.69
Pine+
Grass+
82.40 10.55
63.69 8.01
32.48 6.39
Pine+
Grass
100.43 7.76
64.32 6.44
21.16 3.69
Pine'
Grass+
75.02 14.88
48.13 11.61
14.47 4.20
Pine'
Grass
162.27 28.67
65.40 7.71
22.40 6.04
Grass+
Grass+
7.42 1.08
3.75 0.64
7.42 1.08
Grass'
Grass'
6.73 0.82
2.38 0.31
6.73 0.82
oo


68
Table 4-3. Maximum uptake rate, Imax, (¡xmol P cm'2 s'1) and C^, (jxM P), the minimum
solution concentration from which a nutrient can be absorbed, for Pinus elliottii and
Panicum chamaelonche grown in a hydroponic solution containing 0.32 /M P. Each
value represents the mean of three replicates SE.
Plant species
^max
r
v^min
Pinus elliottii
0.116 0.027
0.080 0.018
Panicum chamaelonche
0.075 0.016
0.028 0.014


44
application on 2 April (Fig. 3-1A). Over the entire growing season, both the proportion
of roots with arbuscules and the activity for benomyl-treated plants did not change
significantly, whereas samples from the control plots had significantly negative slopes
with time for both arbuscule abundance and activity (Table 3-1). Early in the season
ground cover was sparse and the spray was applied directly to the soil. This was
paralleled by a short-term decrease in the proportion of roots with arbuscules (Fig. 3-1A)
as well as metabolic activity (Fig. 3-1B). As ground cover increased through the growing
season, more of the spray was intercepted by foliage leaving less to penetrate through to
the soil. Concomitant with this, the differences between treated and nontreated plots
disappeared. In late summer, as the plants started to senesce, roots of benomyl-treated
plants had more arbuscules and arbuscule activity than nontreated plants. In a concurrent
study, no effect of benomyl on shoot P status was observed at samplings taken in June
and August. There was no apparent relationship between precipitation, application of
benomyl and mycorrhizal response (Fig.3-1C).
Greenhouse Study
Benomyl effects on pine
There were no significant effects of benomyl on inoculated or noninoculated pine
biomass (Fig. 3-2A). Phosphorus content of the needles increased over time for all
treatments from a mean of 320 mg to 450 mg per plant, but this was not related to the
benomyl treatments (data not shown). Similarly, benomyl had no effect on the length or


CHAPTER 4
MYCORRHIZAS AFFECT PLANT COMPETITION FOR PHOSPHORUS
BETWEEN PINUS ELLIOTTII and PANICUM CHAMAELONCHE
Introduction
Soil fertility largely determines the amount of plant biomass an environment can
support (Donald, 1951). In environments with low nutrients, plants are stressed directly
by the lack of adequate nutrients, and they survive primarily by stress tolerance
mechanisms (Grime, 1979). An environment with more nutrients has the potential to
produce more plant biomass, which increases plant growth and also raises the chances
that root nutrient depletion zones of two plants will overlap. As a consequence, plant
competition for nutrients becomes one of the factors governing plant growth and survival.
Environmentally induced stress on a plant, therefore, can be considered a gradient
extending from direct physical stress on an individual plant to stress produced
biologically by plant interactions (Berkowitz et al., 1995; Grime, 1979).
Autecological studies have extensively documented that mycorrhizas can increase
plant tolerance to environmental stresses and contribute to a plants survival and growth
(Sylvia and Williams, 1992). The various mycorrhizal contributions that enhance
individual plant health similarly benefit a plant when competing with neighboring plants.
Much less research has quantitatively addressed the influence of mycorrhizas on the
synecology of plants. Previous studies have demonstrated that mycorrhizas can enhance
a plants competitive ability (Allen and Allen, 1984; Fitter, 1977; Hall, 1978; Hartnett
52


63
P Applied (pM P), Log Scale
Figure 4-2. Panicum chamaelonche (A) shoot-phosphorus concentration, (B) shoot-
phosphorus content and (C) total dry weight in response to different competition
treatments and grown at either 0.32, 3.23 or 32.26 iM P for 18 wk. Each symbol
represents the mean of six replicates + SE. Inoculated grass was not colonized at the end
of the experiment and therefore was not included in the analysis.


87
5000
4000
^ 3000
'o>
q_ 2000
D)
1000
+>
c
0)
+ o
§ 4000
^ 3500
Q.
g 3000
-S 2500
Q.
2000
1500
1000
500
0
Figure Al-2. Plant-P content for (A) slash pine and (B) grass grown in the growth
chamber for 62 d. Each bar represents the mean of a minimum of six replicates SE.


INFLUENCE OF MYCORRHIZAS ON PLANT COMPETITION FOR
PHOSPHORUS BETWEEN SLASH PINE AND GRASS
By
CHRISTIAN THOMAS PEDERSEN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1995

To Karen,
for her love, support, patience
and her sense of humor that carried
us both through this adventure

ACKNOWLEDGMENTS
I thank Dr. David Sylvia for giving my wife and me a warm welcome to Florida
and for his continued support throughout my studies. I also thank the members of my
supervisory committee, Drs. Nicholas Comerford, James Graham, David Mitchell and
Donn Shilling, for their inputs. I would like to especially thank Drs. David Mitchell,
David Hubbell and Suresh Rao for their major contributions to the philosophical aspects
of the Doctorate in Philosophy. Appreciation is extended to the National Science
Foundation for partially funding this research (Grant No. BSR-9019788). The opportunity
to use the laboratory facilities of Drs. Rao and Comerford is gratefully acknowledged.
Mary McLeod, Dongping Dai, Drs. Amiel Jarstfer and Linda Lee deserve thanks for
their invaluable technical guidance and patience throughout the process. Thanks also go
to the many laboratory assistants that have stood by my side and who probably do not
realize how much they have contributed. I appreciate Mike Allens technical support with
my Benlate field work. To the many graduate students who have passed through and the
few that still remain: The interactions we had were valuable to me. Specifically, I would
like to thank Dr. Pauline Grierson, Len Scinto and Dr. Philip Smethurst for their
supportive conversations that put things in perspective. Many thanks also to David
Farmer and Steve Trabue for the late night reality checks on the second floor of McCarty
Hall. Lastly, I am grateful for my familys love and support, which, even though they
were far removed from my research activities, was of great importance.
in

TABLE OF CONTENTS
ACKNOWLEDGMENTS iii
LIST OF TABLES v
LIST OF FIGURES viii
ABSTRACT x
CHAPTERS
1 INTRODUCTION 1
2 MYCORRHIZAS AND PLANT COMPETITION 3
Introduction 3
The Autecology of the Mycorrhizal Symbiosis 5
Nutrient Uptake: The Role of External Hyphae 5
Other Mycorrhizal Effects 14
Synecology 19
Plant Interactions 20
Environmental Conditions and Plant Competition 26
Plant Succession and Community Structure 30
3 LIMITATIONS IN THE USE OF BENOMYL IN
EVALUATING MYCORRHIZAL FUNCTIONING 34
Introduction 34
Materials and Methods 36
Results 41
Discussion 48
IV

4 MYCORRHIZAS AFFECT PLANT COMPETITION FOR
PHOSPHORUS BETWEEN PINE AND GRASS 52
Introduction 52
Materials and Methods 53
Results 60
Discussion 69
5 CONCLUSIONS 75
APPENDICES
1 GROWTH CHAMBER COMPETITION STUDY BETWEEN
PINE AND GRASS 80
Introduction 80
Materials and Methods 80
Results 83
Discussion 93
2 COLONIZATION OF PANICUM CHAMAELONCHE AND CORN BY
DIFFERENT ARBUSCULAR MYCORRHIZAL FUNGI 95
3 PHOSPHORUS GROWTH RESPONSE CURVE FOR
NONMYCORRHIZAL PANICUM CHAMAELONCHE 98
REFERENCE LIST 101
BIOGRAPHICAL SKETCH 122
v

LIST OF TABLES
Table Page
2-1 Summary of nutrient uptake kinetic studies 9
3-1 Test for homogeneity of slopes for the effect of Benlate 50 DF applied
in the field on percent Panicum chamaelonche roots with arbuscules and
their activity over time 43
4-1 Pirns elliottii (pine) and Panicum chamaelonche (grass) treatment
combinations planted in the competition study. Two plants were planted
per pot. The superscripts + and signify an inoculated or
noninoculated plant respectively. Pine was inoculated with Pisolithus
tinctorius and the grass was inoculated with Glomus sp. (INVAM FL329,
formerly FL906) 56
4-2 Ergosterol concentration (/xg g1) of Pinus elliottii roots inoculated with
Pisolithus tinctorius (pine+) or noninoculated (pine'), and grown in
combination with Pinus elliottii (pine) or Panicum chamaelonche (grass)
at 0.32, 3.23 or 32.26 /xM P for 18 wk. Each value represents the mean
of six replicates SE 64
4-3 Initial phosphorus uptake rate, I^ (/xmol P cm'2 s'1) and (/xM P), the
minimum solution concentration from which a nutrient can be absorbed,
for Pinus elliottii and Panicum chamaelonche grown in a hydroponic
solution containing 0.32 /xM P. Each value represents the mean of three
replicates SE 68
Al-1 Tests for single degree of freedom contrasts for root-length density, plant
biomass, plant P content and percent colonization. Each parameter was
analyzed separately for grass and pine 84
vi

A1-2 Mean hyphal length density (m cm'3 of soil) for each competition
treatment presented separately for each compartment (one hyphal and two
plant compartments). Hyphal length is made up of the sum of both AM
and EM fungal hyphae. Each value represents the mean of a minimum of
six replicates SE 85
A1-3 Mean soil-phosphorus content (fig P g'1 of soil) for each competition
treatment presented separately for each compartment (one hyphal and two
plant compartments). Each value represents the mean of a minimum of six
replicates SE 90
Vll

LIST OF FIGURES
Figure
page
2-1 Length of external hyphae spreading from mycorrhizal roots of Trifolium
subterraneum after (a) 28 days and (b) 47 days. Bars represent standard
error of the mean [with permission from (Jakobsen et al., 1992)] 7
3-1 Assessment of arbuscular activity in Panicum chamaelonche roots from
the field site in 1991. (A) Percentage of root length with arbuscules in
benomyl-treated and nontreated plots, (B) Percentage root length with
metabolically active arbuscules in benomyl-treated and nontreated plots
and (C) Precipitation. Each symbol represents the mean of three replicates
SE 42
3-2 Total dry weight of mycorrhizal (M) and nonmycorrhizal (C) plants, (A)
Pirns elliottii and (B) corn in response to 0, 20, 60 or 150 kg benomyl ha'
1 in the greenhouse. Each symbol represents the mean of seven replicates
SE 45
3-3 Soil hyphal length (total) and activity (active) of mycorrhizal (A) Pinus
elliottii and (B) corn plants in response to 0, 20, 60 or 150 kg benomyl
ha'1 in the greenhouse. Each symbol represents the mean of seven
replicates SE 46
3-4 Mycorrhizal colonization of (A) slash pine and (B) corn grown in the
greenhouse in response to 0, 20, 60 or 150 kg benomyl ha'1. Each symbol
represents the mean of seven replicates SE 47
4-1 Pinus elliottii (A) shoot-phosphorus concentration, (B) shoot-phosphorus
content and (C) total dry weight in response to different competition
treatments and grown at either 0.32, 3.23 or 32.26 /xM P for 18 wk. Each
symbol represents the mean of six replicates SE. Inoculated grass was
not colonized at the end of the experiment and therefore was not included
in the analysis
vm
62

4-2 Panicum chamaelonche (A) shoot-phosphorus concentration, (B) shoot-
phosphorus content and (C) total dry weight in response to different
competition treatments and grown at either 0.32, 3.23 or 32.26 /xM P for
18 wk. Each symbol represents the mean of six replicates SE.
Inoculated grass was not colonized at the end of the experiment and
therefore was not included in the analysis 63
4-3 Root-length density of (A) Pinus elliottii and (B) Panicum chamaelonche
in different competition treatments and grown at 0.32, 3.23 or 32.26 /M
P for 18 wk. Each symbol represents the mean of six replicates + SE.
Inoculated grass was not colonized at the end of the experiment and
therefore was not included in the analysis 66
4-4 Relative crowding coefficient (RCC) for (A) Pirns elliottii inoculated with
Pisolithus tinctorius grown in combination with Panicum chamaelonche
and (B) noninoculated Pinus elliottii grown in combination with P.
chamaelonche at either 0.32, 3.23 or 32.26 iM P for 18 wk. Each symbol
represents the mean of six replicates. Mean standard errors were smaller
than the symbols and are therefore not included 67
A1-1 Plant biomass for (A) slash pine and (B) grass grown in the growth
chamber for 62 d. Each bar represents the mean of a minimum of six
replicates SE 86
A1-2 Plant-P content for (A) slash pine and (B) grass grown in the growth
chamber for 62 d. Each bar represents the mean of a minimum of six
replicates SE 87
Al-3 Mycorrhizal colonization of (A) slash pine and (B) grass grown in the
growth chamber for 62 d. Each bar represents the mean of a minimum of
six replicates + SE 88
A1-4 Root-length density for (A) slash pine and (B) grass grown in the growth
chamber for 62 d. Each bar represents the mean of a minimum of six
replicates SE 91
Al-5 Uptake rate of (A) slash pine and (B) grass grown in the growth chamber
for 62 d. Uptake rate was calculated on a unit surface area basis which
included both root and hyphal surface area. Each bar represents the mean
of a minimum of six replicates SE 92
IX

A2-1 Colonization of (A) Panicum chamaelonche and (B) Zea mays inoculated
with root fragments of (1) Acaulospora scrobiculata (S315), (2) Gigaspora
margarita (INVAM FL215), (3) Glomus etunicatum (INVAM FL312) or
(4) Glomus sp. (INVAM FL329, formerly FL906) or spores of (5)
Gigaspora rosea (INVAM FL224) or (6) Scutellospora heterogama
(INVAM FL225). Each bar represents the mean of four replicates
SE 97
A3-1 Nonmycorrhizal Panicum chamaelonche (A) plant biomass and (B) plant
phosphorus content in response to 0.001, 0.003, 0.010, 0.032, 0.100,
0.316, 0.1000, 3.162 or 10.000 mg P L1. Each symbol represents the
mean of seven replicates SE 100
x

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
INFLUENCE OF MYCORRHIZAS ON PLANT COMPETITION FOR
PHOSPHORUS BETWEEN SLASH PINE AND GRASS
By
Christian Thomas Pedersen
December 1995
Chairperson: Dr. D. Sylvia
Major Department: Soil and Water Science
Individual plants benefit from the mycorrhizal condition primarily by improved
nutrient uptake, especially of phosphorus (P), resulting in enhanced plant survival and
growth in resource-limited conditions. On a broader scale, mycorrhizas have the potential
to mediate plant competition and subsequently may be important at the community level.
In the southeastern United States, slash pine (Pinus elliottii Engelm. var. elliottii)
is grown in plantations, where it competes for nutrients with grasses and other
herbaceous vegetation. One objective of my research was to assess mycorrhizal
contribution to intra- and interspecific plant competition for P in the greenhouse between
pine and Panicum chamaelonche Trin., a dominant grass species at a local plantation site.
The grass was inoculated or noninoculated with the arbuscular mycorrhizal (AM) fungus,
Glomus sp. (INVAM FL329, formerly INVAM FL906), and pine was inoculated or
xi

noninoculated with the ectomycorrhizal (EM) fungus, Pisolithus tinctorius (isolate S106).
The effect of P (0.32, 3.23 or 32.26 /xM) on competition also was analyzed in the
greenhouse because resource abundance can affect the outcome of competition and
inorganic P is limiting in pine plantations. Inoculated grasses were not colonized at the
end of the experiment and were excluded from data analyses. When competing with
grass, inoculated pine acquired more P and had a higher total dry weight than
noninoculated pine. Grass shoot-P content was reduced at the 32.26-/zM P level when
grown with pine, irrespective of the pine inoculation treatment.
I evaluated the effects of benomyl over time in the field and at 0, 20, 60 and 150
kg benomyl ha'1 equivalent in the greenhouse. My objective was to determine if benomyl
would be suitable for controlling AM but not EM fungi as part of a larger competition
experiment involving pine and weeds. No effect was observed on pine in the greenhouse.
Colonized root length of benomyl-treated Zea mays L. plants in the greenhouse remained
static and the response was not dose-dependent. In contrast, colonization in the control
plants increased over time. Minimal reduction of grass colonization was observed in the
field, where limitations to effective control were ground cover, timing in relation to
mycorrhizal development and benomyl application as a spray instead of as a soil drench.
Xll

CHAPTER 1
INTRODUCTION
Mycorrhizal contribution to plant nutrient uptake, especially phosphorus (P), has
been extensively studied. Under nutrient-limiting conditions mycorrhizas are able to
enhance plant nutrient uptake by various mechanisms, thereby ameliorating plant stress.
Under these conditions plants compete for limited nutrients and mycorrhizas may modify
the competitive interactions between plants. Little work has addressed the role of
mycorrhizas in this area of plant synecology. The existing studies primarily deal with
arbuscular mycorrhizal (AM) fungi and their contribution to plant interactions. Many of
these studies investigated facilitative mycorrhizal plant associations, where mycorrhizal
fungi transfer nutrients from one plant to another through common hyphal connections.
Only one study addressed the function of ectomycorrhizal (EM) fungi in plant
competition. No studies to date have evaluated the role of mycorrhizas in competitive
interactions between AM and EM plants, which occurs frequently during succession. In
the following chapter, I review the various aspects of mycorrhizal functioning in plant
autoecology and synecology and I detail their role in plant competition.
Most previous studies have been performed under controlled conditions in the
greenhouse without the influence of complex interactions of other environmental
variables. When bringing mycorrhizal questions to the field, one of the more difficult
problems is the creation of a suitable nonmycorrhizal control, since a majority of plants
1

2
are normally mycorrhizal. Fungicides can be useful in distinguishing mycorrhizal effects
from other influences on plants in the field. The fungicide benomyl is selective in that
it has inhibitory effects on AM but not on EM fungi. The selectivity of this fungicide
would be useful in isolating the nutrient uptake mediated by AM fungi in AM and EM
plant competition studies. As part of a larger competition study involving slash pine
(Pinus elliottii) and weeds at a field site northwest of Gainesville, I tested benomyl in the
field and in the greenhouse to determine if it would suitably control AM but not EM
fungi (Chapter 3).
I addressed the direct effect of mycorrhizas on plant competition for P in
greenhouse and growth chamber studies involving slash pine and Panicum chamaelonche,
a dominant weed species at the field site. The main objectives of the greenhouse
competition study, described in Chapter 4, were to determine if (i) mycorrhizas affect
plant competition at the interspecific or intraspecific level, (ii) competition is dependent
on soil nutrient concentration and (iii) competitive abilities are related to differences in
P uptake kinetics. The goals of the growth chamber study, presented in the first section
of the Appendix, were to assess (i) the contribution of mycorrhizal fungal hyphae to total
plant P uptake and (ii) the competitive abilities of the AM and EM fungi with respect to
each other.

CHAPTER 2
MYCORRHIZAS AND PLANT COMPETITION
Introduction
Over the past several decades the perception of mycorrhizas has evolved from
viewing them as a unique biological phenomena to understanding them as integral parts
of ecosystems. Much of the literature on mycorrhizas has addressed issues pertaining to
single plants. More recently, there has been a growing tendency to evaluate the
synecological consequences of the mycorrhizal association. The employment of
techniques such as minirhizotrons (Lussenhop and Fogel, 1993), image analysis (Smith
and Dickson, 1991), root-excluding screens and radioisotope labelling, among others, is
redirecting the field to a broader scale of ecology dealing with plant interactions and
community structure. The challenges faced during the next decade will be even more
complex, with the increasing need to study multi-organismal assemblages and their
functions at the ecosystem level. The next steps towards a more holistic view of
mycorrhizal function will be determined by technological advances that will allow us to
gain knowledge of how microbial systems fit together into a cohesive unit. This
knowledge will provide us with a better understanding of the environment and how to
best manage it in a sustainable manner.
Ecosystem studies necessitate an understanding of the functional associations of
organisms with each other and with their environment. For plants, one of the main
3

4
biological interactions is competition. The term competition will be used here as the
interaction between two organisms requiring the same limiting resource, which results
in the decreased growth, survival or reproductive capacity of one of the two organisms.
Plants mainly compete for light, water and nutrients. Physiological flexibility, within
genetic constraints, allows plants to adapt to changes in resource availability.
Physiological flexibility is enhanced by a plants symbiotic relationship with mycorrhizal
fungi. Modification in physiology can result in alterations of nutrient absorption capacity
(Marschner and Dell, 1994) and water relations (Safir et al., 1972), as well as enhance
light utilization and capture (Krishna et al., 1981). Increased tolerance or resistance to
other environmental stresses, such as plant diseases (Rosendahl and Rosendahl, 1990;
Schnbeck, 1978), high heavy metal concentrations (Denny and Wilkins, 1987) or
xenobiotics (Donnelly et al., 1993), also have been found in mycorrhizal plants.
Although the vast majority of studies with mycorrhizas has been conducted with
terrestrial, mycorrhizas also have been found in wetland plants and may function in
nutrient uptake in vascular aquatic plants (Rickerl et al., 1994; Wigand and Stevenson,
1994).
The objective of this chapter is to review mycorrhizal effects on plant competition
and community structure. However, to prepare the foundation for the synecology of the
system, a review of the autecology of mycorrhizal plants is also presented.

5
The Autecologv of the Mvcorrhizal Symbiosis
Nutrient Uptake: The Role of External Hvphae
During the past decade there has been a shift in mycorrhizal studies from
quantification of the internal phase to assessing the external hyphal phase in soil. The
external component of this symbiosis contributes to enhanced nutrient uptake of the plant
primarily by extending the roots nutrient depletion zone (Sanders and Tinker, 1973).
The depletion zone extends from 0.1-15 mm from the root surface, depending on the soil
type and plant species (Barber, 1995). By computer modelling, Itoh and Barber (1983)
determined that by doubling the length of root hairs, which have a diameter similar to
some mycorrhizal hyphae, plant phosphorus (P) uptake would double. Doubling the root-
P uptake rate, however, only increased P uptake by 15%. The mycorrhizal benefit is
inversely proportional to the root hair length (Schweiger et al., 1992), indicating that
root hairs partially offset mycorrhizal nutrient gains due to improved spatial exploitation.
Various techniques have been developed to measure external hyphae (Sylvia, 1992;
Dodd, 1994). As a primary tool, the use of fine-mesh screens to prevent roots from
penetrating into hyphal compartments has permitted the separation of root and hyphal
contribution to plant nutrient uptake (Ames et al., 1983; Schiiepp et al., 1987), as well
as quantification of hyphal distribution and density.
Spatial exploitation and hvphae
As reviewed previously (Boln, 1991; OKeefe and Sylvia, 1991), and based on
plant uptake theory (Barber, 1995; Nye and Tinker, 1977), the key parameters involved

6
in improved nutrient uptake by mycorrhizal plants include the amount of absorbing
surface area, fungal growth rates, nutrient uptake kinetics and hyphal distribution.
Hyphae can extend far beyond the nutrient depletion zone (primarily P) of roots. Using
an exclusion screen technique, Li et al. (1991a) located hyphae of Glomus mosseae up
to a maximum measured distance of 11.7 cm from roots of Trifolium repens L. after 49
d. Ectomycorrhizal rhizomorphs are likely to extend substantially further.
In a separate comparative study on arbuscular-mycorrhizal (AM) fungi and P
uptake, Acaulospora laevis, Glomus sp. and Scutellospora calospora developed hyphae
up to 11 cm from the roots of the host plant, Trifolium subterraneum L., after 47 d (Fig.
1). However, hyphal densities with increasing distance from the mycorrhizal roots were
not the same for all fungi. Acaulospora laevis had a constant hyphal density up to 11 cm,
while for Glomus sp. it decreased after 3 cm, and for S. calospora the highest hyphal
density was observed closest to the root and declined exponentially thereafter. The hyphal
P uptake rates for the three fungi (calculated average for 28-47 d) were 2.8, 0.8 and 0.6
fmol P m1 s'1, respectively, with considerably higher rates for the initial 28-day period.
The consequence of these differences was a substantial contrast in plant P content among
the mycorrhizal treatments. The previously listed characteristics of absorbing surface
area, fungal growth rates, nutrient uptake kinetics and hyphal distribution indicative of
improved nutrient uptake were all favorable in the A. laevis treatment, which was also
associated with the highest plant P content.
Depending on the mycorrhiza and the initial soil nutrient concentration, the
contribution by hyphae to total plant nutrient uptake (Marschner and Dell, 1994;

Hyphal Length (m g"1 of dry soil)
7
Figure 2-1. Length of external hyphae spreading from mycorrhizal roots of Trifolium
subterraneum after (a) 28 days and (b) 47 days. Bars represent standard error of the
mean [with permission from (Jakobsen et al., 1992)].

8
comprehensive review) has ranged between 7 to 109% for P (George et al., 1992; Li et
ah, 1991a; Li et ah, 1991c; Pearson and Jakobsen, 1993), 16-25% for zinc (Kothari et
ah, 1991) and 53-62% for copper (Li et ah, 1991c). In another study, in contrast to the
control, mycorrhizal plants recovered 1.7 times more 15NH4+ applied 2 cm from the root
compartment and 2.75 times more 15NH4+ when applied at a distance of 5 cm (Johansen
et ah, 1993). This provides evidence for an increasing benefit with greater distance. If
diffusion and mass flow of a nutrient are slower than hyphal transport, a mycorrhizal
benefit could conceivably be derived even for nutrients that are not strongly adsorbed to
soils. This was demonstrated when transfer of 15N03' was increased in mycorrhizal
treatments by over 400% under dry soil conditions (Tobar et ah, 1994). These studies
illustrate the capacity of the external phase of mycorrhizas to increase a plants nutrient
absorption. They also demonstrate that the response differs depending on the soil
environment, the fungi involved and the spatial location of nutrients.
Uptake kinetics
Uptake kinetics can be quite different between mycorrhizal and nonmycorrhizal
plants. Based on uptake models and mycorrhizal characteristics, however, differences in
uptake kinetics appear to be of secondary importance compared to surface area and
spatial distribution (OKeefe and Sylvia, 1991). Few uptake studies comparing
mycorrhizal and nonmycorrhizal plants have been performed since the review of OKeefe
and Sylvia (1991). Most studies have used root weight to standardize uptake parameters
(Table 2-1). However, none have taken into account the surface area or weight

Table 2-1. Summary of nutrient uptake kinetic studies.
Fungus
Host
Nutrient
concentration
OiM)
Km
(pM)
^max
c
^min
(pM)
Reference
Glomus fasciculatum
Lycopersicon esculentum
(root segments)
1-100 kh2po4
1.61-0.35
0.1-0.32*
n.dc
(Cress et al., 1986)
none
w
w
3.9-42
0.10-0.25 Ia
n.d
Glomus mosseae
Glycine max
(whole plant)
30 KH2P04
20
58b
n.d
(Karunaratne et al.,
1986)
none
w
3.5
19b
n.d.
Pisolithus tinctorius
Pinus caribea
(whole plant)
20 Na2HP04
3.89
0.30a
0.32
(Pacheco and
Cambraia, 1992)
none
w
ft
16.44
0.23a
11.98
Glomus
macrocarpon
Zea mays
(root segments)
1.5-1070 Zn
5.3-0.38
0.08-0.47"
n.d.
(Sharma et al., 1992)
none
M

4.5-0.95
0.03-0.55"
n.d.
# imol g fresh weight'1 h'1
b nmol m'2 s'1
c n.d. = not determined

10
contributed by mycorrhizal hyphae, which could alter the calculated kinetic parameters.
Also, in uptake experiments, the use of whole plants rather than root segments would be
more appropriate, since this would incorporate possible source-sink effects of the
plant. The information available suggests that there may be differences in the Km, I,^
and Cmin values of mycorrhizal and nonmycorrhizal plants. The term Km represents the
substrate concentration at which the uptake rate is half of the maximum influx rate, 1^.
The value C,,^ is the minimum solution concentration from which a nutrient can be
absorbed. Studies that have estimated P uptake parameters have generally found a higher
Imax for mycorrhizal plants. Uptake kinetics are dependent on the solution nutrient
concentration (Cress et al., 1979; Sharma et al., 1992), and, consequently, selection of
relevant soil nutrient concentrations in experiments is critical. For example, both P and
Zn appear to have more than one concentration dependent uptake system.
Uptake kinetics may have a major role in mycorrhizal plant survival when
nutrients are limited. In nature, reduced availability of nutrients occurs due to fixation
and biological immobilization. With two competing plant species, the one with the lower
Cmin value would be at a competitive advantage, because it can reduce the nutrient
concentration below the C,^ value of the other organism (Tilman, 1982). In fact,
Pacheco (1992) demonstrated a lower for ectomycorrhizal compared to
nonmycorrhizal pine, which suggests a potential advantage for mycorrhizal plants.
Unavailable nutrients may be released in pulses when microbial activity is temporarily
stimulated due to environmental conditions. Under these circumstances, plants with
differing uptake strategies, such as emphasis on C,^ in one plant versus emphasis on 1^

11
in another, could survive in the same environment due to niche separation. Mycorrhizas
may add flexibility to a plants physiological strategy by allowing it to profit from a
broader range of nutrient uptake mechanisms. Furthermore, mycorrhizas may add an
element of efficiency to soil nutrient exploitation by roots. Campbell et al. (1991)
proposed that plant species primarily acquire resources either by efficiently exploiting a
given resource (precision foraging) or by extensive development of roots and occupation
of a high resource site (scale foraging). Although untested, mycorrhizas may give an
advantage to the plant group with a tendency to efficiently exploit a given soil volume
by accessing nutrients outside of the roots nutrient depletion zone.
Exudates and secretions
Root and hyphal secretions and exudates modify nutrient availability in the soil
(Darrah, 1993; Duff et al., 1994). Several different mechanisms are involved and include
release of enzymes, chelating agents such as organic anions and siderophores, and
changes in rhizosphere pH by C02 from respiration and H+ excretion during uptake of
cations.
Nitrogen and phosphorus are often present in organic forms (Stevenson, 1986),
which are less available than the organic forms. Ectomycorrhizal and ericoid fungi permit
plant utilization of organic N from proteins and peptides (Abuzinadah and Read, 1986;
Abuzinadah and Read, 1989; Bajwa et al., 1985; Bajwa and Read, 1985), which are
otherwise unavailable N sources to plants (Abuzinadah and Read, 1986). Arbuscular-
mycorrhizal fungi do not appear capable of utilizing complex organic-N sources (Frey

12
and Schepp, 1993) and, thus, probably lack any significant protease release.
Phosphatases are produced by plant roots (Duff et al., 1994; Tarafdar and Claassen,
1988; Cumming, 1993) and release P from bound organic forms close to the root.
Phosphatase release by mycorrhizal fungal hyphae also has been demonstrated (Antibus
et al., 1992; Jayachandran et al., 1992; Tarafdar and Marschner, 1994) and may increase
plant uptake of P; however, mycorrhizal contribution to total P uptake by this particular
mechanism has not been quantified yet. Extension of hyphae beyond the root depletion
zone would permit solubilization and uptake of P from these unavailable organic-P forms.
For some nonmycorrhizal plants, mobilization of the organic-P fraction can approach one
third of the total P absorbed (Jungk et al., 1993). Proton release by roots, in part to
compensate for NH4+ uptake, can create a substantially lower rhizosphere pH (Marschner
and Romheld, 1983) and acidification of the soil also has been documented in
mycorrhizal systems (Li et al., 1991b). Although this enhances dissolution of iron (Fe),
and consequently bound P, the rate of protonation is slower than by the chelating
mechanism (Schwertmann, 1991).
The availability of inorganic P bound to aluminum (Al) and Fe minerals can be
increased by organic anions, such as oxalate (Fox et al., 1990), by the processes of
chelation, ligand exchange and dissolution of the metallophosphate complex.
Ectomycorrhiza, which form fungal mats, are capable of substantially altering the
chemical soil environment by increasing the oxalate anion concentration by several orders
of magnitude (Griffiths et al., 1994). Soil-solution phosphate in these mats is strongly
correlated with oxalate concentration. Fungi most likely vary in the quantity of organic

13
acids released, resulting in differences in mineral weathering rates, nutrient release and
subsequent benefit to plants. Chelation of A1 not only releases bound P, but also lowers
the free ion activity and thus reduces A1 toxicity to the plant (Arp and Strucel, 1989).
This same mechanism may apply to other metals. Chelating agents specific to Fe are
termed siderophores and are produced by plant roots (Marschner, 1986), as well as by
several mycorrhizal fungi (Cress et al., 1986; Schuler and Haselwandter, 1988; Watteau
and Berthelin, 1995). Watteau and Berthelin (1995) found mycorrhizal siderophores of
the hydroxylate type to be more effective chelators than organic anions and less specific
for Fe than for Al.
The question of accessibility of organic compounds as carbon (C) sources to
mycorrhizal fungi has been debated (Harley and Smith, 1983). Recently, the
ectomycorrhizal fungi Cenococcum geophilum, Laceara bicolor, Rhizopogon vinicolor
and Suillus lakei were shown to utilize C from hemicellulose, cellulose and less readily
from a humic polymer mix or from Pseudotsuga menziesii needles (Durall et al., 1994).
Tanesaka et al. (1993) reported that several ectomycorrhizal fungi apparently did not
have the ability to degrade complex C substances such as wood. Haselwandter et al.
(1990) found several ericoid and ectomycorrhizal fungi capable of lignin degradation. At
present, the ability to degrade organic matter has not been documented for AM fungi.
Nonetheless, they appear to be efficient at capturing P released by decomposers prior to
its being immobilized again (Joner and Jakobsen, 1994), possibly due to an advantageous
spatial distribution. In general, the question of mycorrhizal fungal hyphae accessing

14
nutrients in forms not available to nonmycorrhizal plants may prove to be less important
than the spatial accessibility of nutrients beyond the roots nutrient depletion zone.
Fungi and plants release polysaccharides resulting in a direct mucilaginous
connection to soil particles in their respective rhizospheres and hyphospheres. This
matrix may enhance aggregate formation, reduce nutrient loss from leaching and reduce
dehydration by increasing waterholding capacity (Chenu, 1993). Furthermore, the
connections to soil particles permit direct transfer of nutrients from soil to root (Uren,
1993), which may be essential under low moisture conditions, very similar to the contact
exchange described by Nye and Tinker (1977).
Other Mvcorrhizal Effects
Water relations
Improved water relations in mycorrhizal plants have been documented extensively
(Nelsen, 1987) and have been associated with improved plant-P status (Nelsen and Safir,
1982), though non-P responses are also reported (Aug et al., 1986; Bethlenfalvay et al.,
1988; Davies, Jr. et al., 1992). Drought tolerance in mycorrhizal plants is enhanced by
increasing plant turgor, leaf water potential, stomatal conductance and root hydraulic
conductivity. In addition, Bethlenfalvay et al. (1988) have suggested that mycorrhizal
fungi are able to acquire soil water at lower water potentials than roots, although Nelsen
(1987) proposed that fungal hyphae gave the plant a spatial advantage by extending the
water depletion zone beyond the root. Recently, Ruiz-Lozano et al. (1995) found
differences in proline concentration in drought-stressed mycorrhizal plants and suggested

15
that changes in osmotic potential may contribute to their improved drought tolerance over
nonmycorrhizal plants. Most studies with AM fungi do not show any major water
transfer via hyphae (George et al., 1992; Nelsen and Safir, 1982), although this is not
always the case (Faber et al., 1991). In contrast, ectomycorrhizal fungi appear able to
directly transfer water to the plant (Boyd et al., 1985), especially through rhizomorphs
(Duddridge etal., 1980).
Carbon costs
The benefits of enhanced nutrient uptake associated with mycorrhizal biomass
production has energy costs associated with it that vary with the symbiosis. The plant-
microbe-soil interactions are unique to each environment and correspondingly the
mycorrhizal response may vary (Sylvia et al., 1993). This may depend on the fungus,
such as differing growth responses observed with 20 isolates of Pisolithus spp. on
Eucalyptus granis (Burgess et al., 1994). Responses also vary with plant species and
cultivars of the same plant species (Krishna et al., 1985; Mrtensson and Rydberg, 1995;
Smith et al., 1992). So, although host specificity perse has not been documented clearly,
host specific responses do exist. These differences may be related to root morphology,
as suggested by Baylis (1975). Negative relationships have been found between
mycorrhizal dependency and root fibrousness (Hetrick et al., 1992; Pope et al., 1983)
or root hair length (Crush, 1974). However, as suggested by Graham et al. (1991), other
undetermined factors aside from root architecture are more likely involved. Carbon cost,
measured as energy expended per unit nutrient absorbed (Tinker et al., 1994), varies

16
between different mycorrhizal associations. The observed variation in mycorrhizal growth
response among closely related plants may relate to differing strategies of plant-C
allocation to the symbiosis (Graham and Eissenstat, 1994), as well as to plant age
(Eissenstat et al., 1993). Pearson and Jakobsen (1993) quantified the P-uptake efficiency
(C utilized/P absorbed) for three different AM fungi. For each unit of P absorbed they
found that Scutellospora calospora and a Glomus sp. utilized 25 and 16 times more C,
respectively, than Glomus caledonium. Total-C partitioning belowground was higher in
the less efficient mycorrhizal fungi, indicating that energy efficiency of the symbiosis
may be one reason for differing plant growth responses to fungi.
When comparing carbon costs of mycorrhizal to nonmycorrhizal plants, 4-36%
more of the total C fixed is allocated belowground due to mycorrhizas (Durall et al.,
1994). To distinguish nutritional from other mycorrhizal effects on plant-C balance,
mycorrhizal plants were grown at high soil-P concentrations and demonstrated a 37%
higher belowground carbon allocation than nonmycorrhizal plants (Peng et al., 1993). Of
this 37%, 51 % was attributed to greater root biomass and 10% to construction costs of
lipid-rich roots most likely associated with the mycorrhizal fungus. Enhanced
photosynthesis in mycorrhizal plants can compensate to varying degrees for this increased
C drain (Dosskey et al., 1990; Kucey and Paul, 1982). Plant root turnover is also
associated with a high C cost, although few studies have assessed the role of mycorrhizas
in controlling this process. Durall et al. (1994) determined that ectomycorrhizal roots
have a lower root turnover rate than nonmycorrhizal roots. In environmental conditions
where nutrient pulses occur, roots with a lower root turnover rate demonstrated a

17
competitive advantage (Campbell and Grime, 1989). This suggests that mycorrhizal
plants may profit from the reduced root turnover rate by having to invest less C into
nutrient absorbing structures.
Plant fitness
Although many of the previous topics dealt with improving plant growth and
stress adaptation, few mycorrhizal studies have directly addressed mycorrhizal influence
on plant fitness, that is the plants ability to increase its numbers proportionately to other
species (Begon et al., 1986). Enhanced efficiency of resource acquisition by mycorrhizal
plants allows more energy to be allocated to growth and reproduction, which potentially
increases plant fitness. The result in the next generation may be expressed in terms of
improved survival, growth rate or reproduction. Mycorrhizal plants have displayed
increased seed number, seed weight and P and N content (Lu and Koide, 1994), with
some benefits still significantly expressed in the second generation of offspring grown
in the absence of mycorrhizal fungi (Koide, T. and Lu, 1992). Increased P status of seed
has been associated with subsequently higher P content and plant biomass (Bolland and
Paynter, 1992). For some plant species, the presence of mycorrhizas enhanced seedling
emergence rate (Hartnett et al., 1994). High P concentration in seed has resulted in
increased number of emerging seedlings and a higher rate of emergence (Thomson and
Bolger, 1993), factors which also have been identified as important predictors of
competitive success in secondary succession (Stockey and Hunt, 1994).

18
Xenobiotics
In many ecosystems plants and mycorrhizal fungi are exposed to a wide variety
of toxic compounds (xenobiotics and in some instances naturally occurring toxic
compounds). Mycorrhizal fungi may effectively mediate and alter the interaction between
plant and xenobiotic compounds. Various papers have assessed or reviewed pesticide
effects on mycorrhizal fungi (Dehn et al., 1990; Trappe et al., 1984). Mycorrhizal fungi
may function in the translocation of herbicides. In one study with apple and three
herbicides (dichlobenil, paraquat and simazine), root dry weight of noninoculated plants
exposed to herbicides was reduced by 46% in contrast to a 63% decrease in mycorrhizal
plants (Hamel et al., 1994). Although no effect on hyphal length was found at the highest
simazine concentration applied, 75% of the mycorrhizal plants died compared to none
in the control treatment. The authors attributed this to facilitated herbicide flow to the
host plant mediated by the mycorrhizal fungus. Uptake and translocation of the herbicide
atrazine was also found in mycorrhizal corn, which is atrazine-tolerant (Nelson and
Khan, 1992). Although the quantity absorbed was small compared to direct root uptake,
the question of how this may affect an atrazine-sensitive plant remains unanswered.
Certain mycorrhizal fungi also have demonstrated the capacity to degrade herbicides such
as atrazine and to a lesser extent 2,4-dichlorophenoxyacetic acid (Donnelly et al., 1993).
This provokes the question as to whether mycorrhizal fungi offer some protection against
xenobiotics. In corn and sorghum certain herbicide safening effects by AM fungi have
been found against the herbicides imazaquin, imazethapyr and pendimethalin (Siqueira
et al., 1991).

19
Metal cations and soil acidity
High metal cation concentrations can be toxic to plants. The high solubility of Al,
due to the acidic nature of Oxisols and Ultisols, is a growth-limiting factor for plants in
many tropical countries. Natural selection of mycorrhizal ecotypes leads to varying
genotypic sensitivity to soil acidity (Robson and Abbott, 1989), as well as to high metal
concentrations (Gildon and Tinker, 1983; Griffioen et al., 1994). Several studies have
found mycorrhizas capable of alleviating toxic effects to plants caused by Al, cadmium,
Cu and Zn (Bradley et al., 1982; Colpaert and Van Assche, 1993; Denny and Wilkins,
1987; Dueck et al., 1986; Koslowsky and Boerner, 1989). Two mechanisms currently
explain this response. Firstly, electronegative sites on the hyphal cell walls bind the
positively charged heavy metal cations (Denny and Wilkins, 1987; Galli et al., 1994).
The observation that under acidic soil conditions heavy metal uptake is increased
(Killham and Firestone, 1983) partly confirms this. It is possible that protonation of
negatively charged sites in the plant or fungal walls results in less binding and greater
uptake of the metal cation. The second path is the immobilization of the cations by
complexation in vacuoles with polyphosphates (Martin et al., 1994) or associated
metallothionein-like peptides (Turnau et al., 1994).
Synecologv
Co-evolution of mycorrhizal fungi and plants has been suggested (Allen, 1991;
Harley and Smith, 1983). Since selection for more fit species occurs continuously, and
a larger proportion of plants show mycorrhizal dependency than not, it follows that there

20
must be some measure of improved fitness derived from mycorrhiza; otherwise the
symbiosis would have been selected against. The alternative is that mycorrhizal fungi are
parasites with maximum adaptability to plant resistance strategies. This, however, is
unlikely considering the exchange of nutrients between the two organisms, which is
characteristic of a true mutualism.
The effects of the mutualism on plant growth and survival influence interactions
beyond the single plant level (Brundrett, 1991; Francis and Read, 1994). Plants rarely
grow alone, except in extreme or anthropogenic environments, and consequently end up
competing for similar resources, especially inorganic nutrients, water and light. Under
conditions limiting growth, mycorrhizal plants have distinct competitive advantages.
Thus, from a holistic and functional perspective, mycorrhizal research reaches its full
value when applied to natural or managed ecosystems where interactions occur. Current
issues pertain to the involvement of mycorrhizas in plant community development,
stabilization and diversity, as well as to questions of environmental sustainability and the
economics of agricultural production systems. A relevant question, then, is to what extent
is the force of this symbiosis manifested in plant communities?
Plant Interactions
During competition, plants utilize several different strategies for optimal resource
capture with many of them overlapping those found in the mycorrhizal symbiosis. The
choice of strategy depends primarily on a sites resources and the amount of disturbance
(Grime, 1979; Tilman, 1982). Literature summarized in the first part of this chapter has

21
shown that mycorrhizas can enhance resource capture. Environmental factors strongly
influence the mycorrhizal benefit derived by a plant and consequently also its competitive
ability.
Resource competition
Competition occurs when a resource is inadequate to meet the needs of the
competitors. Nutrient availability fluctuates with the chemical environment and moisture
content of the soil. Soil heterogeneity frequently compounds the intensity of competition
in some areas, since resources are not evenly distributed. Phosphorus has been the focus
of mycorrhizal research, because it is required by plants in proportionately large
quantities, and yet, in the soil it is easily immobilized chemically and biologically.
Consequently, the use of P also dominates mycorrhizal studies involving competition.
When mycorrhizal plants compete under nutrient-limiting conditions, niche
differentiation may be of considerable importance. Plants competing intraspecifically will
have similar nutrient requirements and acquisition strategies which may vary depending
on plant age. Conversely, in interspecific interactions, some competition may be
alleviated by niche differentiation. For example, a potential growth response associated
with spatial niche separation by roots of two grass species only became evident by
experimentally increasing soil depth (Van Auken et al., 1994). The varying plant
responses to different mycorrhizal species in the literature suggest the involvement of a
combination of the earlier reviewed mechanisms, including hyphal spatial distribution and
access to less available nutrients. However, if the mycorrhizal contribution to nutrient

22
uptake is primarily related to spatial niche differences between roots and hyphae, then
larger soil volumes would be preferable in experiments; otherwise root nutrient depletion
zones quickly overlap and the potential mycorrhizal benefit is not realized (OKeefe and
Sylvia, 1991). As an intermediate approach between pot and field competition studies,
artificial micro- or mesocosms have been used (Campbell et al., 1991; Grime et al.,
1987), which, among other things, allow for the exploration of large soil volumes by
external hyphae, the creation of resource gradients or patches and the longer-term
monitoring of plant growth and reproduction in a regulated environment. Further
consideration should be given to the incorporation of an unsterilized soil control into
experiments. The inclusion of plant pathogens, soil arthropods and microbes which affect
resource abundance and mycorrhizal plant growth (Newsham et al., 1994), as well as
subsequent plant interactions, would provide a more realistic extrapolation of
experimental results to natural phenomena.
A number of mycorrhizal plant competition studies have demonstrated that AM
fungi affect competition to varying degrees (Brown et al., 1992; Francis and Read, 1994;
Fitter, 1977; Hetrick et al., 1989; Hartnett et al., 1993; Newman et al., 1992). Plant
competition between two host plants involving a single species of AM fungus account for
the majority of the data. Apparently only one plant competition study dealt with different
groups or species of mycorrhizal fungi and it is also the only EM plant competition study
(Perry et al., 1989). There is evidence that competitive success is related to mycorrhizal
dependency (Hartnett etal., 1993; Hetrick et al., 1989). Mycorrhizal dependency is very
variable and depends on the particular environment and host plant. Hartnett et al. (1993)

23
and Bth and Hayman (1984) determined that, in a given soil volume, mycorrhizal
benefit for a plant decreases with increasing density of its competitors. Higher plant
density is paralleled by an increase in root and hyphal density in the soil and
proportionately greater overlap of nutrient depletion zones. In intraspecific competition
of inoculated plants of high mycorrhizal dependency, density-related competition was
observed, but this did not occur when mycorrhizal fungi were absent. Inoculated plants
with low mycorrhizal dependency lacked this response, indicating their ability to more
efficiently extract nutrients from the soil than the nonmycorrhizal plants with high
mycorrhizal dependency.
Plants of the same species but different plant age also have been compared for
competitive interactions. Eissenstat and Newman (1990) evaluated the possible advantages
of mycorrhizas to seedling establishment in the presence of an older plant of the same
species. The results indicated that there is not a facilitative but rather a competitive
relationship between the two plants, similar to that observed in the absence of
mycorrhizal fungi. In another study, Franson et al. (1994) found that competition
intensity between an established and a seedling soybean plant was not altered by
increasing the stress on the younger plant.
Plant competitive interactions between mycorrhizal host and nonhost plants have
been investigated in a limited number of studies. It is worth noting that some have
documented a reduction in biomass of nonhost plants when such plants were grown under
mycorrhizal conditions (Allen et al., 1989; Ocampo, 1986). Francis and Read (1994)
found evidence for a chemical factor, which was extracted from soil of mycorrhizal

24
plants, that inhibited root growth of nonhost plants. This suggests that mycorrhizas may
have effects beyond those currently known.
In summary, mycorrhizas can enhance a plants competitive ability, and the effect
is generally associated with increased nutrient uptake. The greatest benefit of mycorrhizas
appears to lie in their ability to buffer the plant from adverse environmental conditions
that reduce resource availability.
Mvcorrhiza-mediated reduction of competition
With most plants possessing similar nutritional requirements, competition is a key
factor in their interactions. The existence of hyphal connections between plants is well
known. Various studies, especially those using root-excluding screens, have
unequivocally demonstrated that nutrient transfer between root zones of a donor and
receiver plant can be mediated by mycorrhizal hyphae (Newman, 1988; Newman et al.,
1992). Although it is possible for hyphae from the receiver mycorrhiza to scavenge
nutrients from the rhizosphere of the donor plant, most likely the majority of transfer is
by direct hyphal connections between plants. For example, radio-labelled C from an
ectomycorrhizal donor plant has been found solely in ectomycorrhizal plants and not in
neighbor AM neighbor plants; by using autoradiography, no visual evidence existed of
a direct interspecific C transfer between intermingling roots of donor and receiver plants
(Read et al., 1985). In another study, 46% of the total C transferred directly from plant
to plant was via mycorrhizal connections, 15% of uptake was indirectly mediated by
mycorrhiza, and 39% was translocated by other processes (Martins, 1993). These

25
fractions could be verified further by comparing nutrient transfer from a mycorrhizal
donor plant to either a myc' mutant (a mutant plant not able to form mycorrhiza) or a
normal mycorrhizal receiver plant. The quantity obtained by the receiver is variable, and
appears to depend on the nutrient involved. Generally, P is not transferred at fast rates
(Newman and Eason, 1993) or in quantities that significantly affect growth (Ikram et al.,
1994). The transfer of N by mycorrhizas has been documented (Newman, 1988), with
most studies utilizing a legume, because of its importance in intercropping systems, as
the donor plant. The quantity of N transferred from the root zone of donor plant to the
receiver plant varies (Bethlenfalvay et al., 1991; Frey and Schiiepp, 1993). By increasing
competitive pressures for N in intercropping systems, mycorrhizal fungi at certain times
may enhance nitrogen fixation (Barea et al., 1989), although this is not always the case
(Reeves, 1992). Both of these studies and others (Hamel and Smith, 1991; Ikram et al.,
1994) have found minimal amounts to no N transferred. The quantitative significance of
mycorrhizal transfer of nutrients to total uptake by the receiver plant still remains
unclear.
The phenomenon of increased survival of certain plant species in mycorrhizal
microcosm studies (Grime et al., 1987) deserves further attention, especially, since no
direct cause was found. Source-sink gradients, such as those created by shading or low-
nutrient status of one plant, have been suggested as the force behind nutrient transfer.
For interplant C transfer, shading of the receiver plant increased C translocation to that
plant (Read et al., 1985). However, shading does not always produce this effect (Franson
et al., 1994; Hirrel and Gerdemann, 1979). In contrast, clipping of leaves to simulate

26
herbivory and to produce a C sink resulted in C transfer away from the clipped plant
(Waters and Borowicz, 1994). In settings where young seedlings compete for nutrients
with established plants, the seedlings become more quickly colonized by the preexisting
mycorrhizal network; however, no further benefit to the seedlings was detected (Franson
et al., 1994; Eissenstat and Newman, 1990). In Grimes (1987) study, 14C-labelling of
one dominant plant resulted in substantially more C being transferred to subdominants
when plants were mycorrhizal compared to nonmycorrhizal. Although competition does
occur in these systems, several plants colonized by the same mycorrhizal type will be
closely tied together by the hyphal network and may benefit from C transfer among
plants.
Environmental Conditions and Plant Competition
Non-resource edaphic factors
Several soil characteristics may indirectly influence the assorted mycorrhizal
mechanisms that enhance a plants competitive ability. Soil acidity is an important factor
influencing soil nutrient availability. Acidic soils are a natural result of soil weathering,
and, as stated earlier, Al toxicity is one of the main associated problems. Mycorrhizas
may enable a plant to survive unfavorable conditions caused by toxic concentrations of
metal cations, including Al (Koslowsky and Boerner, 1989). Although mycorrhizas may
facilitate growth of plants under acidic soil conditions, I am not aware of any studies that
systematically address the effect this may have on plant competition.

27
Soil chemical processes associated with organic matter turnover and mycorrhizas
may also play a yet unstudied role in plant interactions. As organic matter is degraded
by microbes, various compounds, including phenolic materials, are released to the soil.
Phenolics have been implicated in various allelopathic interactions (Rice, 1984).
Researchers have demonstrated both inhibition and stimulation of mycorrhizal fungi by
phenolic compounds (Baar et al., 1994; Boufalis and Pellissier, 1994; Siqueira et al.,
1991). Different microbial responses to phenolics have been attributed to variability in
degradation capacity of the microbes, phenolic concentration, soil characteristics and
availability of inorganic soil nutrients (Blum and Shafer, 1988). Similarly, mycorrhizal
fungi vary in their capacity to chemically alter different forms of phenolic compounds
(Giltrap, 1982; Ramstedtand Soderhall, 1983; Tam and Griffiths, 1993). Garbaye (1994)
hypothesized that phenolic compounds may be degraded by bacteria closely associated
with mycorrhizal fungi, thereby also enhancing the establishment of mycorrhizal fungi.
Although no clear link has been found between mycorrhizal sensitivity to phenolic
compounds and plant competitive ability, the results of a few studies suggest a possible
connection (Leake et al., 1989; Wacker and Safir, 1990). Because mycorrhizal fungi
occur in competitive environments, such as forests, with the potential of allelopathy
(Horsley, 1987; Pellissier, 1994), it is important to determine what growth-limiting
factors, as well as their magnitudes, actually occur. Although it is difficult to distinguish
resource competition from interference competition, several researchers have been
successful in differentiating these two phenomena (Nilsson, 1994; Shilling et al., 1992;
Thus, 1994; Wardleetal., 1994).

28
Associated soil biota
Fitter and Garbaye (1994) have summarized the current information about
belowground interactions of mycorrhizas and rhizosphere microbes. Unfortunately, few
studies have addressed how these interactions affect plant populations or communities.
Mycorrhizas influence the rhizosphere environment by modifying plant exudation and
rhizodeposition (Leyval and Berthelin, 1993), and subsequently affect microbial
composition and metabolic activity in varying degrees. Inversely, certain fluorescent
pseudomonads and spore-forming bacilli, similar to growth-promoting rhizobacteria, may
significantly regulate the mycorrhizal benefit to the plant (Garbaye, 1994; Schreiner and
Koide, 1993); however, mechanisms of action are still largely unknown. These bacteria
have demonstrated some fungal, but not plant, host specificity. With appropriately
matched mycorrhizal fungi and bacteria it is conceivable that a plant would possess a
competitive advantage over other plants without selected associations. Rabatin and Stinner
(1991) reviewed the effects of microfauna, many of which are fungivores, on
mycorrhiza. As an example, Boerner and Harris (1988) conducted a competition study
between mycorrhizal Panicum virgatum and the nonhost Brassica napa, where the
addition of Collembola reduced the competitive ability of the grass, resulting in a
reduction of biomass compared to the mycorrhizal P. virgatum without competition.
Studies of plant disease control by mycorrhizas interacting with plant pathogens
have yielded variable results (Linderman, 1994; Duchesne, 1994). Various mechanisms
have been reported that are unique to the environment, host and microbes involved.
Based on field studies utilizing the fungicide benomyl, Carey et al. (1992) suggested that,

29
aside from direct physiological benefits to the plant, mycorrhizal contributions to plant
health in the field may be a common but subtle phenomenon, because it is buried within
complex interactions.
To make the situation more complex, few studies have included interactions
between mycorrhizas and other plant endophytes (Clay, 1992). The fungal endophyte
Acremonium sp., for example, has reduced colonization and reproduction by Glomus sp.
(Chu-Chou et al., 1992; Guo et al., 1992). Reduction of insect herb ivory has been
attributed to secondary metabolite production by fungal endophytes (Clay, 1991). Another
study found that mycorrhizas may reduce feeding inhibition of an insect herbivore
induced by Acremonium sp. (Barker, 1987). Additionally, nonmycorrhizal endophytes are
capable of altering competitive relationships between plants (Clay et al., 1993) and plant
drought resistance (White, 1992) in ways similar to mycorrhiza. The data suggest that
endophytes are involved in various effects observed in plant studies and consequently
they deserve further consideration.
Herb ivory
Herbivores generally have either an inhibitory or neutral effect on mycorrhizas
(Barbosa et al., 1991; Gehring and Whitham, 1994). Herbivory results in increased plant-
C allocation to the replacement of aboveground parts instead of to maintenance of the
mycorrhizal symbiosis (Jones and Last, 1991). There are also a few studies on the
inverse effect of mycorrhizas on herbivores (Gange et al., 1994; Rabin and Pacovsky,
1985). Generally, mycorrhizas had an inhibitory effect on the herbivorous insects. Gange

30
and West (1994) found that compared to fungicide-treated plants, mycorrhizal plants had
lower soluble neutral sugars, starch, total N, and amino acids (alanine and
tyrosine/valine) and a higher concentration of the anti-feedant chemicals, aucubin and
catalpol. In their study, chewing insects were negatively impacted when feeding on
mycorrhizal plants; however, sucking insects developed better on mycorrhizal plants. The
authors hypothesized that a higher C/N ratio in the mycorrhizal plants allowed more C
to be allocated to plant defense mechanisms, such as secondary plant metabolite
production. Localization of the secondary metabolites may partly account for the
differential response between insect types. Viewed in terms of plant competition, plants
able to efficiently modify their C balance to simultaneously reduce insect pests and still
maintain the mycorrhizal association may have a competitive advantage in the long run.
Plant Succession and Community Structure
Limited information is available on the ecological relevance of mycorrhizas in
plant competition. Plant competition can be viewed in terms of single plant interactions,
but its importance lies at the population and community levels. The interactions occurring
at the ecosystem level are obviously complex and many have been set aside for the sake
of simplicity. As has been suggested by various authors (Brundrett, 1991; Francis and
Read, 1994; Newman, 1988) mycorrhizas are likely involved in plant community
structuring, but the magnitude of their effect is unknown. Increasing the competitive
ability of individuals within a population enhances their ability to capture resources and
improves their fitness. One of the major components determining early succession is

31
plant competition for limited nutrients (Wilson and Shure, 1993). Under nutrient
limitations, resource acquisition enhanced by mycorrhizas occurs at the expense of other
plants, which results in the highly competitive plants becoming more abundant and
dominant in the community. Continuous growth of a plant in the same soil eventually
will select a microbial community well adapted to that environment. Over time the
adapted microbial community can become disadvantageous for growth of that plant
species, but not for others, and, in this manner, may contribute to plant succession
(Bever, 1994; Van der Putten et al., 1993). In these studies it was suggested that this
negative feedback on growth may be related to pathogen buildup. Mycorrhizal fungi were
not considered, because of the assumption that mycorrhizal effects are usually beneficial.
However, if there is a selection for less efficient mycorrhizal fungi occurring over time,
then this may similarly contribute to succession by decreasing a plants C-use efficiency
and its competitive ability. In monocultural settings, a shift of mycorrhizal fungal species
composition over time was identified by Johnson et al. (1992a; 1992b) and Wacker et al.
(1990). In both cases there was an associated decline in plant growth, indicating that
mycorrhizal fungi should not be discarded a priori as a contributing factor to growth
declines.
Succession of ectomycorrhizal fungi from "early" to "late" stage fungi occurs in
undisturbed forest systems (Deacon and Fleming, 1992). Differing fungal resource
requirements, as well as changes in other soil microbial components, have been
postulated to cause the succession (Garbaye, 1994). Recent research indicates that this
succession may be tied closely to factors found in the soil organic matter. Removal of

32
litter and humus in Pinus sylvestris stands increased mycorrhizal fungal species richness
and reverted the species composition to the early successional types (Devries et al.,
1995). In other systems, the increased buildup of organic matter also has been associated
with higher concentrations of phenolic compounds (Kuiters and Sarink, 1986; Leake et
al., 1989), which have demonstrated allelochemical effects. Perhaps resistance to and the
ability to degrade phenolic compounds determines which fungal species are capable of
growing at a certain stage of succession. Leake et al. (1989) demonstrated that ericoid
mycorrhizas were capable of enhancing ericoid plant growth and survival, possibly by
a detoxification mechanism. Whereas AM fungi are found more commonly in mineral
soils, ectomycorrhizal fungi are often associated with environments high in organic
matter and are physiologically adapted to utilizing complex substrates (Francis and Read,
1994). Also, ectomycorrhizal mantles surrounding root tips are capable of protecting
these from potentially toxic compounds. As a consequence, tolerance to adverse
environmental conditions allows the plant to focus more of its energy on resource
acquisition strategies without substantial tradeoffs of energy for other mechanisms,
thereby making it a better competitor.
Plant competition, as affected by mycorrhizal fungi, could be relevant in plant
community structuring and succession. As such, mycorrhizal benefits to single plants may
prove functionally significant at the ecosystem level. In addition, positive interactions in
communities are often neglected (Bertness and Callaway, 1994) and should also be
considered in the discussion of plant interactions mediated by mycorrhizas (Amaranthus
and Perry, 1994). Mycorrhizas can moderate plant competition (Perry et al., 1989) and

33
provide resilience to disturbance (Amaranthus and Perry, 1994). Mycorrhizal connections
between dying and living plants also limit soil nutrient loss by leaching and
immobilization (Eason and Newman, 1990). The network of hyphal bridges connecting
neighboring plants can affect coexistence by increasing species richness and diversity
(Gange et al., 1993; Grime et al., 1987). The current literature indicates that this is
perhaps more likely due to transfer of C than of inorganic nutrients. Furthermore, a
higher plant species diversity has been associated with increased ecosystem stability in
a stressed environment (Tilman and Downing, 1994). Obviously, with the multitude of
effects and interactions mediated by mycorrhiza, a quantification of the net mycorrhizal
influence in ecosystems is a formidable challenge. Still, with the current emphasis on
environmentally sound management of ecosystems, it is important to include them in
considerations of appropriate technologies in managed ecosystems.

CHAPTER 3
LIMITATIONS IN THE USE OF BENOMYL IN EVALUATING
MYCORRHIZAL FUNCTIONING
Introduction
A limitation to mycorrhizal field research is the difficulty of obtaining an
appropriate nonmycorrhizal control, since plants in nature are normally colonized. Soil
fumigation has been used to control mycorrhizal fungi; however, the broad biocidal
effects limit the usefulness of this technique. Fungicides are more specific and alter fewer
biological soil processes. Paul et al. (1989) summarized the ideal properties of a
fungicide used to chemically exclude an organism from an experiment. The fungicide
properties should include: (i) moderate persistence to reduce mechanical disturbance from
the application process, (ii) an appropriate activity spectrum that targets selected
organisms only and (iii) no direct physiological effects on the plant.
The systemic fungicide benomyl, a benzimidazole, has been used frequently to
reduce arbuscular mycorrhizal (AM) activity in experimental treatments (Jalali and
Domsch, 1975; Kough et al., 1987; Fitter and Nichols, 1988; Hartnett et al., 1994;
Newsham et al., 1995; West et al., 1993). Benomyls lack of direct effects on plants and
somewhat selective effects against AM fungi (Zygomycetes) currently make it a better
choice compared to other fungicides (Paul et al., 1989; Sukarno et al., 1993).
Nonetheless, the amount of mycorrhizal control achieved with benomyl has varied.
Reduction of colonization or biomass of mycorrhizal plants has been observed in several
34

35
cases (Evans and Miller, 1988; Fitter and Nichols, 1988; Trappe et al., 1984), but these
results are not always achieved (Koide et al., 1988; Fitter, 1986; Trappe et al., 1984).
Much of this variability is likely attributable to the experimental conditions such as soil
type, method and timing of fungicide application and potentially more complex
interactions occurring within the soil microbial community. For example, benomyl can
inhibit nematodes (Elamayem et al., 1978) and different fungi that do not form
mycorrhizas (Edgington et al., 1971), thereby indirectly altering mycorrhizal effects.
Several studies have addressed the effects of arbuscular mycorrhizas on plant
interactions (Fitter, 1977; Hall, 1978; Newman et al., 1992), and some have utilized
benomyl (Hartnett et al., 1993; Hetrick et al., 1989; Newsham et al., 1995) or other
fungicides (Gange et al., 1993) to create control treatments. Only one study addressed
the influence of EM fungi on plant competition (Perry et al., 1989). Very few studies
have taken place under field conditions, and apparently none have addressed the role of
mycorrhizas in the interactions between AM and EM plants. Benomyls putative selective
effect against AM fungi and neutral effects on EM fungi (Trappe et al., 1984) could be
valuable in sorting out the individual benefits of these two types of mycorrhizal
symbioses to different host plants competing for the same nutrients.
As part of a larger plant competition study between AM and EM plants, the
usefulness of benomyl as a tool to selectively control mycorrhizas was tested. The main
objectives were to: (i) compare the efficacy of benomyl in controlling mycorrhizas in the
greenhouse to that in the field, (ii) differentiate effects of benomyl on external hyphae

36
from those on the internal mycorrhizal phase and (iii) determine if the intensity and
longevity of the fungicides effect was dose-dependent.
\
Materials and Methods
Field Study
The site was located 21 km northwest of Gainesville, Florida and was part of a
larger plant competition study involving slash pine (Pinus elliottii Engelm. var. elliottii))
and weeds. Slash pine had been planted in April 1990 in beds approximately 26 cm in
height and about 2 m in width with rows spaced approximately 213 cm apart. Soil was
a Pomona fine sand (a sandy, siliceous, hyperthermic Ultic Haplaquod). The surface 10
cm of soil had 7 pg P g1 extractable in 2 mM CaCl2 and a soil solution with pH 3.9.
Approximately 3.3% weight was lost upon ignition. The dominant weeds were Panicum
chamaelonche Trin., P. aciculare Dec.ex Poir. in Lam., Andropogon spp., Paspalum
spp. Rubus sp. and Serenoa repens. In December 1991, less than 1 spore of mycorrhizal
fungi g'1 of field soil was detected; the populations consisted of a mix of Glomus sp.,
Gigaspora sp. and Scutellospora sp. In the greenhouse, pot cultures of P. chamaelonche
originating from the field and grown in field soil yielded two AM isolates, Gigaspora
rosea (INVAM FL224) and Scutellospora heterogama (INVAM FL225) which were
submitted to and identified by J. Morton at INVAM.
Two areas (each 18.4 m by 11 m) containing slash pine and weeds were selected
randomly for this study. The control plot received no fungicide sprays. Benlate 50 DF
(E.I. du Pont de Nemours & Co., Inc., Wilmington, DE) was applied to the second area

37
with a C02-pressurized backpack sprayer by covering the area once and then making a
second application perpendicular to the first. The first spray (2 April 1991) was applied
at the rate of 5 kg benomyl ha1 using the equivalent of approximately 150 ml of water
m 2. Subsequent sprays (30 May, 11 July and 19 Sept. 1991) were applied at a rate of
20 kg benomyl ha'1.
Panicum chamaelonche was chosen as the indicator plant of AM fungal activity
because it was a dominant weed species at the site. Samples were taken on 2 April, 4
April, 30 May, 10 June, 2 July, 22 July, 13 August, 10 October 1991. At each sampling,
three plants were selected randomly and removed from each plot. The roots were washed
and cut into lengths of 1 to 2 cm. To determine fungicide effects on colonization and
metabolic activity, 1- to 2-g subsamples of roots were stained at room temperature for
8 h in a solution containing 0.2 M Tris HC1 (pH 7.4), 1 mg ml'1 of iodonitrotetrazolium
violet (INT) and 3 mg ml'1 of NADH (Sylvia, 1988). This was followed by clearing the
roots in a boiling, saturated solution of chloral hydrate for 10 min and subsequent
counterstaining overnight in 0.5% aniline blue in lactoglycerol. The chloral hydrate
treatment proved unnecessary and was eliminated in samplings collected after May. The
roots were destained in lactoglycerol and a minimum of 25 1-cm-long root segments per
plant were laid out parallel to each other on slides. The percentage of root segments with
arbuscules and the percentage of total arbuscules that were active (those staining with
INT) were estimated using bright-field microscopy at 400x magnification. The effect of
benomyl on mycorrhizal development was evaluated using the relationship of time and
either arbuscule abundance or activity. The slopes of linear regression of benomyl-treated

38
versus nontreated plants were compared using the General Linear Model procedure of
SAS (SAS Institute, Inc., 1989).
Greenhouse Study
Both of the following experiments had completely randomized factorial designs
(two mycorrhizal treatments x four benomyl levels) with seven replications each. To
maintain uniform daylength of approximately 12 h, extra light (800 /zmol m'2s'' from
17:00 to 20:00 hr) was provided. Plants in all experiments were fertilized semiweekly
with 3.2 /xM NH4N03, 7.5 /zM Ca(N03)2 7.7 /xM KC1, 1.0 /xM MgS04, 20 nM
NaFeEDTA, 5.0 nM CuS044H20, 240 nM H3B04, 20 nM MnCl24H20, 5 nM
Na2Mo042H20 and 20 nM ZnS047H20. The nutrient solution for corn or pine
contained, respectively, 3.2 nM H3P04 or 0.32 nM H3P04. All data were analyzed by
analysis of variance using the General Linear Model procedure (SAS Institute, Inc.,
1989). Both experiments were repeated once under similar environmental conditions.
Benomvl effects on pine
Slash pine seeds were disinfested for 2 min in a 5.25% sodium hypochlorite
solution with 0.2 ml Liqui-Nox surfactant (Alconox, Inc., New York, NY) and then
rinsed thoroughly with tap water. Plants were raised from seed for 12 d in a growth
chamber [29 C/23 C (day/night), with a 15-h light period and irradiance of 1000 zmol
m 1 s'1] in a vermiculite/sand (1:1) mix. They were then transplanted into sand in 50-ml
pots (5 cm2 of surface area) grown in the greenhouse for 6 wk where they received water

39
only. Pisolithus tinctorius (Pers.) Coker & Couch (isolate S106) was grown with no
shaking in a modified Melin-Norkrans liquid medium (Marx, 1969) containing glucose
instead of sucrose. Just prior to use, fungal mats were washed with tap water, added to
a food processor with water and chopped (Rousseau and Reid, 1990). Eight-week-old
pines were inoculated with the fungus by immersing the washed roots in the suspension
and then grown in the greenhouse in 500 ml of sand in Deepots (28 cm2 of surface
area; McConkey, Co., Sumner, WA). Six weeks after inoculation, 10 ml of a suspension
of Benlate 50 WP in deionized water was applied once at 0, 20, 60 or 150 kg benomyl
ha'1 equivalent (based on pot surface area). Plants were grown from January to March
1993 under a mean photosynthetic photon flux density of 535 panol m'2 s'1 and 17/30C
(min./max.) temperature regime.
Groups of plants were harvested before, and then 2 and 4 wk after benomyl
application. Prior to harvesting the plants, a soil core (15.5-mm diam. by 15-cm deep)
was removed from each pot. Hyphal length and activity were evaluated by a slightly
modified procedure of Sylvia (1988). A thoroughly mixed, 10-g, wet-mass subsample of
soil was added to 500 ml of water and chopped in a Waring blender at the high setting
for 20 seconds. The resulting suspension was allowed to settle for 20 seconds before a
25-ml portion was removed and filtered through a 0.45-pim-pore size membrane (GN-6
Metricel; Gelman, Ann Arbor, MI). The hyphae on the membrane were stained for
6 h with INT solution, destained with tap water, counterstained for 30 min with 0.1 %
trypan blue in lactoglycerol and destained again with tap water. Using a gridline-intercept

40
method, total and active hyphal lengths were determined microscopically at 400x from
20 randomly selected fields on the filter.
Pine needles were removed from seedlings and dried overnight at 65C, and P
content was determined colorimetrically (Murphy and Riley, 1962). Ergosterol (a sterol
found in fungal, but not plant, membranes) content in the root was used to provide a
relative estimate of total fungal biomass present (Martin et al., 1990; Salmanowicz et ah,
1989). Fresh roots were washed, ground in liquid nitrogen and thoroughly mixed. A 0.1-
to 0.3-g subsample was extracted overnight at room temperature with 5 ml of 100%
ethanol. This sample was filtered through a 0.45-/xm syringe filter and then assayed for
free ergosterol by high-pressure liquid chromatography (Waters 715 Ultra WISP, Gilson
115 UV detector). Separation was achieved on a C-18 column (Supelcosil LC-18;
Supelco, Inc., Bellefonte, PA) at 40C using a methanol-water mobile phase (92:8)
flowing at 2 ml min'1 with detection at 282 nm.
Benomvl effects on corn
The effect of benomyl on colonization by the AM fungus Glomus sp. (INVAM
FL329, formerly FL906) was studied in a separate experiment. Germinated corn (Zea
mays L. cv. Silver Queen) seed was planted in sand in Deepots with 5 g of soil
inoculum (83 spores g'1) placed 2 to 3 cm below the seedling. Control plants received a
5-ml suspension of inoculum filtrate obtained by mixing 60 g of soil from a pot culture
with 1.2 L of water and then filtering this through a 10-/xm membrane filter. Benlate 50
WP was applied 19 d after planting to the soil surface at rates of 0, 20, 60 and 150 kg

41
benomyl ha"1 equivalent. Plants were grown from March to May 1993 under a mean
photosynthetic photon flux density of 608 /mol m"2 s'1 and 18/35C (min./max.)
temperature regime.
The plants were sampled before, and then 2, 4 and 6 wk after benomyl
application. The harvest procedures were the same as for pine, with the exception of
estimation of root colonization. Washed root segments (1 to 2 cm) were cleared with
10% KOH for 30 min, rinsed several times with tap water, acidified for 30 min in 5%
HC1 and stained overnight in 0.05% aniline blue in lactoglycerol. Colonization was
determined using a gridline-intersect method (Giovannetti and Mosse, 1980). Although
fungi other than AM existed in this particular system, the differentiation of saprophytic
from characteristic AM fungal hyphae was based on gross morphological differences.
Arbuscular mycorrhizal fungi generally had a somewhat larger hyphal diameter (4
compared to <2 /m), stained darker with aniline blue, were not dematiacious, lacked
septation or clamp connections and demonstrated a less angular growth pattern compared
to other fungi present. Prior to statistical analysis, percentage colonization was
transformed using the arcsine, square root transformation.
Results
Field Study
Initial AM colonization of P. chamaelonche in the field was high, indicating that
root growth and mycorrhizal development commenced earlier than the first fungicide

42
DU
<
2
UJ
z
>
>-
_l
^3
O
\-
Q.
1
CL
<
CL
<
D
)
_l
D
Z)
-)
)
Z3
<
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<0
o
o
CM
CD
o
o
T
CM
n
05
o
T
CO
T
CM
T
CM
r-
T
T-
Sampling Date
Figure 3-1. Assessment of arbuscular activity in Panicum chamaelonche roots from the
field site in 1991. (A) Percentage of root length with arbuscules in benomyl-treated and
nontreated plots, (B) Percentage root length with metabolically active arbuscules in
benomyl-treated and nontreated plots and (C) Precipitation. Each symbol represents the
mean of three replicates SE.

43
Table 3-1. Test for homogeneity of slopes for the effect of Benlate 50 DF applied in the
field on percent Panicum chamaelonche roots with arbuscules and their activity over
time.
Slope over time
Roots with arbuscules
(%)
Roots with active arbuscules
(%)
Control
-0.104 **
-0.164 **
Benlate
-0.012
-0.010
indicates slope value is significantly different from 0 at P < 0.01

44
application on 2 April (Fig. 3-1A). Over the entire growing season, both the proportion
of roots with arbuscules and the activity for benomyl-treated plants did not change
significantly, whereas samples from the control plots had significantly negative slopes
with time for both arbuscule abundance and activity (Table 3-1). Early in the season
ground cover was sparse and the spray was applied directly to the soil. This was
paralleled by a short-term decrease in the proportion of roots with arbuscules (Fig. 3-1A)
as well as metabolic activity (Fig. 3-1B). As ground cover increased through the growing
season, more of the spray was intercepted by foliage leaving less to penetrate through to
the soil. Concomitant with this, the differences between treated and nontreated plots
disappeared. In late summer, as the plants started to senesce, roots of benomyl-treated
plants had more arbuscules and arbuscule activity than nontreated plants. In a concurrent
study, no effect of benomyl on shoot P status was observed at samplings taken in June
and August. There was no apparent relationship between precipitation, application of
benomyl and mycorrhizal response (Fig.3-1C).
Greenhouse Study
Benomyl effects on pine
There were no significant effects of benomyl on inoculated or noninoculated pine
biomass (Fig. 3-2A). Phosphorus content of the needles increased over time for all
treatments from a mean of 320 mg to 450 mg per plant, but this was not related to the
benomyl treatments (data not shown). Similarly, benomyl had no effect on the length or

45
O)
i
o
S
o
i-
X///X O kg benomyl ha1
fcS&l 20 kg benomyl ha'1
I f-hH 60 kg benomyl ha'1
IN>NM 150 kg benomyl ha
-i
Week After Benomyl Application
Figure 3-2. Total dry weight of mycorrhizal (M) and nonmycorrhizal (C) plants, (A)
Pinus elliottii and (B) corn in response to 0, 20, 60 or 150 kg benomyl ha-1 in the
greenhouse. Each symbol represents the mean of seven replicates SE.

46
TOTAL ACTIVE TOTAL ACTIVE TOTAL ACTIVE TOTAL ACTIVE
0 2 4 6
Week After Benomyl Application
Figure 3-3. Soil hyphal length (total) and activity (active) of mycorrhizal (A) Pinus
elliottii and (B) corn plants in response to 0, 20, 60 or 150 kg benomyl ha'1 in the
greenhouse. Each symbol represents the mean of seven replicates SE.

Root Length Colonized (m) Ergosterol (mg g-^ of root dwt.)
47
6
5
4
3
2
1
0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
1
B
i
V//A 0 kg benomyl ha'1
20 kg benomyl ha'1
l l 60 kg benomyl ha'1
150 kg benomyl ha'1
ii
I
(
n
I
i
0 2 4 6
Week After Benomyl Application
Figure 3-4. Mycorrhizal colonization of (A) slash pine and (B) corn grown in the
greenhouse in response to 0, 20, 60 or 150 kg benomyl ha1. Each symbol represents the
mean of seven replicates SE.

48
viability of external hyphae of the ectomycorrhizal fungus (Fig. 3-3A). There was a
difference in EM colonization, as measured by ergosterol concentration, at 4 wk
between the 60 and 150 kg benomyl ha1 treatments (Fig. 3-4A); however, this was not
repeatable.
Benomvl effects on corn
Benomyl at all concentrations arrested further root colonization by the AM
fungus, whereas colonization in the treatment receiving no benomyl continued to increase
over the 6-wk period (Fig. 3-4B). There was no dose-related response in colonization.
Noninoculated plants remained noncolonized. Total biomass of mycorrhizal and
nonmycorrhizal plants was reduced by benomyl by approximately 12% (Fig. 3-2B);
however, this was unrelated to the fungicide concentration applied. The length of external
hyphae of AM fungi or their viability was not affected significantly or consistently by the
different rates of benomyl (Fig. 3-3B). The P concentration of corn leaves decreased
steadily throughout the experiment from 3.64 to 0.62 mg P g'1 without any evidence of
a benomyl effect (data not shown).
Discussion
Corn was used as a substitute for P. chamaelonche due to lack of native plant
material. Benomyl arrested mycorrhizal development of corn in the greenhouse
experiment. This is consistent with the mode of action of benomyl, which entails
inhibition of nuclear division by binding to tubulin (Davidse, 1986). There was no dose-

49
dependent response by the mycorrhizal grass in the greenhouse. The range of
concentrations was based on previous values published in the literature (Trappe et al.,
1984). The sand used in the greenhouse minimized possible adsorption phenomena that
normally occur in field soils. Consequently, all the concentrations tested were above the
threshold required to obtain a maximum inhibition of mycorrhizal development. Not only
was all of the fungicide readily available, but it was also well above the manufacturers
recommended application rate, which together presumably caused a decrease in plant
biomass unrelated to the plants mycorrhizal status. In agreement with previous literature
(Trappe et al., 1984), the effect of benomyl on mycorrhizal pine was neutral although
sometimes an increase in growth has been observed (De la Bastide and Kendrick, 1990;
Pawuk and Barnett, 1981).
Arbuscules were counted in the field, since they are a distinguishing characteristic
of the mycorrhizal fungus, and, more importantly, they represent the site where active
exchange of nutrients between the symbionts occurs. The initial decrease in arbuscule
activity in the field following benomyl application has been documented in a greenhouse
study as well (Sukarno et al., 1993). Since the response to 5 kg benomyl ha'1 was minor
compared to total colonization, the benomyl application rate was increased. At the last
sampling as the plants started to senesce, the increase in arbuscule number and activity
in roots of plants treated with benomyl may be due to a reduction in the impact of
nonmycorrhizal fungi on plant growth and subsequent mycorrhizal functioning. Low
colonization and arbuscule numbers in the greenhouse study made it difficult to obtain
a reliable measure of arbuscule abundance to compare to the results in the field. The lack

50
of AM response to benomyl in the field during most of the growing season may be
attributed to the increased interception of the fungicide by ground cover. Although
benomyl can enter through leaves, systemic translocation is not as efficient generally as
direct application to the target site (Hassall, 1990), in this case, the roots.
Larsen et al. (1994) determined that benomyl applied directly to the leaves of
cucumber had little effect on mycorrhizal efficiency, yet when benomyl was applied to
the soil, complete inhibition of P uptake by hyphae occurred within 5 d. Although no
fungicide effect on fungal alkaline phosphatase activity was found inside the root, the
rapid response, nonetheless, suggests some direct influence on uptake or transport
mechanisms. Kough et al. (1987) and Thingstrup and Rosendahl (1994) have observed
suppressive effects of benomyl on internal fungal enzyme activity in mycorrhizal plants.
Although benomyl demonstrated no significant effect on external hyphal length or
viability in this study, an inhibitory response has been found in another system (Sukarno
et al., 1993).
Benomyl can be an effective tool for inhibiting AM activity in the field; however,
researchers need to be aware of the limitations of this approach. The timing of root
colonization and initial nutrient contribution to mycorrhizal dependent seedlings can be
critical to their survival (Hartnett et al., 1994; Hetrick et al., 1989; Plenchette and
Perrin, 1992). Fungicide applications in the field should be timed according to the plants
optimal benefit from mycorrhizas, which, correspondingly, would provide the full impact
of the fungicide treatment on mycorrhizal functioning (Gange et al., 1993; Newsham et
al., 1995). Furthermore, the frequency of application is determined by fungicide

51
persistence in the soil, which is variable (Ware, 1992) due to degradation and sorption
in different soil environments. In sandy soils where sorption is low, somewhat
comparable to the sand used in the greenhouse study, persistence may be longer,
assuming leaching does not occur, so that an application every 5 to 6 wk may suffice.
Soils with higher levels of organic matter or clay may require more frequent applications
or higher concentrations.
The method of application is also critical. Although benomyl is considered a
systemic fungicide, translocation from leaves to the active site in the roots appears to be
minimal. A soil drench is the optimal method of application (Fitter and Nichols, 1988;
Hassall, 1990; Perrin and Plenchette, 1993). Appropriate preparations should be made
to accommodate the increasing ground cover as treatments are applied later in the
growing season. Tall ground cover may be compensated for by applying a large volume
of water to wash the active ingredient to the soil. Benomyl concentrations applied
experimentally have ranged from 0.5 to 300 kg benomyl ha1 (Trappe et al., 1984).
Treatments of as little as 3 kg ha'1 biweekly in a short turf grass setting have been
adequate to reduce AM colonization by 80% (Rhodes and Larsen, 1981). As a
consequence, the combination of concentration, volume of water used and frequency of
application balanced with the environmental conditions should provide the desired
reduction of mycorrhizal activity.

CHAPTER 4
MYCORRHIZAS AFFECT PLANT COMPETITION FOR PHOSPHORUS
BETWEEN PINUS ELLIOTTII and PANICUM CHAMAELONCHE
Introduction
Soil fertility largely determines the amount of plant biomass an environment can
support (Donald, 1951). In environments with low nutrients, plants are stressed directly
by the lack of adequate nutrients, and they survive primarily by stress tolerance
mechanisms (Grime, 1979). An environment with more nutrients has the potential to
produce more plant biomass, which increases plant growth and also raises the chances
that root nutrient depletion zones of two plants will overlap. As a consequence, plant
competition for nutrients becomes one of the factors governing plant growth and survival.
Environmentally induced stress on a plant, therefore, can be considered a gradient
extending from direct physical stress on an individual plant to stress produced
biologically by plant interactions (Berkowitz et al., 1995; Grime, 1979).
Autecological studies have extensively documented that mycorrhizas can increase
plant tolerance to environmental stresses and contribute to a plants survival and growth
(Sylvia and Williams, 1992). The various mycorrhizal contributions that enhance
individual plant health similarly benefit a plant when competing with neighboring plants.
Much less research has quantitatively addressed the influence of mycorrhizas on the
synecology of plants. Previous studies have demonstrated that mycorrhizas can enhance
a plants competitive ability (Allen and Allen, 1984; Fitter, 1977; Hall, 1978; Hartnett
52

53
et al., 1993; Hetrick et al., 1989). The majority of these studies relate to competition
between arbuscular-mycorrhizal (AM) plants. To my knowledge, only one study has
addressed ectomycorrhizal (EM) effects on plant competition (Perry et al., 1989). Plant
competition between EM and AM plants has not been explored specifically.
The goal of this research was to assess the effect of mycorrhizas on the
competitive ability of slash pine (Pinus elliottii Engelm. var. elliottii), which commonly
is grown for pulpwood in the southeastern United States. Grasses, among other plants,
compete extensively in new slash pine plantations since weed control is practiced
infrequently. The specific objectives of the study were to determine (i) if mycorrhizas
alter the competitive ability of pine when growing with grass and (ii) how this
relationship is modified by phosphorus (P) concentration.
Materials and Methods
Greenhouse Competition Study
All experiments were conducted in acid-washed sand. Acid-washing was
accomplished by treating the sand with 25% HC1 for 24 h, then draining the acid and
rinsing the sand until the pH increased to that of the deionized water being used. Eighty
percent of the sand was in the particle size range of 0.160 to 1 mm, and the majority of
the remaining portion was larger than 1 mm.
Slash pine seeds were disinfested for 2 min in a 5.25% sodium hypochlorite
solution with 0.2 ml Liqui-Nox surfactant (Alconox, Inc., New York, N.Y.) and then
rinsed thoroughly with tap water. Seedlings were raised from seed for 2 wk in a growth

54
chamber [29/23 C (day/night), with a 15-h light period and irradiance of 1000 /mol m'1
s'1] in sand and then transplanted to 50-ml pots (5 cm2 of surface area) and grown in sand
in the greenhouse for 8 wk where they received water only. To inoculate pine, washed
roots were dipped in a slurry of rinsed and chopped Pisolithus tinctorius (Pers.) Coker
& Couch (isolate S106) grown in a liquid suspension culture containing modified Melin-
Norkrans liquid medium (Marx, 1969) using glucose instead of sucrose. Roots of control
plants were dipped in tap water. After a further 6 wk of growth in 500 ml of sand in
Deepots (28 cm2 of surface area; McConkey, Co., Sumner, WA), pine roots were
gently rinsed free of adhering sand particles and planted in the appropriate competition
treatments as described below.
Grass plants of a dominant competing weed species in the field (Panicum
chamaelonche Trin.) were obtained from cultures maintained in sand in the greenhouse.
Plants were started from seed and vegetatively propagated in 150-ml pots (7 cm2 of
surface area). Two months in advance of the experiment, grass plants were inoculated
with pot culture inoculum of Glomus sp. (INVAM FL329, formerly FL906) previously
cultured on sorghum in pasteurized soil. Roots of plants were washed and the plants
transplanted into sand in Deepots containing 5 g of soil inoculum (83 spores g'1) located
2-3 cm below the sand surface. Control plants were transplanted into sand without
inoculum and received a 5-ml suspension of inoculum filtrate obtained by mixing 60 g
of soil from a pot culture with 1.2 L of water and then filtering the mixture through a
10-^tm membrane filter. Just prior to the experiment the grass roots were washed as
described for the method of pine roots.

55
Pine and grass plants were sorted separately into three size classes at the start of
the experiment. Noninoculated pine had no visual indication of colonization, whereas
inoculated pine was heavily colonized. Inoculated grasses had a mean root colonization
of 30% at the start of the experiment. There were no significant differences in biomass
between inoculated and noninoculated plants at the beginning of the experiment.
Intraspecific and interspecific paired combinations of plant species (Table 4-1), inoculated
or not, were made by selecting plants from the same size class. Plants were planted
together in 500 ml of sand. There were six replications per treatment. Plants were grown
in the greenhouse with mean temperatures of 21/34C (min./max) and a mean
photosynthetic photon flux density of 1240 jumol m2 s'1 from January to June 1994. A
repeat of the experiment was run from May to October 1994 with seven replications. The
greenhouse temperature regime was 24/36 (min./max.) with a mean photosynthetic
photon flux density of 1490 /mol m'2 s'1. The plants were fertilized semiweekly with a
solution containing: 660 /xM NH4N03, 660 juM (NH4)2S04, 616 xM KC1, 80 /xm MgS04,
54 pM NaFeEDTA, 600 ¡xM CaCl2, 0.25 xM CuS04, 14 /xM H3B03, 40 xM NaMo04,
2.75 /xM MnCl and 1.25 /xM ZnS04. Phosphorus was supplied at either 0.32, 3.23 or
32.26 xM H3P04. In the repeat of this experiment the 0.32 xM H3P04 treatment was
replaced with 323.58 /xM H3P04, since plant growth was very slow at the lowest P
concentration. Soil solution pH was measured from several pots during the experiment
by thoroughly watering pots with deionized water and collecting the leachate.
Plants were removed from the pots after 129 d, and the roots of individual plants
were separated carefully from each other. The exception was the intraspecific grass

56
Table 4-1. Pirns elliottii (pine) and Panicum chamaelonche (grass) treatment
combinations planted in the competition study. Two plants were planted per pot. The
superscripts + and signify an inoculated or noninoculated plant respectively. Pine
was inoculated with Pisolithus tinctorius and the grass was inoculated with Glomus sp.
(INVAM FL329, formerly FL906).
Plant Competition Treatments
Intraspecific
Interspecific
pine+ x pine+
pine+ x grass+
pine' x pine
pine+ x grass'
grass+ x grass+
pine' x grass+
grass' x grass'
pine' x grass

57
combination where the roots were treated as one unit and then half the value allotted to
each plant. Root wet and dry mass were determined. An estimate of root length was
obtained using calculations of specific root length (cm root g"1 of root fresh weight) for
pine and grass from a previous experiment and expressed here as root-length density (cm
root cm'3 of soil). For grass, root colonization was determined using a gridline-intersect
method for the AM treatments (Giovannetti and Mosse, 1980) after clearing the roots for
30 min in 10% KOH and staining in 0.05% aniline blue overnight. For pine, root
ergosterol concentration was used as an estimate of EM fungal biomass (Martin et al.,
1990; Salmanowicz et al., 1989). Fresh pine roots were washed, ground in liquid
nitrogen and thoroughly mixed. A 0.1- to 0.3-g subsample was extracted overnight at
room temperature with 5 ml of 100% ethanol. This sample was filtered through a 0.45
im-syringe filter and then assayed for free ergosterol by high-pressure liquid
chromatography (Waters 715 Ultra WISP, Gilson 115 UV detector). Separation was
carried out using a C-18 column (Supelcosil LC-18; Supelco Inc., Bellefonte, PA) at
40C with a methanol-water mobile phase (92:8) flowing at 2 ml min1, with detection
at 282 nm.
Shoots were analyzed separately from roots. Shoot wet mass was determined and
dry mass was measured after drying overnight at 65C. The shoots were ground and then
ashed at 500C for a minimum of 4 h. Phosphorus analysis of the shoot tissue was
performed using the method of Murphy and Riley (1962).

58
To compare the competitive abilities of the two plant species, the relative
crowding coefficient (RCC; Harper, 1977) was calculated. To avoid subjective pairing
of plants between treatments all possible combinations were used to calculate the RCC.
The following is a sample calculation of the RCC for grass total dry weight when
growing with pine:
grass (interspecific)
pine (interspecific)
RCC (shoot P, mg P) =
grass (intraspecific)
pine (intraspecific)
Data for grass and pine were analyzed separately. To determine if plant
competition was affected by the plant species, data for each plant species were subjected
to analysis of variance and statistically planned contrasts using the General Linear Model
(SAS Institute, Inc., 1989). Data for colonization were transformed to arcsine square
roots prior to analysis (Steel and Torrie, 1980). The least-squares means statement within
SAS was used to compare means.
Determination of P Uptake Kinetics for Pine and Grass
Pine and grass plants were inoculated with their respective mycorrhizal fungi or
noninoculated. Pine plants were grown in Deepots for a further 24 wk after inoculation
with P. tinctorius. Grasses were inoculated 3 wk prior to transferal to 1-L Erlenmeyer
flasks by applying to each plant root system a minimum of 20 spores of a mixed culture
of Gigaspora rosea (INVAM FL224) and Scutellospora heterogama (INVAM FL225),

59
cultured on P. chamaelonche in field soil in the greenhouse. The AM fungal species were
isolated from P. chamaelonche growing in a Spodosol at a field site 21 km northwest of
Gainesville. The grasses were replanted together in a 15-L pot of sand. Noninoculated
plants were treated in the same manner, except that no spores were added to the roots.
Three grasses and pines, inoculated or noninoculated, were selected, and their
roots were gently rinsed free of adhering sand. Each plant was transferred to a single 1-L
Erlenmeyer flask covered with aluminum foil. Plants were grown in a growth chamber
[29/23 C (day/night), with a 15-h light period and irradiance of 1000 /xmol m'1 s'1] in
a continuously aerated nutrient solution with the following nutrient composition: 660 /xM
NH4N03, 616 /xM KC1, 800 /xM MgS04, 54 /xM NaFeEDTA, 600 /xM Ca(N03)2 4H20,
0.75 /xM CuS04, 52 /xM H3B03, 120 /xM NaMo04, 8.25 /xM MnCl and 3.75 /xM ZnS04.
Phosphorus was supplied at 3.23 /xM H3P04. The solution was changed semiweekly. At
the start of the experiment a minimum 4-wk acclimatization period was given allowing
external hyphae to regrow from the colonized roots.
To quantify uptake kinetics, root systems were rinsed with deionized water and
placed in additional deionized water for 1 h. One liter of fresh nutrient solution, identical
to the one used previously, was added to 1-L acid-washed Erlenmeyer flasks. At the start
of the experiment, plant roots were gently patted dry with paper towels, placed in the
nutrient solution and weighed. At regular intervals, 23 ml of solution for P analysis were
removed and immediately filtered through 0.45-/xm syringe filters. The solution was
replaced with sufficient deionized water to bring the system back to its original starting
weight. Twenty milliliters of sample removed for P analysis were evaporated to dryness.

60
Twenty milliliters of concentrated HC1 were added to the sample and also evaporated to
dryness. Phosphorus was determined colorimetrically by a slightly modified procedure
of Murphy and Riley (1962). Reagent, quantitatively diluted with deionized water, was
added directly to the samples. Since some solutions were at the detection limit, less
reagent was added to later samples in order to concentrate them. The resulting P-
depletiOn curve was fit using a curve-fitting procedure (SigmaPlot; Jandell Scientific, San
Rafael, California), and uptake calculations were made based upon this idealized curve.
Total surface area of roots and hyphae were determined using image analysis software
(Mocha; Jandell Scientific, San Rafael, California) or gridline-intersect methods
(Giovannetti and Mosse, 1980). The maximum uptake rate, 1^, was calculated based on
the root surface area and the quantity of P absorbed from the nutrient solution during the
first 45 min. The minimum solution concentration from which a nutrient can be
absorbed, C^, was considered the asymptotic value where the solution P concentration
no longer decreased.
Results
Greenhouse Competition Study
No colonization was found in the inoculated grass plants at the end of the
experiment. Therefore these treatments were excluded from further analyses. Also, the
ubiquitous EM fungus, Thelephora terrestris (Ehrh.) Fr., was found growing on the
noninoculated pine (pine ) treatments, but not in the inoculated pine (pine+) treatments.
The mean soil solution pH was 4.0.

61
Pine shoot-P concentration increased in all treatments with increasing level of
applied P (Fig. 4-1 A). A higher shoot-P concentration was observed in pine+ compared
to the pine' (P < 0.001). In the interspecific competition treatments where pine was
grown with grass, pine+ had an elevated shoot-P content compared to pine' (Fig. 4-1B).
The difference became more apparent with increasing P level. In the treatments where
pine was grown with pine, pine' and pine+ acquired similar quantities of P at all levels
of applied P. Total dry weight of pine was not affected by the level of applied P (Fig.
4-1C). Overall pine+ had a higher total dry weight than pine' (P = 0.07) and more so
when grown with grass (P < 0.01). Pines grown with other pines had a lower dry
weight than when grown with grass (P < 0.01). Colonization was also higher in the
pine+ treatments inoculated with P. tinctorius than in the pine' treatments that became
colonized with T. terrestris (Table 4-2).
Similar trends were observed in the repeat of the experiment, although differences
were smaller and not always significant. Pine+ grown with grass had a 70% higher
shoot-P level compared to pine' (P < 0.05), but only at the 32.26-/M P level. The total
pine+ biomass was 31% larger than pine' at 32.26-/xM P (P < 0.05), and only when
grown with grass.
For grass shoot-P concentration there was a significant interaction between the
level of applied P and the competition treatment (P < 0.05). At the 32.26-/M P level,
the shoot-P concentration of grass when grown with pine+ was lower than when grown
with pine' (Fig. 4-2A). The grass intraspecific competition treatment at this P level was
higher than both pine treatments. The shoot-P content at the 32.26-/xM P level was also

Total Dry Weight (g) Shoot-P Content (mg P) Shoot-P Cone. (|jg P g'1)
0.32 3.22 32.26
P Applied (pM P), Log Scale
Figure 4-1. Pirns elliottii (A) shoot-phosphorus concentration, (B) shoot-phosphorus
content and (C) total dry weight in response to different competition treatments and
grown at either 0.32, 3.23 or 32.26 /M P for 18 wk. Each symbol represents the mean
of six replicates SE. Inoculated grass was not colonized at the end of the experiment
and therefore was not included in the analysis.

63
P Applied (pM P), Log Scale
Figure 4-2. Panicum chamaelonche (A) shoot-phosphorus concentration, (B) shoot-
phosphorus content and (C) total dry weight in response to different competition
treatments and grown at either 0.32, 3.23 or 32.26 iM P for 18 wk. Each symbol
represents the mean of six replicates + SE. Inoculated grass was not colonized at the end
of the experiment and therefore was not included in the analysis.

64
Table 4-2. Ergosterol concentration (fig g'1) of Pinus elliottii roots inoculated with
Pisolithus tinctorius (pine+) or noninoculated (pine), and grown in combination with
Pinus elliottii (pine) or Panicum chamaelonche (grass) at either 0.32, 3.23 or 32.26 /xM
P for 18 wk. Each value represents the mean of six replicates SE.
Competition
Treatment
Phosphorus added (fiM P)
0.32
3.23
32.26
Pine+ x pine+
181 20
282 34
297 33
Pine' x pine'
129 13
104 11
150 11
Pine+ x grass'
192 8
297 46
260 26
Pine' x grass'
137 21
139 12
140 17

65
lower in the treatments where grass competed with pine (Fig. 4-2B). Total dry weight
of grass was not significantly different at any level of applied P or for any competition
treatment (Fig. 4-2C). In the repeat of the experiment there were no differences in shoot-
P content between the different grass competition treatments, except at the 322.58-/xM
P concentration where grass grown with pine had a 39% higher shoot-P content (P <
0.02) than when grown with another grass. Grass total dry weight at that P concentration
was higher in the interspecific treatment with pine than in the intraspecific treatment with
grass (P < 0.01).
Pine+ had a higher root length than pine' over all treatments (P < 0.001), even
though there were no differences in biomass between inoculated and noninoculated plants
at the beginning of the experiment (Fig. 4-3A). Pine root length did not change with the
level of P applied. In the repeat of the experiment, response of pine root length did not
differ between the competition treatments or between the 0.32- and 3.22-^iM P levels
(data not shown). When grass was grown with grass, there was an increase in grass root
length at the 32.26-/xM P level (Fig. 4-3B) which was paralleled by an increase in shoot-
P content. At the 32.26-/xM P level, grass growing with grass had a higher root length
than grass in the interspecific treatments. When grass was grown with pine+, there was
an increase in grass root length from the 0.32- to the 3.22-^M P level, whereas grass
root length for pine' was not different between P levels. In the repeat of the experiment,
root length increased between the 0.32- and 3.22-/M P levels for grass grown with grass
only (data not shown).

66
0.32 3.22 32.26
P Applied (pM P), Log Scale
Figure 4-3. Root-length density of (A) Pinus elliottii and (B) Panicum chamaelonche in
different competition treatments and grown at 0.32, 3.23 or 32.26 /xM P for 18 wk. Each
symbol represents the mean of six replicates SE. Inoculated grass was not colonized
at the end of the experiment and therefore was not included in the analysis.

3
67
- 2
C
O
O
E
o 1
o
O)
i.
O
O
5 3
2
Q)
0
2 -
0.32 3.22 32.26
P Applied (pM P), Log Scale
Figure 4-4. Relative crowding coefficient (RCC) for total dry weight of (A) Pinus elliottii
inoculated with Pisolithus tinctorius grown in combination with Panicum chamaelonche
and (B) noninoculated Pinus elliottii grown in combination with P. chamaelonche at
either 0.32, 3.23 or 32.26 /uM P for 18 wk. Each symbol represents the mean of six
replicates SE. Mean standard errors were smaller than the symbols and are therefore
not included.

68
Table 4-3. Maximum uptake rate, Imax, (¡xmol P cm'2 s'1) and C^, (jxM P), the minimum
solution concentration from which a nutrient can be absorbed, for Pinus elliottii and
Panicum chamaelonche grown in a hydroponic solution containing 0.32 /M P. Each
value represents the mean of three replicates SE.
Plant species
^max
r
v^min
Pinus elliottii
0.116 0.027
0.080 0.018
Panicum chamaelonche
0.075 0.016
0.028 0.014

69
At each level of P, pine+ had a higher RCC than grass (Fig. 4-4A). In contrast,
pine' was more competitive than grass only above the 0.32-/*M P level (Fig. 4-4B). In
the repeat of the experiment, grass RCC between 32.26 and 322.58 /M P rose by 279
and 144% when competing with pine' and pine+ respectively. At this same P level the
RCC for pine' and pine+ dropped by 75 and 27% respectively.
Determination of P Uptake Kinetics for Pine and Grass
Again, no colonization was observed in the grass+ plants, even though I attempted
to use indigenous fungi, so the treatment was excluded from analysis. On a root surface
area basis, 1^ was not different between pine+ and pine'; however, Imax based on total
surface area, which included mycorrhizal hyphae, was much lower in the pine+.
Consequently only the values for the nonmycorrhizal pine and grass are shown. A higher
Imax value was observed for pine, whereas grass had a lower C^ value (Table 4-3).
Discussion
Inoculation of slash pine with P. tinctorius enhanced P acquisition of pine when
grown with nonmycorrhizal grass. This response is dependent on at least two conditions
of the experimental design, namely soil volume and nutrient availability. In large soil
volumes mycorrhizal fungi are able to enhance plant nutrient uptake by accessing areas
beyond the roots nutrient depletion zone. This mechanism is much less important in
smaller volumes of soil, such as in this experiment, since roots and fungal hyphae
proliferate throughout the pot, essentially making the entire volume a single nutrient

70
depletion zone. Since an acid-washed sand was used, soluble inorganic nutrients were the
only source of nutrients available to both the roots and mycorrhizal fungi. This made the
potential ability to utilize nutrients in different forms inconsequential. As a result,
differences in P acquisition most likely were related to a combination of differences in
absorbing surface area and uptake rates. Although plant density also may affect the
outcome of plant competition (Hartnett et al., 1993; Taylor and Aarssen, 1989) this was
not tested.
Previous researchers comparing uptake by mycorrhizal and nonmycorrhizal plants
have observed a higher uptake rate for mycorrhizal plants (Cress et al., 1979;
Karunaratne et al., 1986; Pacheco and Cambraia, 1992). However, these estimates
generally are reported on a root weight or root length basis only. If the estimates
included hyphal surface area the uptake rates would be greatly reduced for the
mycorrhizal plants. In the current study, pine+ and pine' had similar uptake rates if based
on root surface area alone. The lower uptake rate of pine+ compared to pine', based on
total surface area, strongly suggests that hyphal nutrient depletion zones were
overlapping. If inadequate mixing of the nutrient solution occurs, the rate-limiting step
for uptake would be the replenishment of P absorbed inside the dense mass of hyphae.
The P uptake kinetics of nonmycorrhizal pine and grass determined hydroponically in a
0.32-piM P solution were different, but the variability was relatively high due to the low
number of replications. The higher 1^ demonstrated by pine would permit pine to
sequester more P than grass, which would give the pine a competitive advantage over
grass under the regular fertilization schedule followed here. The lower Cmin for grass

71
would be more advantageous where low nutrient concentrations persist over longer time
periods, such as in the field but not in this greenhouse study where the time between
fertilization was relatively brief. These observations suggest that differences in P uptake
kinetics partially may be responsible for the outcome of competition.
Pine and grass root growth rates vary with P concentration, resulting in different
absorbing surface areas. The poor relationship I observed between root length and shoot-
P content indicates that root length is not the only factor contributing to the pines
competitive interaction with grass. Nonetheless, the increase in root length in the pine+
compared to the pine' treatments suggests that mycorrhizal fungi increased pine root
length and thereby enhanced nutrient uptake. As a result the competitive ability of pine
was increased compared to grass.
The exact nature of the relationship between intensity of competition and resource
abundance is still under debate, but it depends on the environmental conditions and plant
species involved (Di Tommaso and Aarssen, 1991; Grace, 1995). Tilman (1982) stated
that competition increases with decreasing resource availability. An alternate viewpoint
is espoused by Grime (1979) who maintained that competition intensity increases with
increasing habitat fertility. By definition competition is expressed as effects on plant
biomass, survival or reproduction. In this study plants did not demonstrate any dry
weight response to P application, which suggests that P was not the only nutrient limiting
plant growth. At the 0.32-/xM P level plant growth was marginal as a result of inadequate
P in the system. Although dry weight was not altered by the different levels of applied
P, P uptake by both pine and grass was affected. Since nutrient uptake is part of the

72
mechanism leading to differences in plant biomass, it is very likely that this would
influence plant competition. Differences between P status of grass and pine indicate that
P capture by pine reduced the amount of P taken up by grass, specifically at the 32.26-
pM P level. Pine, based on its higher RCC, appeared to be more competitive than grass
when the two competed with each other. When grown with grass the enhanced P uptake
of pine+ corresponded with a larger total dry weight compared to pine', indicating that
inoculation with P. tinctorius did alter the competitive ability of pine.
Although not validated in a repeated experiment, grass at 322.58 M P had a 7.5
or 4.6 times larger RCC than pine+ or pine', respectively. A change in competitive
dominance between the species Rumex acetosella and Poa pratensis with changes in soil
fertility also has been documented (Fowler, 1982). Dual-phasic, P uptake kinetics
dependent on solution-P concentration have been found in fungi (Jennings, 1995), plants
(Barber, 1972) and in mycorrhizal roots (Cress et al., 1979). If a dual-phasic uptake
system exists for each of the plant species, then a higher affinity enzyme system in one
of the species could provide a possible explanation for plant dominance based on uptake
ability.
When pine was grown with pine, competition was equally intense if the plants
were inoculated with P. tinctorius or colonized with T. terrestris. Since the pine' plants
were not uniformly colonized with T. terrestris, I was not able to determine if the EM
inoculation treatments substantially altered the intensity of intraspecific competition of
pine with and without mycorrhizas. However, in an EM competition study by Perry et

73
al. (1989), biomass of plants in intraspecific competition (12 trees pot1) was altered by
different EM fungi, indicating that competition intensity varies with the fungal species.
The somewhat reduced response in the repeat of this experiment may be related
to temperature differences between the two experiments. The first one ended in June and
the second ended in October, which resulted in not only a 2C higher maximum
temperature in the second study, but a longer daily exposure to higher temperatures as
well. The mean colonization of pine+ in the repeat was 46% lower compared to the first
run of the experiment. A decrease of mycorrhizal effectiveness has been observed at
temperatures of 34 to 35C for certain Pisolithus tinctorius isolates (Marx et al., 1970)
grown on pine, as well as for Glomus spp. (Fabig et al., 1989) grown on several grass
hosts. The lack of colonization in the grass plants may be related to the high soil acidity
(pH 4.0) and the lack of buffering capacity of the sand. Activity of Glomus spp. is
optimal above pH 5.3 (Abbott and Robson, 1985; Wang et al., 1985).
The conclusion of this study is that P. tinctorius can increase P acquisition of pine
when grown with grass, which consequently could lead to an increase in competitive
ability. The controlled conditions used in this experiment allow the isolation of specific
variables that affect a plants competitive ability. The actual proportion of a plants total
competitive ability contributed by the mycorrhizal component can only be determined
under field conditions where soil chemical, physical and biological parameters modify
plant interactions and the mycorrhizal response. Yet, our ability to isolate the different
components of a plants competitive ability and determine the relative importance of each

74
component is limited precisely by the interwoven nature of the plant and soil complex.
When this is accomplished, we will be closer to determining the magnitude of
mycorrhizal effects on the ecology or economy of an ecosystem.

CHAPTER 5
CONCLUSION
Simultaneously evaluating arbuscular mycorrhizal (AM) and ectomycorrhizal
(EM) effects on plant competition for nutrients involves discerning the complex
interactions between four different organisms. To do this under controlled experimental
conditions requires an understanding of the growth requirements of each species. One of
the main problems in my studies was the difficulty in obtaining colonization in the AM
grass treatments. This was likely related to the artificial environmental conditions created
for the experiments. The main factors distinguishing the field soil from the acid-washed
sand were the presence of greater buffering capacity and organic matter in the soil, and
differing microbial composition and nutrient regimes. Since Panicum chamaelonche was
highly colonized in the field compared to the greenhouse, it is likely that modification
of one or several of these factors would increase colonization.
Distinguishing the effects of one mycorrhizal type from another can be
accomplished by the use of fungicides. In the research presented here the use of benomyl
as a tool to control the AM fungal component was tested. The following conclusions can
be drawn from this research.
75

76
1. Benomyl can successfully inhibit development of an AM fungus under controlled
conditions in the greenhouse with no side effects on the EM fungus, Pisolithus
tinctorius.
2. Early in the season with low ground cover in the field, benomyl caused a slight
reduction in arbuscule activity. Later as ground cover increased, systemic
translocation of benomyl from shoot to roots of grasses apparently was
insufficient to reduce mycorrhizal colonization, even at high benomyl
concentrations.
3. Soil drench of benomyl would be a more effective method to place the fungicide
directly at the target site, namely the roots.
4. Sufficient water should be used to permit penetration of benomyl into the soil as
ground cover increases.
Previous studies have demonstrated that mycorrhizas can enhance a plants
competitive ability. The role of mycorrhizas in competition between EM and AM plants
and the effects of different P levels have not been explored specifically. The greenhouse
study I conducted to address these interactions yielded the following results.
1. Both the reduction in P acquisition of grass when grown with pine compared to
another grass at the 32.26-piM P level amd the higher relative crowding
coefficient for total dry weight indicate that pine is more competitive than grass
under the conditions tested.

77
2. Inoculation of slash pine with P. tinctorius enhanced both P uptake and total dry
weight and hence the competitive ability of pine when competing with
nonmycorrhizal grass.
3. When grown in intraspecific competition, no difference was observed in the
competitive ability of pine colonized with P. tinctorius or Thelephora terrestris.
4. The different P levels added did not affect grass or pine biomass which suggests
that P was not the only limiting factor to growth.
5. A higher I,,^ value for pine and the lower Cmjn for grass suggest that differing P
uptake kinetics can contribute to competitive interactions.
Several additional factors would have to be elucidated to draw conclusions from
these results about pine and grass interactions in the field. In a separate field competition
study involving slash pine and weeds (primarily grasses) pine growth was substantially
decreased in contrast to the greenhouse where pine exhibited a higher competitive ability
than grass. In the Spodosol at the field site, organic forms of P are the major source of
P, which is released during periodic pulses of nutrient cycling triggered by increases in
soil moisture. This contrasts with the inorganic P used in the greenhouse study, which
was applied at frequent and regular intervals and thus maintained a relatively consistent
P concentration in the system. Also, the buffering capacity of the field soil was absent
in the greenhouse, and this would modify plant-soil-microbe interactions by altering
nutrient availability and flux. As a consequence differing pine and grass P uptake kinetics
expressed in the greenhouse would not necessarily provide the same competitive

78
advantages in the field. The use of a field soil in subsequent studies would incorporate,
at least in part, these effects.
In the hydroponic study on P-uptake an attempt was made to measure the effects
of mycorrhizas on P uptake kinetics. The results involving mycorrhizas were inconclusive
since mycorrhizal plants with a higher total absorbing surface area demonstrated a lower
Imax value than the nonmycorrhizal plants. Based on visual observations this is due to
extensive hyphal development in the mycorrhizal treatments most likely resulting in
overlapping depletion zones of roots and hyphae. Allowing hyphae to regrow for a period
of 2 wk instead of 4 wk prior to P uptake measurement probably would have avoided the
problem. Although not quantified in the hydroponic study, mycorrhizal fungi may have
different Imax and C,^ values from the host plant. If the fungus has a higher I^ or a
lower than the host plant, as well as the competing plant, this would confer a
competitive advantage to the host plant.
In the field, root-length density measured down to a depth of 87 cm was several
fold higher for grass than for pine. The 500-mL soil volume used in the greenhouse
experiment created a root-bound condition which did not fully permit this difference to
be expressed. Much of the contribution of mycorrhizas is due to their accessing nutrients
beyond the roots nutrient depletion zone; however, since hyphae and roots were able to
access nutrients in most of the soil volume in pots, this probably did not contribute a
competitive advantage in my study. The spatial advantage mycorrhizal hyphae would
provide to a host plant by their presence directly at the site of nutrient mineralization,
such as in and around organic matter, also was not expressed. Although external

79
mycorrhizal hyphae contribute to nutrient uptake and thus plant competitive ability in the
field due to their spatial distribution, part of this component could be reduced by
hyperparasitism by parasitic fungi and the effects of fungal feeding by Arthropods.
Incorporation of a larger nonsterilized, soil volume in future competition studies would
allow more components of a plants competitive ability to function.
The simplification of the soil environment achieved by using an acid-washed sand
allowed the isolation of specific mycorrhizal effects that influence plant competition.
Ideally, the next step in this process would be to address the competition between
mycorrhizal plants in field soil or directly in the field while acknowledging the
limitations imposed by the complexity and heterogeneity of field soil conditions.

APPENDIX 1
GROWTH CHAMBER COMPETITION STUDY BETWEEN PINUS ELLIOTTII
AND PANICUM CHAMAELONCHE
Introduction
A competition study involving pine (Pinus elliottii Engelm. var. elliottii)) and
grass {Panicum chamaelonche Trin.) was set up to determine: (i) the contribution of
mycorrhizal fungal hyphae to total plant P uptake and (ii) the competitive abilities of
arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) fungi with respect to each other.
This experiment was conducted twice. No colonization was obtained in the inoculated
grass treatments the first time this experiment was run, and therefore no competition
resulted between plants.
Materials and Methods
The study was initiated as part of a larger field investigation into competition for
nutrients between pine and grass at a field site 21 km northwest of Gainesville, Florida.
Pine and grass were grown in acid-washed sand and inoculated as in the Material and
Methods of Chapter 4. Grasses were inoculated 14 wk prior to the start of the experiment
by applying to each plant root system a minimum of 20 spores of a mixed culture of
Gigaspora rosea (INVAM FL224) and Scutellospora heterogama (INVAM FL225) as in
the Materials and Methods section of Chapter 4. At the start of the experiment, grass had
a mean root colonization of 13%. Pine roots were visually inspected to prevent inclusion
80

81
of noninoculated pine colonized by Thelephora terrestris. At the start of the experiment
plant roots were washed free of all sand and divided into three size classes. Different
combinations of plant species, inoculated or noninoculated, were made by selecting pairs
of plants from the same size class to create the competition treatments listed in Table 4-1
of Chapter 4. There was a minimum of six replications per treatment.
Growth boxes were constructed with two plant compartments (416 g of dry sand
each) on opposite sides of a hyphal compartment (225 g of dry sand). The plant
compartments were separated from the hyphal compartment by root-excluding nylon
screens (Tetko, Inc., Depew, N.Y.) with a mesh size of either 15 /m for the grass or
40 fj.m for the pine. The internal dimensions of each plant compartment were 4 x 9 x
11.5 cm (width x length x depth) and 2.5 x 9 x 11.5 cm for the hyphal compartment.
Plant fresh weights were measured at the start of the experiment. Eight pine and grass
plants, inoculated or noninoculated, were used to determine plant water content and
initial P status. After planting, water was added to reach 10% of the soil gravimetric
water content and the boxes were then weighed. Deionized water was added to maintain
this weight during the experiment. Plant compartments were fertilized separately from
the hyphal compartments. In weeks 1, 2, 3 and 6, plant compartments were fertilized
three times weekly with 1.4 /moles P as NaH2P04 along with 10 ml of nutrient solution
containing 2.8 mM NH4N03, 2.8 mM Ca(N03)2, 2.6 mM KC1, 3.4 mM MgS04, 230 /M
NaFeEDTA, 3.2 /M CuS04, 221 /M H3B03, 510 /M NaMo04, 35.1 /M MnCl and
15.9 /M ZnS04. During weeks 4 and 5 they received 15.1 /moles P at each fertilization.
Starting in the fourth week, hyphal compartments received 8.4 /moles P. To prevent

82
massflow of nutrients from the hyphal to the plant compartment, water used to bring the
boxes to their original weight was added only to the plant compartment. Plants were
grown in a growth chamber with mean temperatures of 23/29C (dark/light cycle,
respectively) and a mean photosynthetic photon flux density of 1000 mol m'2 s'1 at plant
height. Plant shading was minimized by the distance between plants. Boxes were
randomized each time plants were watered in the growth chamber.
Plants were harvested after 62 d. The surface sand layer containing a crusted algal
mat approximately 5-mm thick was removed and treated separately. Sand was removed
from roots first by shaking and then by rinsing the roots with water. All root pieces were
collected. A sample of subsurface sand was retained for further analysis. Plant tissue-P
status, colonization and root and hyphal surface areas were measured as in the Materials
and Methods of Chapter 4. Root and hyphal surface areas were measured separately for
the plant and hyphal compartments. For absorbing surface area calculations involving
intraspecifically competing mycorrhizal plants, half of the total hyphal surface area from
the hyphal compartment was added to the surface area of the plant on each side. Plant
P-uptake rate was calculated as the change in plant P content during the time of the
experiment based on combined root and hyphal surface area. The mean total hyphal
density for each compartment and treatment was calculated.
Sand for P analysis was thoroughly mixed prior to analysis. A 4.5- to 5.5-g
sample in 20-ml, borosilicate vials was treated overnight with 5 ml of concentrated HC1.
This was evaporated and 10 ml of 0.1 N HC1 were added. Phosphorus was analyzed after
24 h following the procedure of Murphy and Riley (1960). Surface and subsurface soil

83
samples from the plant and hyphal compartments were treated separately by the same
procedure.
Data for grass and pine were analyzed separately. To determine if plant
competition was affected by the plant species, data for each plant species were tested by
single degree of freedom contrasts using the General Linear Model procedure of SAS
(SAS Institute, Inc., 1989). For the contrasts, target plants were analyzed based on
mycorrhizal status, type of competition (intraspecific /interspecific) or mycorrhizal status
of the competing neighbor plant. Data for colonization were arcsine, square root-
transformed prior to analysis (Steel and Torrie, 1980). The least-squares means statement
within SAS was used to compare means.
Results
Pine+ exhibited greater plant biomass compared to the pine' treatments (Fig. Al-
1A, Table Al-1). There was no difference between intraspecific and interspecific
competition in the pine' treatments irrespective of the mycorrhizal status of grass. No
difference was observed for grass response to intra- or interspecific competition either
inoculated or noninoculated (Fig. A1-1B).
Similar to pine biomass, pine+ had a higher plant-P content than pine' (Fig. Al-
2A, Table Al-1). Grass+ had a lower P content than grass' when competing with both
pine+ and pine' (Fig. A1-2B). No such difference was found in the intraspecific
treatments where grass competed with grass.

Table Al-1. Tests for single degree of freedom contrasts for root-length density, plant biomass, plant
P content and percent colonization. Each parameter was analyzed separately for grass and pine.
Root-Length Density
(m cm"3 of soil)
Plant
Biomass
(g)
Plant P
Content
(mg P g1)
Colonization
(%)
Uptake Rate
(fmol P cm'2 s'1)
Contrasts for grass:
Grass+ / grass'
.06
n.s.
.02
.001
.05
(Grass+ / grass') over all pine
.06
n.s.
.03
.02
n.s.
(Pine+ / pine ) over all grass
.04
n.s.
n.s.
n.s.
n.s.
Intra- / interspecific
n.s.
n.s.
n.s.
.05
n.s.
Contrasts for pine:
Pine+ / pine'
<.001
.001
.01
.006
.03
(Pine+ / pine ) over all grass
<.001
<.001
.06
.01
n.s.
(Grass+ / grass') over all pine
n.s.
n.s.
n.s.
n.s.
n.s.
Intra- / interspecific
n.s.
n.s.
.07
n.s.
n.s.
oo
4^

Table A1-2. Mean hyphal length density (m cm'3 of soil) for each competition treatment presented
separately for each compartment (one hyphal and two plant compartments). Hyphal length is made up
of the sum of both AM and EM hyphae. Each value represents the mean of a minimum of six replicates
SE.
Compartment
A B
Plant Compartment A
Hyphal Compartment
Plant Compartment B
Pine+
Pine+
131.26 13.58
113.12 15.87
131.26 15.58
Pine'
Pine'
94.82 8.69
80.73 12.36
94.82 + 8.69
Pine+
Grass+
82.40 10.55
63.69 8.01
32.48 6.39
Pine+
Grass
100.43 7.76
64.32 6.44
21.16 3.69
Pine'
Grass+
75.02 14.88
48.13 11.61
14.47 4.20
Pine'
Grass
162.27 28.67
65.40 7.71
22.40 6.04
Grass+
Grass+
7.42 1.08
3.75 0.64
7.42 1.08
Grass'
Grass'
6.73 0.82
2.38 0.31
6.73 0.82
oo

Plant Biomass (g)
86
Figure Al-1. Plant biomass for (A) slash pine and (B) grass grown in the growth
chamber for 62 d. Each bar represents the mean of a minimum of six replicates SE.

87
5000
4000
^ 3000
'o>
q_ 2000
D)
1000
+>
c
0)
+ o
§ 4000
^ 3500
Q.
g 3000
-S 2500
Q.
2000
1500
1000
500
0
Figure Al-2. Plant-P content for (A) slash pine and (B) grass grown in the growth
chamber for 62 d. Each bar represents the mean of a minimum of six replicates SE.

88
O
o
O)
O)
£
o
L_
*->
(O
O
O)
k_
LU
C
O
+3
03
N
'E
o
o
o
-*->
o
o
£
V)
(0
ro
L_
O
800
700
600
500
400
300
200
100
0
8
7
6
5
4
3
2
1
0
B
* #
i*
4*
+ + X + X *f
^ ^
* o'*'
r ,& <* + +
<>
^ AV oN O
Figure Al-3. Mycorrhizal colonization of (A) slash pine and (B) grass grown in the
growth chamber for 62 d. Each bar represents the mean of a minimum of six replicates
SE.

89
Pine' was colonized by Thelephora terrestris and had a higher level of colonization
than pine+ over all competition treatments (Fig. A1-3A, Table Al-1). In the intraspecific
pine treatments, more external hyphae were present in the pine+ than in pine' treatment
(Table Al-2). Grasses were colonized in the grass+ but not in the grass' treatments (Fig.
A1-3B). Lower colonization of grass+ was observed when competing with pine than
when competing with another grass. Hyphal length density was higher overall in the pine
than in grass. Hyphae contributed between 10 to 16% and 68 to 83% of the total
absorbing surface area for grass and pine respectively (data not shown). When total
hyphal length was separated by fungal type, AM hyphal length in the plant compartments
containing grass was 3.59 0.51 and 3.82 0.68 m cm'3 of soil in the pine+ and pine'
treatments, respectively, compared to 7.35 1.09 m cm'3 of soil with grass alone. At
the lowest hyphal density measured in a compartment, the theoretical inter-hyphal
distance was equal to 23.2 on [calculated using the formula: 2/(Lvtt)0 5 from Baldwin and
Nye (1974); hyphal length density (LJ= 238 cm of hyphae cm'3 of soil]. The theoretical
two-dimensional depletion zone would be approximately 83.1 un [determined with the
formula: 2(Dt)0 5 (Baldwin and Nye, 1974); assuming a diffusion coefficient (D) = lxl0'8
cm2 s'1 and time (t)=2 d].
Root-length density of pine+ was greater over all competition treatments than that
of pine', independent of intra- or interspecific competition (Fig A1-4A). Grass* was
significantly different from grass' (P < 0.06) over all treatments (Table Al-1). The
major source of this difference was the higher root-length density of grass' when
competing with pine+ compared to the other competition treatments (Fig. A1-4B).

Table Al-3. Mean soil P content (/xg P g'1 of soil) for each competition treatment presented separately for each compartment (one
hyphal and two plant compartments). Values represent the mean of a minimum of six replicates SE.
Compartment
A B
Plant Compartment A
Hyphal Compartment
Plant Compartment B
Pine+
Pine+
1758 99
2543 224
1758 99
Pine'
Pine'
2210 122
2971 61
2210 122
Pine+
Grass+
2009 68
2321 155
1865 + 44
Pine+
Grass'
2091 238
2622 204
1722 203
Pine'
Grass+
2172 228
3233 201
2077 249
Pine"
Grass'
2426 174
2529 194
1580 169
Grass+
Grass+
2142 261
2732 255
2142 261
Grass'
Grass'
1827 150
2477 223
1827 150
s

Root-Length Density (m cm'3 of soil)
0.15
Figure Al-4. Root-length density for (A) slash pine and (B) grass grown in the growth
chamber for 62 d. Each bar represents the mean of a minimum of six replicates SE.

92
Figure Al-5. Uptake rate of (A) slash pine and (B) grass grown in the growth chamber
for 62 d. Uptake rate was calculated on a unit surface area basis which included both
root and hyphal surface area. Each bar represents the mean of a minimum of six
replicates SE.

94
uptake on a unit surface area basis, which was expressed by the uptake rate calculations.
Pine uptake rate was substantially lower then grass which was due to the much higher
pine absorbing surface area attributable to external hyphae of mycorrhizal fungi. Since
the observed differences are not completely explained by this, it is likely that not all
hyphal surface area was active in uptake.
Even though AM fungi of grass, had access to the additional P in the hyphal
compartment, grass+ growth was not stimulated. In fact, grass+ had a lower P content
than grass' when competing with pine, inoculated or not. Colonization of grass+ and AM
hyphal lengths were lower when competing with pine. It is not clear why this reduction
in AM growth occurred; however, it is possible that EM fungi may have exhibited some
form of antibiosis. Due to the distance between plants it is less likely that the effect was
induced by pine.
A total P budget based on soil and plant P content was not calculated since the
pine" plants became colonized and the AM fungi did not contribute to increased P uptake
from the hyphal compartment.
In conclusion, inoculation of pine with P. tinctorius enhanced pine P uptake over
the pine' treatment, which was colonized by T. terrestris. Competition between pine and
grass could not be addressed adequately due to the lack of AM contribution to grass
uptake.

93
Uptake rates for both pine+ and grass+, based on the combined surface area of
roots and hyphae, were lower than for the respective noninoculated treatments (Fig. Al-
5, Table Al-1). Uptake rate based on root surface area alone increased the difference
(data not shown). There were no differences in uptake rates for either pine or grass
between their respective intra- and interspecific competition treatments.
Soil-P content for grass+ was higher than grass', whereas pine+ had slightly less
P in the soil than pine' (Table Al-3).
Discussion
The higher plant biomass and P content of pine+, compared to pine', were
associated with increased root-length density in the pine+ treatments. The mycorrhizal
fungus apparently contributed to increased growth of pine+ which is supported by the
lower soil-P content in the pine+ compartments. Although pine' was found to have higher
levels of colonization than pine+, the EM fungus, P. tinctorius, was more effective than
T. terrestris at increasing plant growth. It was not possible to determine the additional
quantity of P contributed by mycorrhizal fungi since the nonmycorrhizal control was lost
when it was colonied by T. terrestris. The lower uptake rate in pine+ plants with a higher
surface area may have occurred for two reasons: (i) P depletion zones overlapped due
to a high density of absorbing surface area or (ii) not all of the surface area used in the
calculation was involved in nutrient absorption.
Sample calculations with hyphae alone demonstrate that hyphal depletion zones
overlapped even at the lowest hyphal density measured. This would result in less P

APPENDIX 2
COLONIZATION OF PANICUM CHAMAELONCHE AND CORN BY DIFFERENT
ARBUSCULAR MYCORRHIZAL FUNGI
The objective of this experiment was to determine the ability of different
arbuscular mycorrhizal (AM) fungi to form mycorrhizas with either corn (Zea mays L.
cv. Silver Queen) or Panicum chamaelonche Trin. under the conditions in the
greenhouse.
Panicum chamaelonche plants were obtained from cultures maintained in sand in
the greenhouse. Plants were started from seed collected from the field and vegetatively
propagated in 150-ml pots (7 cm2 of surface area). Corn plants were grown in 150-ml
pots for 2 wk prior to inoculation. At the start of the experiment plant roots were washed
free of sand. Four plants of each species were inoculated with either a minimum of 0.5
g of onion root fragments colonized by Acaulospora scrobiculata (S315), Gigaspora
margarita (INVAM FL215), Glomus etunicatum (INVAM FL312), or Glomus sp.
(INVAM FL329, formerly FL906), or a minimum of 20 spores of Gigaspora rosea
(INVAM FL224) or Scutellospora heterogama (INVAM FL225). The latter two had been
isolated from pot cultures of P. chamaelonche originating from the field and grown in
field soil in the greenhouse. There were 4 replicates for each treatment. Plants had the
same environmental conditions as in the Materials and Methods of Chapter 4 and were
fertilized with the same nutrient solution at the 32.26 iM NaH2P04 level. After 9 wk,
plants were harvested and colonization was determined using the same technique used in
95

96
the Materials and Methods of Chapter 4. This experiment was not repeated. Colonization
of corn by AM fungi was lower than that for P. chamaelonche, possibly due to the way
the two plant species were started. Both plant species in the treatments with two
Gigaspora spp. and A. scrobiculata were more highly colonized than plants in the other
treatments, suggesting that these AM fungi may be better suited to the particular
combination of soil and greenhouse environment.

97
Figure A2-1. Colonization of (A) Panicum chamaelonche and (B) Zea mays inoculated
with root fragments of (1) Acaulospora scrobiculata (S315), (2) Gigaspora margarita
(INVAM FL215), (3) Glomus etunicatum (INVAM FL312) or (4) Glomus sp. (INVAM
FL329, formerly FL906) or spores of (5) Gigaspora rosea (INVAM FL224) or (6)
Scutellospora heterogama (INVAM FL225). Each bar represents the mean of four
replicates SE.

APPENDIX 3
PHOSPHORUS GROWTH RESPONSE CURVE FOR NONMYCORRHIZAL
PANICUM CHAMAELONCHE
The objective of the following experiment was to determine the growth response
curve of Panicum chamaelonche Trin. to phosphorus (P) and to find three appropriate
levels of P to apply in the greenhouse competition experiment in Chapter 4. This
experiment was only performed once.
Grass plants were started from seed and grown in the greenhouse in 150-ml pots
(7 cm2 of surface area) in sand which had been acid-washed as in the Materials and
Methods of Chapter 4. Plants were fertilized semiweekly with nutrient solution containing
660 /xM NH4N03, 660 pM (NH4)2S04, 3.23 xM NaH2P04, 616 pM KC1, 80 pm MgS04,
54 pM NaFeEDTA, 600 pM CaCl2, 0.25 xM CuS04, 14 xM H3B03, 40 pM NaMo04,
2.75 juM MnCl and 1.25 pM ZnS04. After 5 wk, plants were transplanted into 500 g of
sand in Deepots. Fifty 50 ml of the above nutrient solution containing either 0.001,
0.003, 0.010, 0.032, 0.100, 0.316 1.000, 3.162 or 10.000 mg P/L from NaH2P04 was
added to each pot biweekly. Pots were leached once per week with deionized water to
remove any excess salts and P. The environmental conditions were the same as in the
Materials and Methods section of Chapter 4. Plants were harvested after 13 wk of
growth. Plants were dried at 65C and plant biomass and total P were determined using
the same methods as in the Materials and Methods section of Chapter 4. Plant response
98

99
is shown in Fig. A2-1A and B. The plant growth response at 0.1, 1.0 and 10 mg P L'1
would represent plant growth at low, medium and high nutrient regimes.

Plant Biomass (g) Plant-P Content (mg P)
100
Figure A3-1. Nonmycorrhizal Panicum chamaelonche (A) plant biomass and (B) plant
phosphorus content in response to 0.001, 0.003 0.010, 0.032, 0.100, 0.316, 0.1000,
3.162 or 10.000 mg P L1. Each symbol represents the mean of seven replicates SE.

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BIOGRAPHICAL SKETCH
I was born on April 29, 1958 in Queens, New York. I grew up bilingually
learning both Swiss-German as well as English. At the age of 13 my family moved to
Switzerland, where I attended the public school system. I completed a four-year degree
from the College of Business Administration and Economics in Zurich. In 1980, I
returned to the United States where I completed a B.S. in horticulture from The
Pennsylvania State University in 1983 specializing in vegetable and fruit production.
After various jobs from agricultural consulting to landscaping, I took on a position as
assistant winemaker at Bucks Country Vineyards in Pennsylvania. Surrounded by
fermenting musts and grape diseases I took an interest in microbiology. Not to forsake
plants in the process, I decided to return for graduate studies in soil microbiology.
Specifically, my work dealt with tissue-cultured asparagus and mycorrhizas. I completed
my M.S. in the Department of Botany and Plant Pathology at the Michigan State
University in 1990. From there, to round out my background, I continued towards a
Ph.D. in the Soil and Water Science Department at the University of Florida.
122

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Jj Cr-ei
David M. Sylvia, Chair
Professor of Soil and Water Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of
Professor of Soil and Water Science
Philosophy.
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
H. Graham
essor of Soil and Water Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
David J. Miichell
Professor of Plant Pathology
I certify that I have read this study and that in 'my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, iAjscopq and qualit
as a dissertation for the degree of Doctor of PbitoSopITyT
Donn G. Shilling
Professor of Agronomy

This dissertation was submitted to the Graduate Faculty of the College of
Agriculture and to the Graduate School and was accepted as partial fulfillment of the
requirements for the degree of Doctor of Philosophy.
December 1995
Dean, College of Agriculture
Dean, Graduate School



APPENDIX 3
PHOSPHORUS GROWTH RESPONSE CURVE FOR NONMYCORRHIZAL
PANICUM CHAMAELONCHE
The objective of the following experiment was to determine the growth response
curve of Panicum chamaelonche Trin. to phosphorus (P) and to find three appropriate
levels of P to apply in the greenhouse competition experiment in Chapter 4. This
experiment was only performed once.
Grass plants were started from seed and grown in the greenhouse in 150-ml pots
(7 cm2 of surface area) in sand which had been acid-washed as in the Materials and
Methods of Chapter 4. Plants were fertilized semiweekly with nutrient solution containing
660 /xM NH4N03, 660 pM (NH4)2S04, 3.23 xM NaH2P04, 616 pM KC1, 80 pm MgS04,
54 pM NaFeEDTA, 600 pM CaCl2, 0.25 xM CuS04, 14 xM H3B03, 40 pM NaMo04,
2.75 juM MnCl and 1.25 pM ZnS04. After 5 wk, plants were transplanted into 500 g of
sand in Deepots. Fifty 50 ml of the above nutrient solution containing either 0.001,
0.003, 0.010, 0.032, 0.100, 0.316 1.000, 3.162 or 10.000 mg P/L from NaH2P04 was
added to each pot biweekly. Pots were leached once per week with deionized water to
remove any excess salts and P. The environmental conditions were the same as in the
Materials and Methods section of Chapter 4. Plants were harvested after 13 wk of
growth. Plants were dried at 65C and plant biomass and total P were determined using
the same methods as in the Materials and Methods section of Chapter 4. Plant response
98


120
Tobar R., R. Azcn, and J.M. Barea. 1994. Improved nitrogen uptake and transport
from 15N-labelled nitrate by external hyphae of arbuscular mycorrhiza under
water-stressed conditions. New Phytol. 126:119-122.
Trappe J.M., R. Molina, and M.A. Castellano. 1984. Reactions of mycorrhizal fungi and
mycorrhizal formation to pesticides. Annu. Rev. Phytopathol. 22:331-359.
Turnau K., I. Kottke, J. Dexheimer, and B. Botton. 1994. Element distribution in
mycelium of Pisolithus arrhizus treated with cadmium dust. Ann. Bot. 74:137-142.
Uren N.C. 1993. Mucilage secretion and its interaction with soil, and contact reduction.
Plant Soil 156:79-82.
Van Auken O.W., J.K. Bush, and D.D. Diamond. 1994. Changes in growth of two C4
grasses (Schizachyrium scoparium and Paspalum plicatulum) in monoculture and mixture:
influcence of soil depth. Am. J. Bot. 81:15-20.
Van der Putten W.H., C. Van Dijk, and B.A.M. Peters. 1993. Plant-specific soil-borne
diseases contribute to succession in foredune vegetation. Nature 362:53-55.
Wacker T.L. and G.E. Safir. 1990. Effects of ferulic acid on Glomus fasciculatum and
associated effects on phosphorus uptake and growth of asparagus (Asparagus officinalis
L.). J. Chem. Ecol. 16:901-909.
Wacker T.L., G.R. Safir, and C.T. Stephens. 1990. Evidence for succession of
mycorrhizal fungi in Michigan asparagus fields. Acta Hort. 271:273-279.
Wang, G.M., D.P. Stribley, and P.B. Tinker. 1985. Soil pH and vesicular-arbuscular
mycorrhizas. p. 219-224. In A.H. Fitter (ed.) Ecological interactions in soil: Plants,
microbes and animals. Blackwell Scientific Publications, Oxford, United Kingdom.
Wardle D.A., K.S. Nicholson, M. Ahmed, and A. Rahman. 1994. Interference effects
of the invasive plant Carduus nutans L. against the nitrogen fixation ability of Trifolium
repens L. Plant Soil 163:287-297.
Ware, G. 1992. Reviews of Environmental Contamination and Toxicology. Springer
Verlag, New York, N.Y.
Waters J.R. and V.A. Borowicz. 1994. Effect of clipping, benomyl, and genet on 14C
transfer between mycorrhizal plants. Oikos 71:246-252.
Watteau F. and J. Berthelin. 1995. Microbial dissolution of iron and aluminium from soil
minerals: efficiency and specificity of hydroxamate siderophores compared to aliphatic
acids. Eur. J. Soil Biol. 30:1-9.


79
mycorrhizal hyphae contribute to nutrient uptake and thus plant competitive ability in the
field due to their spatial distribution, part of this component could be reduced by
hyperparasitism by parasitic fungi and the effects of fungal feeding by Arthropods.
Incorporation of a larger nonsterilized, soil volume in future competition studies would
allow more components of a plants competitive ability to function.
The simplification of the soil environment achieved by using an acid-washed sand
allowed the isolation of specific mycorrhizal effects that influence plant competition.
Ideally, the next step in this process would be to address the competition between
mycorrhizal plants in field soil or directly in the field while acknowledging the
limitations imposed by the complexity and heterogeneity of field soil conditions.


50
of AM response to benomyl in the field during most of the growing season may be
attributed to the increased interception of the fungicide by ground cover. Although
benomyl can enter through leaves, systemic translocation is not as efficient generally as
direct application to the target site (Hassall, 1990), in this case, the roots.
Larsen et al. (1994) determined that benomyl applied directly to the leaves of
cucumber had little effect on mycorrhizal efficiency, yet when benomyl was applied to
the soil, complete inhibition of P uptake by hyphae occurred within 5 d. Although no
fungicide effect on fungal alkaline phosphatase activity was found inside the root, the
rapid response, nonetheless, suggests some direct influence on uptake or transport
mechanisms. Kough et al. (1987) and Thingstrup and Rosendahl (1994) have observed
suppressive effects of benomyl on internal fungal enzyme activity in mycorrhizal plants.
Although benomyl demonstrated no significant effect on external hyphal length or
viability in this study, an inhibitory response has been found in another system (Sukarno
et al., 1993).
Benomyl can be an effective tool for inhibiting AM activity in the field; however,
researchers need to be aware of the limitations of this approach. The timing of root
colonization and initial nutrient contribution to mycorrhizal dependent seedlings can be
critical to their survival (Hartnett et al., 1994; Hetrick et al., 1989; Plenchette and
Perrin, 1992). Fungicide applications in the field should be timed according to the plants
optimal benefit from mycorrhizas, which, correspondingly, would provide the full impact
of the fungicide treatment on mycorrhizal functioning (Gange et al., 1993; Newsham et
al., 1995). Furthermore, the frequency of application is determined by fungicide


99
is shown in Fig. A2-1A and B. The plant growth response at 0.1, 1.0 and 10 mg P L'1
would represent plant growth at low, medium and high nutrient regimes.


22
uptake is primarily related to spatial niche differences between roots and hyphae, then
larger soil volumes would be preferable in experiments; otherwise root nutrient depletion
zones quickly overlap and the potential mycorrhizal benefit is not realized (OKeefe and
Sylvia, 1991). As an intermediate approach between pot and field competition studies,
artificial micro- or mesocosms have been used (Campbell et al., 1991; Grime et al.,
1987), which, among other things, allow for the exploration of large soil volumes by
external hyphae, the creation of resource gradients or patches and the longer-term
monitoring of plant growth and reproduction in a regulated environment. Further
consideration should be given to the incorporation of an unsterilized soil control into
experiments. The inclusion of plant pathogens, soil arthropods and microbes which affect
resource abundance and mycorrhizal plant growth (Newsham et al., 1994), as well as
subsequent plant interactions, would provide a more realistic extrapolation of
experimental results to natural phenomena.
A number of mycorrhizal plant competition studies have demonstrated that AM
fungi affect competition to varying degrees (Brown et al., 1992; Francis and Read, 1994;
Fitter, 1977; Hetrick et al., 1989; Hartnett et al., 1993; Newman et al., 1992). Plant
competition between two host plants involving a single species of AM fungus account for
the majority of the data. Apparently only one plant competition study dealt with different
groups or species of mycorrhizal fungi and it is also the only EM plant competition study
(Perry et al., 1989). There is evidence that competitive success is related to mycorrhizal
dependency (Hartnett etal., 1993; Hetrick et al., 1989). Mycorrhizal dependency is very
variable and depends on the particular environment and host plant. Hartnett et al. (1993)


CHAPTER 5
CONCLUSION
Simultaneously evaluating arbuscular mycorrhizal (AM) and ectomycorrhizal
(EM) effects on plant competition for nutrients involves discerning the complex
interactions between four different organisms. To do this under controlled experimental
conditions requires an understanding of the growth requirements of each species. One of
the main problems in my studies was the difficulty in obtaining colonization in the AM
grass treatments. This was likely related to the artificial environmental conditions created
for the experiments. The main factors distinguishing the field soil from the acid-washed
sand were the presence of greater buffering capacity and organic matter in the soil, and
differing microbial composition and nutrient regimes. Since Panicum chamaelonche was
highly colonized in the field compared to the greenhouse, it is likely that modification
of one or several of these factors would increase colonization.
Distinguishing the effects of one mycorrhizal type from another can be
accomplished by the use of fungicides. In the research presented here the use of benomyl
as a tool to control the AM fungal component was tested. The following conclusions can
be drawn from this research.
75


REFERENCE LIST
Abbott L.K. and A.D. Robson. 1985. The effect of soil pH on the formation of VA
mycorrhizas by two species of Glomus. Aust. J. Soil Res. 23:253-261.
Abuzinadah R.A. and D.J. Read. 1986a. The role of proteins in the nitrogen nutrition
of ectomycorrhizal plants. I. Utilization of peptides and proteins by ectomycorrhizal
fungi. New Phytol. 103:481-493.
Abuzinadah R.A. and D.J. Read. 1986b. The role of proteins in the nitrogen nutrition
of ectomycorrhizal plants. III. Protein utilization by Betula, Picea, and Pinus in
mycorrhizal association with Hebeloma crustuliniforme. New Phytol. 103:506-514.
Abuzinadah R.A. and D.J. Read. 1989. The role of proteins in the nitrogen nutrition of
ectomycorrhizal plants V. Nitrogen transfer in birch (Betula pndula) grown in
association with mycorrhizal and non-mycorrhizal fungi. New Phytol. 112:61-68.
Allen E.B. and M.F. Allen. 1984. Competition between plants of different successional
stages: mycorrhizae as regulators. Can. J. Bot. 62:2625-2629.
Allen M.F. 1991. The Ecology of Mycorrhizae. Cambridge University Press, New York,
N.Y.
Allen M.F., E.B. Allen, and C.F. Friese. 1989. Responses of the non-mycotrophic plant
Salsola kali to invasion by vesicular-arbuscular mycorrhizal fungi. New Phytol.
111:45-49.
Amaranthus M.P. and D.A. Perry. 1994. The functioning of ectomycorrhizal fungi in
the field: Linkages in space and time. Plant Soil 159:133-140.
Ames R.N., C.P.P. Reid, L.K. Porter, and C. Cambardella. 1983. Hyphal uptake and
transport of nitrogen from two 15N-labeled sources by Glomus mosseae, a
vesicular-arbuscular mycorrhizal fungus. New Phytol. 95:381-396.
Antibus R.K., R.L. Sinsabaugh, and A.E. Linkins. 1992. Phosphatase activities and
phosphorus uptake from inositol phosphate by ectomycorrhizal fungi. Can. J. Bot.
70:794-801.
101


55
Pine and grass plants were sorted separately into three size classes at the start of
the experiment. Noninoculated pine had no visual indication of colonization, whereas
inoculated pine was heavily colonized. Inoculated grasses had a mean root colonization
of 30% at the start of the experiment. There were no significant differences in biomass
between inoculated and noninoculated plants at the beginning of the experiment.
Intraspecific and interspecific paired combinations of plant species (Table 4-1), inoculated
or not, were made by selecting plants from the same size class. Plants were planted
together in 500 ml of sand. There were six replications per treatment. Plants were grown
in the greenhouse with mean temperatures of 21/34C (min./max) and a mean
photosynthetic photon flux density of 1240 jumol m2 s'1 from January to June 1994. A
repeat of the experiment was run from May to October 1994 with seven replications. The
greenhouse temperature regime was 24/36 (min./max.) with a mean photosynthetic
photon flux density of 1490 /mol m'2 s'1. The plants were fertilized semiweekly with a
solution containing: 660 /xM NH4N03, 660 juM (NH4)2S04, 616 xM KC1, 80 /xm MgS04,
54 pM NaFeEDTA, 600 ¡xM CaCl2, 0.25 xM CuS04, 14 /xM H3B03, 40 xM NaMo04,
2.75 /xM MnCl and 1.25 /xM ZnS04. Phosphorus was supplied at either 0.32, 3.23 or
32.26 xM H3P04. In the repeat of this experiment the 0.32 xM H3P04 treatment was
replaced with 323.58 /xM H3P04, since plant growth was very slow at the lowest P
concentration. Soil solution pH was measured from several pots during the experiment
by thoroughly watering pots with deionized water and collecting the leachate.
Plants were removed from the pots after 129 d, and the roots of individual plants
were separated carefully from each other. The exception was the intraspecific grass


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Jj Cr-ei
David M. Sylvia, Chair
Professor of Soil and Water Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of
Professor of Soil and Water Science
Philosophy.
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
H. Graham
essor of Soil and Water Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
David J. Miichell
Professor of Plant Pathology
I certify that I have read this study and that in 'my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, iAjscopq and qualit
as a dissertation for the degree of Doctor of PbitoSopITyT
Donn G. Shilling
Professor of Agronomy


48
viability of external hyphae of the ectomycorrhizal fungus (Fig. 3-3A). There was a
difference in EM colonization, as measured by ergosterol concentration, at 4 wk
between the 60 and 150 kg benomyl ha1 treatments (Fig. 3-4A); however, this was not
repeatable.
Benomvl effects on corn
Benomyl at all concentrations arrested further root colonization by the AM
fungus, whereas colonization in the treatment receiving no benomyl continued to increase
over the 6-wk period (Fig. 3-4B). There was no dose-related response in colonization.
Noninoculated plants remained noncolonized. Total biomass of mycorrhizal and
nonmycorrhizal plants was reduced by benomyl by approximately 12% (Fig. 3-2B);
however, this was unrelated to the fungicide concentration applied. The length of external
hyphae of AM fungi or their viability was not affected significantly or consistently by the
different rates of benomyl (Fig. 3-3B). The P concentration of corn leaves decreased
steadily throughout the experiment from 3.64 to 0.62 mg P g'1 without any evidence of
a benomyl effect (data not shown).
Discussion
Corn was used as a substitute for P. chamaelonche due to lack of native plant
material. Benomyl arrested mycorrhizal development of corn in the greenhouse
experiment. This is consistent with the mode of action of benomyl, which entails
inhibition of nuclear division by binding to tubulin (Davidse, 1986). There was no dose-


57
combination where the roots were treated as one unit and then half the value allotted to
each plant. Root wet and dry mass were determined. An estimate of root length was
obtained using calculations of specific root length (cm root g"1 of root fresh weight) for
pine and grass from a previous experiment and expressed here as root-length density (cm
root cm'3 of soil). For grass, root colonization was determined using a gridline-intersect
method for the AM treatments (Giovannetti and Mosse, 1980) after clearing the roots for
30 min in 10% KOH and staining in 0.05% aniline blue overnight. For pine, root
ergosterol concentration was used as an estimate of EM fungal biomass (Martin et al.,
1990; Salmanowicz et al., 1989). Fresh pine roots were washed, ground in liquid
nitrogen and thoroughly mixed. A 0.1- to 0.3-g subsample was extracted overnight at
room temperature with 5 ml of 100% ethanol. This sample was filtered through a 0.45
im-syringe filter and then assayed for free ergosterol by high-pressure liquid
chromatography (Waters 715 Ultra WISP, Gilson 115 UV detector). Separation was
carried out using a C-18 column (Supelcosil LC-18; Supelco Inc., Bellefonte, PA) at
40C with a methanol-water mobile phase (92:8) flowing at 2 ml min1, with detection
at 282 nm.
Shoots were analyzed separately from roots. Shoot wet mass was determined and
dry mass was measured after drying overnight at 65C. The shoots were ground and then
ashed at 500C for a minimum of 4 h. Phosphorus analysis of the shoot tissue was
performed using the method of Murphy and Riley (1962).


36
from those on the internal mycorrhizal phase and (iii) determine if the intensity and
longevity of the fungicides effect was dose-dependent.
\
Materials and Methods
Field Study
The site was located 21 km northwest of Gainesville, Florida and was part of a
larger plant competition study involving slash pine (Pinus elliottii Engelm. var. elliottii))
and weeds. Slash pine had been planted in April 1990 in beds approximately 26 cm in
height and about 2 m in width with rows spaced approximately 213 cm apart. Soil was
a Pomona fine sand (a sandy, siliceous, hyperthermic Ultic Haplaquod). The surface 10
cm of soil had 7 pg P g1 extractable in 2 mM CaCl2 and a soil solution with pH 3.9.
Approximately 3.3% weight was lost upon ignition. The dominant weeds were Panicum
chamaelonche Trin., P. aciculare Dec.ex Poir. in Lam., Andropogon spp., Paspalum
spp. Rubus sp. and Serenoa repens. In December 1991, less than 1 spore of mycorrhizal
fungi g'1 of field soil was detected; the populations consisted of a mix of Glomus sp.,
Gigaspora sp. and Scutellospora sp. In the greenhouse, pot cultures of P. chamaelonche
originating from the field and grown in field soil yielded two AM isolates, Gigaspora
rosea (INVAM FL224) and Scutellospora heterogama (INVAM FL225) which were
submitted to and identified by J. Morton at INVAM.
Two areas (each 18.4 m by 11 m) containing slash pine and weeds were selected
randomly for this study. The control plot received no fungicide sprays. Benlate 50 DF
(E.I. du Pont de Nemours & Co., Inc., Wilmington, DE) was applied to the second area


This dissertation was submitted to the Graduate Faculty of the College of
Agriculture and to the Graduate School and was accepted as partial fulfillment of the
requirements for the degree of Doctor of Philosophy.
December 1995
Dean, College of Agriculture
Dean, Graduate School


37
with a C02-pressurized backpack sprayer by covering the area once and then making a
second application perpendicular to the first. The first spray (2 April 1991) was applied
at the rate of 5 kg benomyl ha1 using the equivalent of approximately 150 ml of water
m 2. Subsequent sprays (30 May, 11 July and 19 Sept. 1991) were applied at a rate of
20 kg benomyl ha'1.
Panicum chamaelonche was chosen as the indicator plant of AM fungal activity
because it was a dominant weed species at the site. Samples were taken on 2 April, 4
April, 30 May, 10 June, 2 July, 22 July, 13 August, 10 October 1991. At each sampling,
three plants were selected randomly and removed from each plot. The roots were washed
and cut into lengths of 1 to 2 cm. To determine fungicide effects on colonization and
metabolic activity, 1- to 2-g subsamples of roots were stained at room temperature for
8 h in a solution containing 0.2 M Tris HC1 (pH 7.4), 1 mg ml'1 of iodonitrotetrazolium
violet (INT) and 3 mg ml'1 of NADH (Sylvia, 1988). This was followed by clearing the
roots in a boiling, saturated solution of chloral hydrate for 10 min and subsequent
counterstaining overnight in 0.5% aniline blue in lactoglycerol. The chloral hydrate
treatment proved unnecessary and was eliminated in samplings collected after May. The
roots were destained in lactoglycerol and a minimum of 25 1-cm-long root segments per
plant were laid out parallel to each other on slides. The percentage of root segments with
arbuscules and the percentage of total arbuscules that were active (those staining with
INT) were estimated using bright-field microscopy at 400x magnification. The effect of
benomyl on mycorrhizal development was evaluated using the relationship of time and
either arbuscule abundance or activity. The slopes of linear regression of benomyl-treated


105
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Pirns sylvestris L. New Phytol. 123:325-333.
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A2-1 Colonization of (A) Panicum chamaelonche and (B) Zea mays inoculated
with root fragments of (1) Acaulospora scrobiculata (S315), (2) Gigaspora
margarita (INVAM FL215), (3) Glomus etunicatum (INVAM FL312) or
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(INVAM FL225). Each bar represents the mean of four replicates
SE 97
A3-1 Nonmycorrhizal Panicum chamaelonche (A) plant biomass and (B) plant
phosphorus content in response to 0.001, 0.003, 0.010, 0.032, 0.100,
0.316, 0.1000, 3.162 or 10.000 mg P L1. Each symbol represents the
mean of seven replicates SE 100
x


Ill
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83
samples from the plant and hyphal compartments were treated separately by the same
procedure.
Data for grass and pine were analyzed separately. To determine if plant
competition was affected by the plant species, data for each plant species were tested by
single degree of freedom contrasts using the General Linear Model procedure of SAS
(SAS Institute, Inc., 1989). For the contrasts, target plants were analyzed based on
mycorrhizal status, type of competition (intraspecific /interspecific) or mycorrhizal status
of the competing neighbor plant. Data for colonization were arcsine, square root-
transformed prior to analysis (Steel and Torrie, 1980). The least-squares means statement
within SAS was used to compare means.
Results
Pine+ exhibited greater plant biomass compared to the pine' treatments (Fig. Al-
1A, Table Al-1). There was no difference between intraspecific and interspecific
competition in the pine' treatments irrespective of the mycorrhizal status of grass. No
difference was observed for grass response to intra- or interspecific competition either
inoculated or noninoculated (Fig. A1-1B).
Similar to pine biomass, pine+ had a higher plant-P content than pine' (Fig. Al-
2A, Table Al-1). Grass+ had a lower P content than grass' when competing with both
pine+ and pine' (Fig. A1-2B). No such difference was found in the intraspecific
treatments where grass competed with grass.


16
between different mycorrhizal associations. The observed variation in mycorrhizal growth
response among closely related plants may relate to differing strategies of plant-C
allocation to the symbiosis (Graham and Eissenstat, 1994), as well as to plant age
(Eissenstat et al., 1993). Pearson and Jakobsen (1993) quantified the P-uptake efficiency
(C utilized/P absorbed) for three different AM fungi. For each unit of P absorbed they
found that Scutellospora calospora and a Glomus sp. utilized 25 and 16 times more C,
respectively, than Glomus caledonium. Total-C partitioning belowground was higher in
the less efficient mycorrhizal fungi, indicating that energy efficiency of the symbiosis
may be one reason for differing plant growth responses to fungi.
When comparing carbon costs of mycorrhizal to nonmycorrhizal plants, 4-36%
more of the total C fixed is allocated belowground due to mycorrhizas (Durall et al.,
1994). To distinguish nutritional from other mycorrhizal effects on plant-C balance,
mycorrhizal plants were grown at high soil-P concentrations and demonstrated a 37%
higher belowground carbon allocation than nonmycorrhizal plants (Peng et al., 1993). Of
this 37%, 51 % was attributed to greater root biomass and 10% to construction costs of
lipid-rich roots most likely associated with the mycorrhizal fungus. Enhanced
photosynthesis in mycorrhizal plants can compensate to varying degrees for this increased
C drain (Dosskey et al., 1990; Kucey and Paul, 1982). Plant root turnover is also
associated with a high C cost, although few studies have assessed the role of mycorrhizas
in controlling this process. Durall et al. (1994) determined that ectomycorrhizal roots
have a lower root turnover rate than nonmycorrhizal roots. In environmental conditions
where nutrient pulses occur, roots with a lower root turnover rate demonstrated a


109
Gildon A. and P.B. Tinker. 1983. Interactions of vesicular-arbuscular mycorrhizal
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N. Z. J. Agrie. Res. 21:509-515.
Hamel C., F. Morin, A. Fortin, R.L. Granger, and D.L. Smith. 1994. Mycorrhizal
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119:1255-1260.


13
acids released, resulting in differences in mineral weathering rates, nutrient release and
subsequent benefit to plants. Chelation of A1 not only releases bound P, but also lowers
the free ion activity and thus reduces A1 toxicity to the plant (Arp and Strucel, 1989).
This same mechanism may apply to other metals. Chelating agents specific to Fe are
termed siderophores and are produced by plant roots (Marschner, 1986), as well as by
several mycorrhizal fungi (Cress et al., 1986; Schuler and Haselwandter, 1988; Watteau
and Berthelin, 1995). Watteau and Berthelin (1995) found mycorrhizal siderophores of
the hydroxylate type to be more effective chelators than organic anions and less specific
for Fe than for Al.
The question of accessibility of organic compounds as carbon (C) sources to
mycorrhizal fungi has been debated (Harley and Smith, 1983). Recently, the
ectomycorrhizal fungi Cenococcum geophilum, Laceara bicolor, Rhizopogon vinicolor
and Suillus lakei were shown to utilize C from hemicellulose, cellulose and less readily
from a humic polymer mix or from Pseudotsuga menziesii needles (Durall et al., 1994).
Tanesaka et al. (1993) reported that several ectomycorrhizal fungi apparently did not
have the ability to degrade complex C substances such as wood. Haselwandter et al.
(1990) found several ericoid and ectomycorrhizal fungi capable of lignin degradation. At
present, the ability to degrade organic matter has not been documented for AM fungi.
Nonetheless, they appear to be efficient at capturing P released by decomposers prior to
its being immobilized again (Joner and Jakobsen, 1994), possibly due to an advantageous
spatial distribution. In general, the question of mycorrhizal fungal hyphae accessing


102
Arp A.P. and I. Strucel. 1989. Water uptake by black spruce seedlings from rooting
media (solution, sand, peat) treated with inorganic and oxalated aluminum. Water, Air,
Soil Pollut. 44:57-70.
Aug R.M., K.A. Schekel, and R.L. Wample. 1986. Greater leaf conductance of
well-watered VA mycorrhizal rose plants is not related to phosphorus nutrition. New
Phytol. 103:107-116.
Baar J., W.A. Ozinga, I.L. Sweers, and T.W. Kuyper. 1994. Stimulatory and inhibitory
effects of needle litter and grass extracts on the growth of some ectomycorrhizal fungi.
Soil Biol. Biochem. 26:1073-1079.
Bth E. and D.S. Hayman. 1984. Effect of soil volume and plant density on
mycorrhizal infection and growth response. Plant Soil 77:373-376.
Bajwa R., S. Abuarghub, and D.J. Read. 1985. The biology of mycorrhiza in the
Ericaceae. X. The utilization of proteolytic enzymes by the mycorrhizal endophyte and
by mycorrhizal plants. New Phytol. 101:469-486.
Bajwa R. and D.J. Read. 1985. The biology of mycorrhiza in the Ericaceae IX. Peptides
as nitrogen sources for the ericoid endophyte and for mycorrhizal and nonmycorrhizal
plants. New Phytol. 101:459-467.
Baldwin, J.P. and P.H. Nye. 1974. A model to calculate the uptake by a developing root
system or root hair system of solutes with concentration variable diffusion coefficients.
Plant Soil 40:703-706.
Barber D.A. 1972. "Dual isotherms" for the absorption of ions by plant tissues. New
Phytol. 71:255-262.
Barber S.A. 1995. Soil Nutrient Availability: A Mechanistic Approach. John Wiley &
Sons, Inc., New York, N.Y.
Barbosa P., Krischik V.A. and C.G. Jones. 1991. Microbial Mediation of
Plant-Herbivore Interactions. John Wiley & Sons, Inc., New York, N.Y.
Barea J.M., R. Azcn, and C. Azcn-Aguilar. 1989. Time course of N2-fixation (15N)
in the field by clover growing alone or in mixture with ryegrass to improve pasture
productivity, and inoculated with vesicular-arbuscular mycorrhizal fungi. New Phytol.
112:399-404.
Barker G.M. 1987. Mycorrhizal infection influences Acremonium-induced resistance to
Argentine stem weevil in ryegrass. Proc. N.Z. Weed Pest Control Conf. 199-203.


73
al. (1989), biomass of plants in intraspecific competition (12 trees pot1) was altered by
different EM fungi, indicating that competition intensity varies with the fungal species.
The somewhat reduced response in the repeat of this experiment may be related
to temperature differences between the two experiments. The first one ended in June and
the second ended in October, which resulted in not only a 2C higher maximum
temperature in the second study, but a longer daily exposure to higher temperatures as
well. The mean colonization of pine+ in the repeat was 46% lower compared to the first
run of the experiment. A decrease of mycorrhizal effectiveness has been observed at
temperatures of 34 to 35C for certain Pisolithus tinctorius isolates (Marx et al., 1970)
grown on pine, as well as for Glomus spp. (Fabig et al., 1989) grown on several grass
hosts. The lack of colonization in the grass plants may be related to the high soil acidity
(pH 4.0) and the lack of buffering capacity of the sand. Activity of Glomus spp. is
optimal above pH 5.3 (Abbott and Robson, 1985; Wang et al., 1985).
The conclusion of this study is that P. tinctorius can increase P acquisition of pine
when grown with grass, which consequently could lead to an increase in competitive
ability. The controlled conditions used in this experiment allow the isolation of specific
variables that affect a plants competitive ability. The actual proportion of a plants total
competitive ability contributed by the mycorrhizal component can only be determined
under field conditions where soil chemical, physical and biological parameters modify
plant interactions and the mycorrhizal response. Yet, our ability to isolate the different
components of a plants competitive ability and determine the relative importance of each


Hyphal Length (m g"1 of dry soil)
7
Figure 2-1. Length of external hyphae spreading from mycorrhizal roots of Trifolium
subterraneum after (a) 28 days and (b) 47 days. Bars represent standard error of the
mean [with permission from (Jakobsen et al., 1992)].


71
would be more advantageous where low nutrient concentrations persist over longer time
periods, such as in the field but not in this greenhouse study where the time between
fertilization was relatively brief. These observations suggest that differences in P uptake
kinetics partially may be responsible for the outcome of competition.
Pine and grass root growth rates vary with P concentration, resulting in different
absorbing surface areas. The poor relationship I observed between root length and shoot-
P content indicates that root length is not the only factor contributing to the pines
competitive interaction with grass. Nonetheless, the increase in root length in the pine+
compared to the pine' treatments suggests that mycorrhizal fungi increased pine root
length and thereby enhanced nutrient uptake. As a result the competitive ability of pine
was increased compared to grass.
The exact nature of the relationship between intensity of competition and resource
abundance is still under debate, but it depends on the environmental conditions and plant
species involved (Di Tommaso and Aarssen, 1991; Grace, 1995). Tilman (1982) stated
that competition increases with decreasing resource availability. An alternate viewpoint
is espoused by Grime (1979) who maintained that competition intensity increases with
increasing habitat fertility. By definition competition is expressed as effects on plant
biomass, survival or reproduction. In this study plants did not demonstrate any dry
weight response to P application, which suggests that P was not the only nutrient limiting
plant growth. At the 0.32-/xM P level plant growth was marginal as a result of inadequate
P in the system. Although dry weight was not altered by the different levels of applied
P, P uptake by both pine and grass was affected. Since nutrient uptake is part of the


40
method, total and active hyphal lengths were determined microscopically at 400x from
20 randomly selected fields on the filter.
Pine needles were removed from seedlings and dried overnight at 65C, and P
content was determined colorimetrically (Murphy and Riley, 1962). Ergosterol (a sterol
found in fungal, but not plant, membranes) content in the root was used to provide a
relative estimate of total fungal biomass present (Martin et al., 1990; Salmanowicz et ah,
1989). Fresh roots were washed, ground in liquid nitrogen and thoroughly mixed. A 0.1-
to 0.3-g subsample was extracted overnight at room temperature with 5 ml of 100%
ethanol. This sample was filtered through a 0.45-/xm syringe filter and then assayed for
free ergosterol by high-pressure liquid chromatography (Waters 715 Ultra WISP, Gilson
115 UV detector). Separation was achieved on a C-18 column (Supelcosil LC-18;
Supelco, Inc., Bellefonte, PA) at 40C using a methanol-water mobile phase (92:8)
flowing at 2 ml min'1 with detection at 282 nm.
Benomvl effects on corn
The effect of benomyl on colonization by the AM fungus Glomus sp. (INVAM
FL329, formerly FL906) was studied in a separate experiment. Germinated corn (Zea
mays L. cv. Silver Queen) seed was planted in sand in Deepots with 5 g of soil
inoculum (83 spores g'1) placed 2 to 3 cm below the seedling. Control plants received a
5-ml suspension of inoculum filtrate obtained by mixing 60 g of soil from a pot culture
with 1.2 L of water and then filtering this through a 10-/xm membrane filter. Benlate 50
WP was applied 19 d after planting to the soil surface at rates of 0, 20, 60 and 150 kg


97
Figure A2-1. Colonization of (A) Panicum chamaelonche and (B) Zea mays inoculated
with root fragments of (1) Acaulospora scrobiculata (S315), (2) Gigaspora margarita
(INVAM FL215), (3) Glomus etunicatum (INVAM FL312) or (4) Glomus sp. (INVAM
FL329, formerly FL906) or spores of (5) Gigaspora rosea (INVAM FL224) or (6)
Scutellospora heterogama (INVAM FL225). Each bar represents the mean of four
replicates SE.


CHAPTER 2
MYCORRHIZAS AND PLANT COMPETITION
Introduction
Over the past several decades the perception of mycorrhizas has evolved from
viewing them as a unique biological phenomena to understanding them as integral parts
of ecosystems. Much of the literature on mycorrhizas has addressed issues pertaining to
single plants. More recently, there has been a growing tendency to evaluate the
synecological consequences of the mycorrhizal association. The employment of
techniques such as minirhizotrons (Lussenhop and Fogel, 1993), image analysis (Smith
and Dickson, 1991), root-excluding screens and radioisotope labelling, among others, is
redirecting the field to a broader scale of ecology dealing with plant interactions and
community structure. The challenges faced during the next decade will be even more
complex, with the increasing need to study multi-organismal assemblages and their
functions at the ecosystem level. The next steps towards a more holistic view of
mycorrhizal function will be determined by technological advances that will allow us to
gain knowledge of how microbial systems fit together into a cohesive unit. This
knowledge will provide us with a better understanding of the environment and how to
best manage it in a sustainable manner.
Ecosystem studies necessitate an understanding of the functional associations of
organisms with each other and with their environment. For plants, one of the main
3


29
aside from direct physiological benefits to the plant, mycorrhizal contributions to plant
health in the field may be a common but subtle phenomenon, because it is buried within
complex interactions.
To make the situation more complex, few studies have included interactions
between mycorrhizas and other plant endophytes (Clay, 1992). The fungal endophyte
Acremonium sp., for example, has reduced colonization and reproduction by Glomus sp.
(Chu-Chou et al., 1992; Guo et al., 1992). Reduction of insect herb ivory has been
attributed to secondary metabolite production by fungal endophytes (Clay, 1991). Another
study found that mycorrhizas may reduce feeding inhibition of an insect herbivore
induced by Acremonium sp. (Barker, 1987). Additionally, nonmycorrhizal endophytes are
capable of altering competitive relationships between plants (Clay et al., 1993) and plant
drought resistance (White, 1992) in ways similar to mycorrhiza. The data suggest that
endophytes are involved in various effects observed in plant studies and consequently
they deserve further consideration.
Herb ivory
Herbivores generally have either an inhibitory or neutral effect on mycorrhizas
(Barbosa et al., 1991; Gehring and Whitham, 1994). Herbivory results in increased plant-
C allocation to the replacement of aboveground parts instead of to maintenance of the
mycorrhizal symbiosis (Jones and Last, 1991). There are also a few studies on the
inverse effect of mycorrhizas on herbivores (Gange et al., 1994; Rabin and Pacovsky,
1985). Generally, mycorrhizas had an inhibitory effect on the herbivorous insects. Gange


31
plant competition for limited nutrients (Wilson and Shure, 1993). Under nutrient
limitations, resource acquisition enhanced by mycorrhizas occurs at the expense of other
plants, which results in the highly competitive plants becoming more abundant and
dominant in the community. Continuous growth of a plant in the same soil eventually
will select a microbial community well adapted to that environment. Over time the
adapted microbial community can become disadvantageous for growth of that plant
species, but not for others, and, in this manner, may contribute to plant succession
(Bever, 1994; Van der Putten et al., 1993). In these studies it was suggested that this
negative feedback on growth may be related to pathogen buildup. Mycorrhizal fungi were
not considered, because of the assumption that mycorrhizal effects are usually beneficial.
However, if there is a selection for less efficient mycorrhizal fungi occurring over time,
then this may similarly contribute to succession by decreasing a plants C-use efficiency
and its competitive ability. In monocultural settings, a shift of mycorrhizal fungal species
composition over time was identified by Johnson et al. (1992a; 1992b) and Wacker et al.
(1990). In both cases there was an associated decline in plant growth, indicating that
mycorrhizal fungi should not be discarded a priori as a contributing factor to growth
declines.
Succession of ectomycorrhizal fungi from "early" to "late" stage fungi occurs in
undisturbed forest systems (Deacon and Fleming, 1992). Differing fungal resource
requirements, as well as changes in other soil microbial components, have been
postulated to cause the succession (Garbaye, 1994). Recent research indicates that this
succession may be tied closely to factors found in the soil organic matter. Removal of


38
versus nontreated plants were compared using the General Linear Model procedure of
SAS (SAS Institute, Inc., 1989).
Greenhouse Study
Both of the following experiments had completely randomized factorial designs
(two mycorrhizal treatments x four benomyl levels) with seven replications each. To
maintain uniform daylength of approximately 12 h, extra light (800 /zmol m'2s'' from
17:00 to 20:00 hr) was provided. Plants in all experiments were fertilized semiweekly
with 3.2 /xM NH4N03, 7.5 /zM Ca(N03)2 7.7 /xM KC1, 1.0 /xM MgS04, 20 nM
NaFeEDTA, 5.0 nM CuS044H20, 240 nM H3B04, 20 nM MnCl24H20, 5 nM
Na2Mo042H20 and 20 nM ZnS047H20. The nutrient solution for corn or pine
contained, respectively, 3.2 nM H3P04 or 0.32 nM H3P04. All data were analyzed by
analysis of variance using the General Linear Model procedure (SAS Institute, Inc.,
1989). Both experiments were repeated once under similar environmental conditions.
Benomvl effects on pine
Slash pine seeds were disinfested for 2 min in a 5.25% sodium hypochlorite
solution with 0.2 ml Liqui-Nox surfactant (Alconox, Inc., New York, NY) and then
rinsed thoroughly with tap water. Plants were raised from seed for 12 d in a growth
chamber [29 C/23 C (day/night), with a 15-h light period and irradiance of 1000 zmol
m 1 s'1] in a vermiculite/sand (1:1) mix. They were then transplanted into sand in 50-ml
pots (5 cm2 of surface area) grown in the greenhouse for 6 wk where they received water


119
Sylvia D.M., D.O. Wilson, J.H. Graham, J.J. Maddox, P.P. Millner, J.B. Morton,
H.D. Skipper, S.F. Wright, and A.G. Jarstfer. 1993. Evaluation of vesicular-arbuscular
mycorrhizal fungi in diverse plants and soils. Soil Biol. Biochem. 25:705-713.
Tam P.C.F. and D.A. Griffiths. 1993. Mycorrhizal associations in Hong-Kong Fagaceae
V. The role of polyphenols. Mycorrhiza 3:165-170.
Tanesaka E., H. Masuda, and K. Kinugawa. 1993. Wood degrading ability of
Basidiomycetes that are wood decomposers, litter decomposers, or mycorrhizal
symbionts. Mycologia 85:347-354.
Tarafdar J.C. and N. Claassen. 1988. Organic phosphorus compounds as a phosphorus
source for higher plants through the activity of phosphatases produced by plant roots and
microorganisms. Biol. Frtil. Soils 5:308-312.
Tarafdar J.C. and H. Marschner. 1994. Phosphatase activity in the rhizosphere and
hyphosphere of VA mycorrhizal wheat supplied with inorganic and organic phosphorus.
Soil Biol. Biochem. 26:387-395.
Taylor D.R. and L.W. Aarssen. 1989. On the density dependence of replacement-series
competition experiments. J. Ecol. 77:975-988.
Thingstrup I. and S. Rosendahl. 1994. Quantification of fungal activity in arbuscular
mycorrhizal symbiosis by polyacrylamide gel electrophoresis and densitometry of malate
dehydrogenase. Soil Biol. Biochem. 26:1483-1489.
Thomson C.J. and T.P. Bolger. 1993. Effects of seed phosphorus concentration on the
emergence and growth of subterranean clover (Trifolium subterraneum). Plant Soil
156:285-288.
Thus H. 1994. The effect of phytotoxins on competitive outcome in a model system.
Ecology 75:1959-1964.
Tilman D. 1982. Resource Competition and Community Structure. Princeton University
Press, Princeton, N.J.
Tilman D. and J.A. Downing. 1994. Biodiversity and stability in grasslands. Nature
367:363-365.
Tinker P.B., D.M. Durall, and M.D. Jones. 1994. Carbon use efficiency in mycorrhizas:
theory and sample calculations. New Phytol. 128:115-122.


78
advantages in the field. The use of a field soil in subsequent studies would incorporate,
at least in part, these effects.
In the hydroponic study on P-uptake an attempt was made to measure the effects
of mycorrhizas on P uptake kinetics. The results involving mycorrhizas were inconclusive
since mycorrhizal plants with a higher total absorbing surface area demonstrated a lower
Imax value than the nonmycorrhizal plants. Based on visual observations this is due to
extensive hyphal development in the mycorrhizal treatments most likely resulting in
overlapping depletion zones of roots and hyphae. Allowing hyphae to regrow for a period
of 2 wk instead of 4 wk prior to P uptake measurement probably would have avoided the
problem. Although not quantified in the hydroponic study, mycorrhizal fungi may have
different Imax and C,^ values from the host plant. If the fungus has a higher I^ or a
lower than the host plant, as well as the competing plant, this would confer a
competitive advantage to the host plant.
In the field, root-length density measured down to a depth of 87 cm was several
fold higher for grass than for pine. The 500-mL soil volume used in the greenhouse
experiment created a root-bound condition which did not fully permit this difference to
be expressed. Much of the contribution of mycorrhizas is due to their accessing nutrients
beyond the roots nutrient depletion zone; however, since hyphae and roots were able to
access nutrients in most of the soil volume in pots, this probably did not contribute a
competitive advantage in my study. The spatial advantage mycorrhizal hyphae would
provide to a host plant by their presence directly at the site of nutrient mineralization,
such as in and around organic matter, also was not expressed. Although external


33
provide resilience to disturbance (Amaranthus and Perry, 1994). Mycorrhizal connections
between dying and living plants also limit soil nutrient loss by leaching and
immobilization (Eason and Newman, 1990). The network of hyphal bridges connecting
neighboring plants can affect coexistence by increasing species richness and diversity
(Gange et al., 1993; Grime et al., 1987). The current literature indicates that this is
perhaps more likely due to transfer of C than of inorganic nutrients. Furthermore, a
higher plant species diversity has been associated with increased ecosystem stability in
a stressed environment (Tilman and Downing, 1994). Obviously, with the multitude of
effects and interactions mediated by mycorrhiza, a quantification of the net mycorrhizal
influence in ecosystems is a formidable challenge. Still, with the current emphasis on
environmentally sound management of ecosystems, it is important to include them in
considerations of appropriate technologies in managed ecosystems.


18
Xenobiotics
In many ecosystems plants and mycorrhizal fungi are exposed to a wide variety
of toxic compounds (xenobiotics and in some instances naturally occurring toxic
compounds). Mycorrhizal fungi may effectively mediate and alter the interaction between
plant and xenobiotic compounds. Various papers have assessed or reviewed pesticide
effects on mycorrhizal fungi (Dehn et al., 1990; Trappe et al., 1984). Mycorrhizal fungi
may function in the translocation of herbicides. In one study with apple and three
herbicides (dichlobenil, paraquat and simazine), root dry weight of noninoculated plants
exposed to herbicides was reduced by 46% in contrast to a 63% decrease in mycorrhizal
plants (Hamel et al., 1994). Although no effect on hyphal length was found at the highest
simazine concentration applied, 75% of the mycorrhizal plants died compared to none
in the control treatment. The authors attributed this to facilitated herbicide flow to the
host plant mediated by the mycorrhizal fungus. Uptake and translocation of the herbicide
atrazine was also found in mycorrhizal corn, which is atrazine-tolerant (Nelson and
Khan, 1992). Although the quantity absorbed was small compared to direct root uptake,
the question of how this may affect an atrazine-sensitive plant remains unanswered.
Certain mycorrhizal fungi also have demonstrated the capacity to degrade herbicides such
as atrazine and to a lesser extent 2,4-dichlorophenoxyacetic acid (Donnelly et al., 1993).
This provokes the question as to whether mycorrhizal fungi offer some protection against
xenobiotics. In corn and sorghum certain herbicide safening effects by AM fungi have
been found against the herbicides imazaquin, imazethapyr and pendimethalin (Siqueira
et al., 1991).


110
Hamel C. and D.L. Smith. 1991. Interspecific N-transfer and plant development in a
mycorrhizal field-grown mixture. Soil Biol. Biochem. 23:661-665.
Harley J.L. and S.E. Smith. 1983. Mycorrhizal Symbiosis. Academic Press, New York,
N.Y.
Harper J.L. 1977. Population Biology of Plants. Academic Press, New York, N.Y.
Hartnett D.C., B.A.D. Hetrick, G.W.T. Wilson, and D.J. Gibson. 1993. Mycorrhizal
influence on intra- and interspecific neighbour interactions among co-occurring prairie
grasses. J. Ecol. 81:787-795.
Hartnett D.C., R.J. Samenus, L.E. Fischer, and B.A.D. Hetrick. 1994. Plant
demographic responses to mycorrhizal symbiosis in tallgrass prairie. Oecologia 99:21-26.
Haselwandter K., O. Bobleter, and D.J. Read. 1990. Degradation of 14C-labelled lignin
and dehydropolymer of coniferyl alcohol by ericoid and ectomycorrhizal fungi. Arch.
Mikrobiol. 153:352-354.
Hassall K.A. 1990. The Biochemistry and Uses of Pesticides: Structure, Metabolism,
Mode of Action and Uses in Crop Protection. Macmillan Press Ltd., New York, N.Y.
Hetrick B.A.D., G.W.T. Wilson, and T.S. Cox. 1992. Mycorrhizal dependence of
modern wheat varieties, landraces, and ancestors. Can. J. Bot. 70:2032-2040.
Hetrick B.A.D., G.W.T. Wilson, and D.C. Hartnett. 1989. Relationship between
mycorrhizal dependence and competitive ability of two tallgrass prairie grasses. Can. J.
Bot. 67:2608-2615.
Hirrel M.C. and J.W. Gerdemann. 1979. Enhanced carbon transfer between onions
infected with a vesicular-arbuscular mycorrhizal fungus. New Phytol. 83:731-738.
Horsley, S.B. 1987. Allelopathic interference with regeneration of the Allegheny
hardwood forest, p. 205-212. In G.R. Waller (ed.) Allelochemicals: Role in Agriculture
and Forestry. American Chemical Society, Washington, D.C.
Ikram A., E.S. Jensen, and I. Jakobsen. 1994. No significant transfer of N and P from
Pueraria phaseoloides to Hevea brasiliensis via hyphal links of arbuscular mycorrhiza.
Soil Biol. Biochem. 26:1541-1547.
Itoh S. and S.A. Barber. 1983. A numerical solution of whole plant nutrient uptake for
woil-root systems with root hairs. Plant Soil 70:403-413.


89
Pine' was colonized by Thelephora terrestris and had a higher level of colonization
than pine+ over all competition treatments (Fig. A1-3A, Table Al-1). In the intraspecific
pine treatments, more external hyphae were present in the pine+ than in pine' treatment
(Table Al-2). Grasses were colonized in the grass+ but not in the grass' treatments (Fig.
A1-3B). Lower colonization of grass+ was observed when competing with pine than
when competing with another grass. Hyphal length density was higher overall in the pine
than in grass. Hyphae contributed between 10 to 16% and 68 to 83% of the total
absorbing surface area for grass and pine respectively (data not shown). When total
hyphal length was separated by fungal type, AM hyphal length in the plant compartments
containing grass was 3.59 0.51 and 3.82 0.68 m cm'3 of soil in the pine+ and pine'
treatments, respectively, compared to 7.35 1.09 m cm'3 of soil with grass alone. At
the lowest hyphal density measured in a compartment, the theoretical inter-hyphal
distance was equal to 23.2 on [calculated using the formula: 2/(Lvtt)0 5 from Baldwin and
Nye (1974); hyphal length density (LJ= 238 cm of hyphae cm'3 of soil]. The theoretical
two-dimensional depletion zone would be approximately 83.1 un [determined with the
formula: 2(Dt)0 5 (Baldwin and Nye, 1974); assuming a diffusion coefficient (D) = lxl0'8
cm2 s'1 and time (t)=2 d].
Root-length density of pine+ was greater over all competition treatments than that
of pine', independent of intra- or interspecific competition (Fig A1-4A). Grass* was
significantly different from grass' (P < 0.06) over all treatments (Table Al-1). The
major source of this difference was the higher root-length density of grass' when
competing with pine+ compared to the other competition treatments (Fig. A1-4B).


54
chamber [29/23 C (day/night), with a 15-h light period and irradiance of 1000 /mol m'1
s'1] in sand and then transplanted to 50-ml pots (5 cm2 of surface area) and grown in sand
in the greenhouse for 8 wk where they received water only. To inoculate pine, washed
roots were dipped in a slurry of rinsed and chopped Pisolithus tinctorius (Pers.) Coker
& Couch (isolate S106) grown in a liquid suspension culture containing modified Melin-
Norkrans liquid medium (Marx, 1969) using glucose instead of sucrose. Roots of control
plants were dipped in tap water. After a further 6 wk of growth in 500 ml of sand in
Deepots (28 cm2 of surface area; McConkey, Co., Sumner, WA), pine roots were
gently rinsed free of adhering sand particles and planted in the appropriate competition
treatments as described below.
Grass plants of a dominant competing weed species in the field (Panicum
chamaelonche Trin.) were obtained from cultures maintained in sand in the greenhouse.
Plants were started from seed and vegetatively propagated in 150-ml pots (7 cm2 of
surface area). Two months in advance of the experiment, grass plants were inoculated
with pot culture inoculum of Glomus sp. (INVAM FL329, formerly FL906) previously
cultured on sorghum in pasteurized soil. Roots of plants were washed and the plants
transplanted into sand in Deepots containing 5 g of soil inoculum (83 spores g'1) located
2-3 cm below the sand surface. Control plants were transplanted into sand without
inoculum and received a 5-ml suspension of inoculum filtrate obtained by mixing 60 g
of soil from a pot culture with 1.2 L of water and then filtering the mixture through a
10-^tm membrane filter. Just prior to the experiment the grass roots were washed as
described for the method of pine roots.


3
67
- 2
C
O
O
E
o 1
o
O)
i.
O
O
5 3
2
Q)
0
2 -
0.32 3.22 32.26
P Applied (pM P), Log Scale
Figure 4-4. Relative crowding coefficient (RCC) for total dry weight of (A) Pinus elliottii
inoculated with Pisolithus tinctorius grown in combination with Panicum chamaelonche
and (B) noninoculated Pinus elliottii grown in combination with P. chamaelonche at
either 0.32, 3.23 or 32.26 /uM P for 18 wk. Each symbol represents the mean of six
replicates SE. Mean standard errors were smaller than the symbols and are therefore
not included.


Root-Length Density (m cm'3 of soil)
0.15
Figure Al-4. Root-length density for (A) slash pine and (B) grass grown in the growth
chamber for 62 d. Each bar represents the mean of a minimum of six replicates SE.


104
Bradley R.D., A.J. Burt, and D.J. Read. 1982. The biology of mycorrhizae in the
Ericaceae. VIII.The role of mycorrhizal infection in heavy metal resistance. New
Phytol. 91:197-209.
Brown M.S., R. Ferrera-Cerrato, and G.J. Bethlenfalvay. 1992. Mycorrhiza-mediated
nutrient distribution between associated soybean and corn plants evaluated by the
diagnosis and recommendation integrated system (DRIS). Symbiosis 12:83-94.
Brundrett, M.C. 1991. Mycorrhizas in natural ecosystems, p. 171-213. In M. Begon,
A.H. Fitter and A. MacFadyen (ed.) Advances in Ecological Research vol. 21. Academic
Press, New York, N.Y.
Burgess T., B. Dell, and N. Malajczuk. 1994. Variation in mycorrhizal development and
growth stimulation by 20 Pisolithus isolates inoculated on to Eucalyptus granis W Hill
ex Maiden. New Phytol. 127:731-739.
Campbell B.D. and J.P. Grime. 1989. A comparative study of plant responsiveness to
the duration of episodes of mineral nutrient enrichment. New Phytol. 112:261-267.
Campbell B.D., J.P. Grime, and J.M.L. Mackey. 1991. A trade-off between scale and
precision in resource foraging. Oecologia 87:532-538.
Campbell B.D., J.P. Grime, J.M.L. Mackey, and A. Jalili. 1991. The quest for a
mechanistic understanding of resource competition in plant communities: the role of
experiments. Funct. Ecol. 5:241-253.
Carey P.D., A.H. Fitter, and A.R. Watkinson. 1992. A field study using the fungicide
benomyl to investigate the effect of mycorrhizal fungi on plant fitness. Oecologia
90:550-555.
Chenu C. 1993. Clay- or sand-polysaccharide associations as models for the interface
betweeen micro-organisms and soil: water related properties and microstructure.
Geoderma 56:143-156.
Chu-Chou M., B. Guo, Z.Q. An, J.W. Hendrix, R.S. Ferriss, M.R. Siegel, C.T.
Dougherty, and P.B. Burrus. 1992. Suppression of mycorrhizal fungi in fescue by the
Acremonium coenophialum endophyte. Soil Biol. Biochem. 24:633-637.
Clay, K. 1991. Fungal endophytes, grasses and herbivores, p. 199-226. In P. Barbosa,
V.A. Krischik and C.G. Jones (ed.) Microbial Mediation of Plant-Herbivore Interactions.
John Wiley & Sons, Inc. New York, N.Y.
Clay, K. 1992. Mycophyllas and Mycorrhizas. p. 13-25. In Mycorrhizas and
Ecosystems. C.A.B. International, Wallingford, U.K.


77
2. Inoculation of slash pine with P. tinctorius enhanced both P uptake and total dry
weight and hence the competitive ability of pine when competing with
nonmycorrhizal grass.
3. When grown in intraspecific competition, no difference was observed in the
competitive ability of pine colonized with P. tinctorius or Thelephora terrestris.
4. The different P levels added did not affect grass or pine biomass which suggests
that P was not the only limiting factor to growth.
5. A higher I,,^ value for pine and the lower Cmjn for grass suggest that differing P
uptake kinetics can contribute to competitive interactions.
Several additional factors would have to be elucidated to draw conclusions from
these results about pine and grass interactions in the field. In a separate field competition
study involving slash pine and weeds (primarily grasses) pine growth was substantially
decreased in contrast to the greenhouse where pine exhibited a higher competitive ability
than grass. In the Spodosol at the field site, organic forms of P are the major source of
P, which is released during periodic pulses of nutrient cycling triggered by increases in
soil moisture. This contrasts with the inorganic P used in the greenhouse study, which
was applied at frequent and regular intervals and thus maintained a relatively consistent
P concentration in the system. Also, the buffering capacity of the field soil was absent
in the greenhouse, and this would modify plant-soil-microbe interactions by altering
nutrient availability and flux. As a consequence differing pine and grass P uptake kinetics
expressed in the greenhouse would not necessarily provide the same competitive


23
and Bth and Hayman (1984) determined that, in a given soil volume, mycorrhizal
benefit for a plant decreases with increasing density of its competitors. Higher plant
density is paralleled by an increase in root and hyphal density in the soil and
proportionately greater overlap of nutrient depletion zones. In intraspecific competition
of inoculated plants of high mycorrhizal dependency, density-related competition was
observed, but this did not occur when mycorrhizal fungi were absent. Inoculated plants
with low mycorrhizal dependency lacked this response, indicating their ability to more
efficiently extract nutrients from the soil than the nonmycorrhizal plants with high
mycorrhizal dependency.
Plants of the same species but different plant age also have been compared for
competitive interactions. Eissenstat and Newman (1990) evaluated the possible advantages
of mycorrhizas to seedling establishment in the presence of an older plant of the same
species. The results indicated that there is not a facilitative but rather a competitive
relationship between the two plants, similar to that observed in the absence of
mycorrhizal fungi. In another study, Franson et al. (1994) found that competition
intensity between an established and a seedling soybean plant was not altered by
increasing the stress on the younger plant.
Plant competitive interactions between mycorrhizal host and nonhost plants have
been investigated in a limited number of studies. It is worth noting that some have
documented a reduction in biomass of nonhost plants when such plants were grown under
mycorrhizal conditions (Allen et al., 1989; Ocampo, 1986). Francis and Read (1994)
found evidence for a chemical factor, which was extracted from soil of mycorrhizal


4
biological interactions is competition. The term competition will be used here as the
interaction between two organisms requiring the same limiting resource, which results
in the decreased growth, survival or reproductive capacity of one of the two organisms.
Plants mainly compete for light, water and nutrients. Physiological flexibility, within
genetic constraints, allows plants to adapt to changes in resource availability.
Physiological flexibility is enhanced by a plants symbiotic relationship with mycorrhizal
fungi. Modification in physiology can result in alterations of nutrient absorption capacity
(Marschner and Dell, 1994) and water relations (Safir et al., 1972), as well as enhance
light utilization and capture (Krishna et al., 1981). Increased tolerance or resistance to
other environmental stresses, such as plant diseases (Rosendahl and Rosendahl, 1990;
Schnbeck, 1978), high heavy metal concentrations (Denny and Wilkins, 1987) or
xenobiotics (Donnelly et al., 1993), also have been found in mycorrhizal plants.
Although the vast majority of studies with mycorrhizas has been conducted with
terrestrial, mycorrhizas also have been found in wetland plants and may function in
nutrient uptake in vascular aquatic plants (Rickerl et al., 1994; Wigand and Stevenson,
1994).
The objective of this chapter is to review mycorrhizal effects on plant competition
and community structure. However, to prepare the foundation for the synecology of the
system, a review of the autecology of mycorrhizal plants is also presented.


59
cultured on P. chamaelonche in field soil in the greenhouse. The AM fungal species were
isolated from P. chamaelonche growing in a Spodosol at a field site 21 km northwest of
Gainesville. The grasses were replanted together in a 15-L pot of sand. Noninoculated
plants were treated in the same manner, except that no spores were added to the roots.
Three grasses and pines, inoculated or noninoculated, were selected, and their
roots were gently rinsed free of adhering sand. Each plant was transferred to a single 1-L
Erlenmeyer flask covered with aluminum foil. Plants were grown in a growth chamber
[29/23 C (day/night), with a 15-h light period and irradiance of 1000 /xmol m'1 s'1] in
a continuously aerated nutrient solution with the following nutrient composition: 660 /xM
NH4N03, 616 /xM KC1, 800 /xM MgS04, 54 /xM NaFeEDTA, 600 /xM Ca(N03)2 4H20,
0.75 /xM CuS04, 52 /xM H3B03, 120 /xM NaMo04, 8.25 /xM MnCl and 3.75 /xM ZnS04.
Phosphorus was supplied at 3.23 /xM H3P04. The solution was changed semiweekly. At
the start of the experiment a minimum 4-wk acclimatization period was given allowing
external hyphae to regrow from the colonized roots.
To quantify uptake kinetics, root systems were rinsed with deionized water and
placed in additional deionized water for 1 h. One liter of fresh nutrient solution, identical
to the one used previously, was added to 1-L acid-washed Erlenmeyer flasks. At the start
of the experiment, plant roots were gently patted dry with paper towels, placed in the
nutrient solution and weighed. At regular intervals, 23 ml of solution for P analysis were
removed and immediately filtered through 0.45-/xm syringe filters. The solution was
replaced with sufficient deionized water to bring the system back to its original starting
weight. Twenty milliliters of sample removed for P analysis were evaporated to dryness.


32
litter and humus in Pinus sylvestris stands increased mycorrhizal fungal species richness
and reverted the species composition to the early successional types (Devries et al.,
1995). In other systems, the increased buildup of organic matter also has been associated
with higher concentrations of phenolic compounds (Kuiters and Sarink, 1986; Leake et
al., 1989), which have demonstrated allelochemical effects. Perhaps resistance to and the
ability to degrade phenolic compounds determines which fungal species are capable of
growing at a certain stage of succession. Leake et al. (1989) demonstrated that ericoid
mycorrhizas were capable of enhancing ericoid plant growth and survival, possibly by
a detoxification mechanism. Whereas AM fungi are found more commonly in mineral
soils, ectomycorrhizal fungi are often associated with environments high in organic
matter and are physiologically adapted to utilizing complex substrates (Francis and Read,
1994). Also, ectomycorrhizal mantles surrounding root tips are capable of protecting
these from potentially toxic compounds. As a consequence, tolerance to adverse
environmental conditions allows the plant to focus more of its energy on resource
acquisition strategies without substantial tradeoffs of energy for other mechanisms,
thereby making it a better competitor.
Plant competition, as affected by mycorrhizal fungi, could be relevant in plant
community structuring and succession. As such, mycorrhizal benefits to single plants may
prove functionally significant at the ecosystem level. In addition, positive interactions in
communities are often neglected (Bertness and Callaway, 1994) and should also be
considered in the discussion of plant interactions mediated by mycorrhizas (Amaranthus
and Perry, 1994). Mycorrhizas can moderate plant competition (Perry et al., 1989) and


81
of noninoculated pine colonized by Thelephora terrestris. At the start of the experiment
plant roots were washed free of all sand and divided into three size classes. Different
combinations of plant species, inoculated or noninoculated, were made by selecting pairs
of plants from the same size class to create the competition treatments listed in Table 4-1
of Chapter 4. There was a minimum of six replications per treatment.
Growth boxes were constructed with two plant compartments (416 g of dry sand
each) on opposite sides of a hyphal compartment (225 g of dry sand). The plant
compartments were separated from the hyphal compartment by root-excluding nylon
screens (Tetko, Inc., Depew, N.Y.) with a mesh size of either 15 /m for the grass or
40 fj.m for the pine. The internal dimensions of each plant compartment were 4 x 9 x
11.5 cm (width x length x depth) and 2.5 x 9 x 11.5 cm for the hyphal compartment.
Plant fresh weights were measured at the start of the experiment. Eight pine and grass
plants, inoculated or noninoculated, were used to determine plant water content and
initial P status. After planting, water was added to reach 10% of the soil gravimetric
water content and the boxes were then weighed. Deionized water was added to maintain
this weight during the experiment. Plant compartments were fertilized separately from
the hyphal compartments. In weeks 1, 2, 3 and 6, plant compartments were fertilized
three times weekly with 1.4 /moles P as NaH2P04 along with 10 ml of nutrient solution
containing 2.8 mM NH4N03, 2.8 mM Ca(N03)2, 2.6 mM KC1, 3.4 mM MgS04, 230 /M
NaFeEDTA, 3.2 /M CuS04, 221 /M H3B03, 510 /M NaMo04, 35.1 /M MnCl and
15.9 /M ZnS04. During weeks 4 and 5 they received 15.1 /moles P at each fertilization.
Starting in the fourth week, hyphal compartments received 8.4 /moles P. To prevent


76
1. Benomyl can successfully inhibit development of an AM fungus under controlled
conditions in the greenhouse with no side effects on the EM fungus, Pisolithus
tinctorius.
2. Early in the season with low ground cover in the field, benomyl caused a slight
reduction in arbuscule activity. Later as ground cover increased, systemic
translocation of benomyl from shoot to roots of grasses apparently was
insufficient to reduce mycorrhizal colonization, even at high benomyl
concentrations.
3. Soil drench of benomyl would be a more effective method to place the fungicide
directly at the target site, namely the roots.
4. Sufficient water should be used to permit penetration of benomyl into the soil as
ground cover increases.
Previous studies have demonstrated that mycorrhizas can enhance a plants
competitive ability. The role of mycorrhizas in competition between EM and AM plants
and the effects of different P levels have not been explored specifically. The greenhouse
study I conducted to address these interactions yielded the following results.
1. Both the reduction in P acquisition of grass when grown with pine compared to
another grass at the 32.26-piM P level amd the higher relative crowding
coefficient for total dry weight indicate that pine is more competitive than grass
under the conditions tested.


Total Dry Weight (g) Shoot-P Content (mg P) Shoot-P Cone. (|jg P g'1)
0.32 3.22 32.26
P Applied (pM P), Log Scale
Figure 4-1. Pirns elliottii (A) shoot-phosphorus concentration, (B) shoot-phosphorus
content and (C) total dry weight in response to different competition treatments and
grown at either 0.32, 3.23 or 32.26 /M P for 18 wk. Each symbol represents the mean
of six replicates SE. Inoculated grass was not colonized at the end of the experiment
and therefore was not included in the analysis.


65
lower in the treatments where grass competed with pine (Fig. 4-2B). Total dry weight
of grass was not significantly different at any level of applied P or for any competition
treatment (Fig. 4-2C). In the repeat of the experiment there were no differences in shoot-
P content between the different grass competition treatments, except at the 322.58-/xM
P concentration where grass grown with pine had a 39% higher shoot-P content (P <
0.02) than when grown with another grass. Grass total dry weight at that P concentration
was higher in the interspecific treatment with pine than in the intraspecific treatment with
grass (P < 0.01).
Pine+ had a higher root length than pine' over all treatments (P < 0.001), even
though there were no differences in biomass between inoculated and noninoculated plants
at the beginning of the experiment (Fig. 4-3A). Pine root length did not change with the
level of P applied. In the repeat of the experiment, response of pine root length did not
differ between the competition treatments or between the 0.32- and 3.22-^iM P levels
(data not shown). When grass was grown with grass, there was an increase in grass root
length at the 32.26-/xM P level (Fig. 4-3B) which was paralleled by an increase in shoot-
P content. At the 32.26-/xM P level, grass growing with grass had a higher root length
than grass in the interspecific treatments. When grass was grown with pine+, there was
an increase in grass root length from the 0.32- to the 3.22-^M P level, whereas grass
root length for pine' was not different between P levels. In the repeat of the experiment,
root length increased between the 0.32- and 3.22-/M P levels for grass grown with grass
only (data not shown).


42
DU
<
2
UJ
z
>
>-
_l
^3
O
\-
Q.
1
CL
<
CL
<
D
)
_l
D
Z)
-)
)
Z3
<
UJ
<0
o
o
CM
CD
o
o
T
CM
n
05
o
T
CO
T
CM
T
CM
r-
T
T-
Sampling Date
Figure 3-1. Assessment of arbuscular activity in Panicum chamaelonche roots from the
field site in 1991. (A) Percentage of root length with arbuscules in benomyl-treated and
nontreated plots, (B) Percentage root length with metabolically active arbuscules in
benomyl-treated and nontreated plots and (C) Precipitation. Each symbol represents the
mean of three replicates SE.


4-2 Panicum chamaelonche (A) shoot-phosphorus concentration, (B) shoot-
phosphorus content and (C) total dry weight in response to different
competition treatments and grown at either 0.32, 3.23 or 32.26 /xM P for
18 wk. Each symbol represents the mean of six replicates SE.
Inoculated grass was not colonized at the end of the experiment and
therefore was not included in the analysis 63
4-3 Root-length density of (A) Pinus elliottii and (B) Panicum chamaelonche
in different competition treatments and grown at 0.32, 3.23 or 32.26 /M
P for 18 wk. Each symbol represents the mean of six replicates + SE.
Inoculated grass was not colonized at the end of the experiment and
therefore was not included in the analysis 66
4-4 Relative crowding coefficient (RCC) for (A) Pirns elliottii inoculated with
Pisolithus tinctorius grown in combination with Panicum chamaelonche
and (B) noninoculated Pinus elliottii grown in combination with P.
chamaelonche at either 0.32, 3.23 or 32.26 iM P for 18 wk. Each symbol
represents the mean of six replicates. Mean standard errors were smaller
than the symbols and are therefore not included 67
A1-1 Plant biomass for (A) slash pine and (B) grass grown in the growth
chamber for 62 d. Each bar represents the mean of a minimum of six
replicates SE 86
A1-2 Plant-P content for (A) slash pine and (B) grass grown in the growth
chamber for 62 d. Each bar represents the mean of a minimum of six
replicates SE 87
Al-3 Mycorrhizal colonization of (A) slash pine and (B) grass grown in the
growth chamber for 62 d. Each bar represents the mean of a minimum of
six replicates + SE 88
A1-4 Root-length density for (A) slash pine and (B) grass grown in the growth
chamber for 62 d. Each bar represents the mean of a minimum of six
replicates SE 91
Al-5 Uptake rate of (A) slash pine and (B) grass grown in the growth chamber
for 62 d. Uptake rate was calculated on a unit surface area basis which
included both root and hyphal surface area. Each bar represents the mean
of a minimum of six replicates SE 92
IX


24
plants, that inhibited root growth of nonhost plants. This suggests that mycorrhizas may
have effects beyond those currently known.
In summary, mycorrhizas can enhance a plants competitive ability, and the effect
is generally associated with increased nutrient uptake. The greatest benefit of mycorrhizas
appears to lie in their ability to buffer the plant from adverse environmental conditions
that reduce resource availability.
Mvcorrhiza-mediated reduction of competition
With most plants possessing similar nutritional requirements, competition is a key
factor in their interactions. The existence of hyphal connections between plants is well
known. Various studies, especially those using root-excluding screens, have
unequivocally demonstrated that nutrient transfer between root zones of a donor and
receiver plant can be mediated by mycorrhizal hyphae (Newman, 1988; Newman et al.,
1992). Although it is possible for hyphae from the receiver mycorrhiza to scavenge
nutrients from the rhizosphere of the donor plant, most likely the majority of transfer is
by direct hyphal connections between plants. For example, radio-labelled C from an
ectomycorrhizal donor plant has been found solely in ectomycorrhizal plants and not in
neighbor AM neighbor plants; by using autoradiography, no visual evidence existed of
a direct interspecific C transfer between intermingling roots of donor and receiver plants
(Read et al., 1985). In another study, 46% of the total C transferred directly from plant
to plant was via mycorrhizal connections, 15% of uptake was indirectly mediated by
mycorrhiza, and 39% was translocated by other processes (Martins, 1993). These


26
herbivory and to produce a C sink resulted in C transfer away from the clipped plant
(Waters and Borowicz, 1994). In settings where young seedlings compete for nutrients
with established plants, the seedlings become more quickly colonized by the preexisting
mycorrhizal network; however, no further benefit to the seedlings was detected (Franson
et al., 1994; Eissenstat and Newman, 1990). In Grimes (1987) study, 14C-labelling of
one dominant plant resulted in substantially more C being transferred to subdominants
when plants were mycorrhizal compared to nonmycorrhizal. Although competition does
occur in these systems, several plants colonized by the same mycorrhizal type will be
closely tied together by the hyphal network and may benefit from C transfer among
plants.
Environmental Conditions and Plant Competition
Non-resource edaphic factors
Several soil characteristics may indirectly influence the assorted mycorrhizal
mechanisms that enhance a plants competitive ability. Soil acidity is an important factor
influencing soil nutrient availability. Acidic soils are a natural result of soil weathering,
and, as stated earlier, Al toxicity is one of the main associated problems. Mycorrhizas
may enable a plant to survive unfavorable conditions caused by toxic concentrations of
metal cations, including Al (Koslowsky and Boerner, 1989). Although mycorrhizas may
facilitate growth of plants under acidic soil conditions, I am not aware of any studies that
systematically address the effect this may have on plant competition.


66
0.32 3.22 32.26
P Applied (pM P), Log Scale
Figure 4-3. Root-length density of (A) Pinus elliottii and (B) Panicum chamaelonche in
different competition treatments and grown at 0.32, 3.23 or 32.26 /xM P for 18 wk. Each
symbol represents the mean of six replicates SE. Inoculated grass was not colonized
at the end of the experiment and therefore was not included in the analysis.


17
competitive advantage (Campbell and Grime, 1989). This suggests that mycorrhizal
plants may profit from the reduced root turnover rate by having to invest less C into
nutrient absorbing structures.
Plant fitness
Although many of the previous topics dealt with improving plant growth and
stress adaptation, few mycorrhizal studies have directly addressed mycorrhizal influence
on plant fitness, that is the plants ability to increase its numbers proportionately to other
species (Begon et al., 1986). Enhanced efficiency of resource acquisition by mycorrhizal
plants allows more energy to be allocated to growth and reproduction, which potentially
increases plant fitness. The result in the next generation may be expressed in terms of
improved survival, growth rate or reproduction. Mycorrhizal plants have displayed
increased seed number, seed weight and P and N content (Lu and Koide, 1994), with
some benefits still significantly expressed in the second generation of offspring grown
in the absence of mycorrhizal fungi (Koide, T. and Lu, 1992). Increased P status of seed
has been associated with subsequently higher P content and plant biomass (Bolland and
Paynter, 1992). For some plant species, the presence of mycorrhizas enhanced seedling
emergence rate (Hartnett et al., 1994). High P concentration in seed has resulted in
increased number of emerging seedlings and a higher rate of emergence (Thomson and
Bolger, 1993), factors which also have been identified as important predictors of
competitive success in secondary succession (Stockey and Hunt, 1994).


12
and Schepp, 1993) and, thus, probably lack any significant protease release.
Phosphatases are produced by plant roots (Duff et al., 1994; Tarafdar and Claassen,
1988; Cumming, 1993) and release P from bound organic forms close to the root.
Phosphatase release by mycorrhizal fungal hyphae also has been demonstrated (Antibus
et al., 1992; Jayachandran et al., 1992; Tarafdar and Marschner, 1994) and may increase
plant uptake of P; however, mycorrhizal contribution to total P uptake by this particular
mechanism has not been quantified yet. Extension of hyphae beyond the root depletion
zone would permit solubilization and uptake of P from these unavailable organic-P forms.
For some nonmycorrhizal plants, mobilization of the organic-P fraction can approach one
third of the total P absorbed (Jungk et al., 1993). Proton release by roots, in part to
compensate for NH4+ uptake, can create a substantially lower rhizosphere pH (Marschner
and Romheld, 1983) and acidification of the soil also has been documented in
mycorrhizal systems (Li et al., 1991b). Although this enhances dissolution of iron (Fe),
and consequently bound P, the rate of protonation is slower than by the chelating
mechanism (Schwertmann, 1991).
The availability of inorganic P bound to aluminum (Al) and Fe minerals can be
increased by organic anions, such as oxalate (Fox et al., 1990), by the processes of
chelation, ligand exchange and dissolution of the metallophosphate complex.
Ectomycorrhiza, which form fungal mats, are capable of substantially altering the
chemical soil environment by increasing the oxalate anion concentration by several orders
of magnitude (Griffiths et al., 1994). Soil-solution phosphate in these mats is strongly
correlated with oxalate concentration. Fungi most likely vary in the quantity of organic


58
To compare the competitive abilities of the two plant species, the relative
crowding coefficient (RCC; Harper, 1977) was calculated. To avoid subjective pairing
of plants between treatments all possible combinations were used to calculate the RCC.
The following is a sample calculation of the RCC for grass total dry weight when
growing with pine:
grass (interspecific)
pine (interspecific)
RCC (shoot P, mg P) =
grass (intraspecific)
pine (intraspecific)
Data for grass and pine were analyzed separately. To determine if plant
competition was affected by the plant species, data for each plant species were subjected
to analysis of variance and statistically planned contrasts using the General Linear Model
(SAS Institute, Inc., 1989). Data for colonization were transformed to arcsine square
roots prior to analysis (Steel and Torrie, 1980). The least-squares means statement within
SAS was used to compare means.
Determination of P Uptake Kinetics for Pine and Grass
Pine and grass plants were inoculated with their respective mycorrhizal fungi or
noninoculated. Pine plants were grown in Deepots for a further 24 wk after inoculation
with P. tinctorius. Grasses were inoculated 3 wk prior to transferal to 1-L Erlenmeyer
flasks by applying to each plant root system a minimum of 20 spores of a mixed culture
of Gigaspora rosea (INVAM FL224) and Scutellospora heterogama (INVAM FL225),


19
Metal cations and soil acidity
High metal cation concentrations can be toxic to plants. The high solubility of Al,
due to the acidic nature of Oxisols and Ultisols, is a growth-limiting factor for plants in
many tropical countries. Natural selection of mycorrhizal ecotypes leads to varying
genotypic sensitivity to soil acidity (Robson and Abbott, 1989), as well as to high metal
concentrations (Gildon and Tinker, 1983; Griffioen et al., 1994). Several studies have
found mycorrhizas capable of alleviating toxic effects to plants caused by Al, cadmium,
Cu and Zn (Bradley et al., 1982; Colpaert and Van Assche, 1993; Denny and Wilkins,
1987; Dueck et al., 1986; Koslowsky and Boerner, 1989). Two mechanisms currently
explain this response. Firstly, electronegative sites on the hyphal cell walls bind the
positively charged heavy metal cations (Denny and Wilkins, 1987; Galli et al., 1994).
The observation that under acidic soil conditions heavy metal uptake is increased
(Killham and Firestone, 1983) partly confirms this. It is possible that protonation of
negatively charged sites in the plant or fungal walls results in less binding and greater
uptake of the metal cation. The second path is the immobilization of the cations by
complexation in vacuoles with polyphosphates (Martin et al., 1994) or associated
metallothionein-like peptides (Turnau et al., 1994).
Synecologv
Co-evolution of mycorrhizal fungi and plants has been suggested (Allen, 1991;
Harley and Smith, 1983). Since selection for more fit species occurs continuously, and
a larger proportion of plants show mycorrhizal dependency than not, it follows that there


108
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Gehring C.A. and T.G. Whitham. 1994. Interactions between aboveground herbivores
and the mycorrhizal mutualists of plants. Trends Ecol. Evol. 9:251-255.
George E., K.U. Hussler, D. Vetterlein, E. Gorgus, and H. Marschner. 1992. Water
and nutrient translocation by hyphae of Glomus mosseae. Can. J. Bot. 70:2130-2137.


noninoculated with the ectomycorrhizal (EM) fungus, Pisolithus tinctorius (isolate S106).
The effect of P (0.32, 3.23 or 32.26 /xM) on competition also was analyzed in the
greenhouse because resource abundance can affect the outcome of competition and
inorganic P is limiting in pine plantations. Inoculated grasses were not colonized at the
end of the experiment and were excluded from data analyses. When competing with
grass, inoculated pine acquired more P and had a higher total dry weight than
noninoculated pine. Grass shoot-P content was reduced at the 32.26-/zM P level when
grown with pine, irrespective of the pine inoculation treatment.
I evaluated the effects of benomyl over time in the field and at 0, 20, 60 and 150
kg benomyl ha'1 equivalent in the greenhouse. My objective was to determine if benomyl
would be suitable for controlling AM but not EM fungi as part of a larger competition
experiment involving pine and weeds. No effect was observed on pine in the greenhouse.
Colonized root length of benomyl-treated Zea mays L. plants in the greenhouse remained
static and the response was not dose-dependent. In contrast, colonization in the control
plants increased over time. Minimal reduction of grass colonization was observed in the
field, where limitations to effective control were ground cover, timing in relation to
mycorrhizal development and benomyl application as a spray instead of as a soil drench.
Xll


CHAPTER 1
INTRODUCTION
Mycorrhizal contribution to plant nutrient uptake, especially phosphorus (P), has
been extensively studied. Under nutrient-limiting conditions mycorrhizas are able to
enhance plant nutrient uptake by various mechanisms, thereby ameliorating plant stress.
Under these conditions plants compete for limited nutrients and mycorrhizas may modify
the competitive interactions between plants. Little work has addressed the role of
mycorrhizas in this area of plant synecology. The existing studies primarily deal with
arbuscular mycorrhizal (AM) fungi and their contribution to plant interactions. Many of
these studies investigated facilitative mycorrhizal plant associations, where mycorrhizal
fungi transfer nutrients from one plant to another through common hyphal connections.
Only one study addressed the function of ectomycorrhizal (EM) fungi in plant
competition. No studies to date have evaluated the role of mycorrhizas in competitive
interactions between AM and EM plants, which occurs frequently during succession. In
the following chapter, I review the various aspects of mycorrhizal functioning in plant
autoecology and synecology and I detail their role in plant competition.
Most previous studies have been performed under controlled conditions in the
greenhouse without the influence of complex interactions of other environmental
variables. When bringing mycorrhizal questions to the field, one of the more difficult
problems is the creation of a suitable nonmycorrhizal control, since a majority of plants
1


28
Associated soil biota
Fitter and Garbaye (1994) have summarized the current information about
belowground interactions of mycorrhizas and rhizosphere microbes. Unfortunately, few
studies have addressed how these interactions affect plant populations or communities.
Mycorrhizas influence the rhizosphere environment by modifying plant exudation and
rhizodeposition (Leyval and Berthelin, 1993), and subsequently affect microbial
composition and metabolic activity in varying degrees. Inversely, certain fluorescent
pseudomonads and spore-forming bacilli, similar to growth-promoting rhizobacteria, may
significantly regulate the mycorrhizal benefit to the plant (Garbaye, 1994; Schreiner and
Koide, 1993); however, mechanisms of action are still largely unknown. These bacteria
have demonstrated some fungal, but not plant, host specificity. With appropriately
matched mycorrhizal fungi and bacteria it is conceivable that a plant would possess a
competitive advantage over other plants without selected associations. Rabatin and Stinner
(1991) reviewed the effects of microfauna, many of which are fungivores, on
mycorrhiza. As an example, Boerner and Harris (1988) conducted a competition study
between mycorrhizal Panicum virgatum and the nonhost Brassica napa, where the
addition of Collembola reduced the competitive ability of the grass, resulting in a
reduction of biomass compared to the mycorrhizal P. virgatum without competition.
Studies of plant disease control by mycorrhizas interacting with plant pathogens
have yielded variable results (Linderman, 1994; Duchesne, 1994). Various mechanisms
have been reported that are unique to the environment, host and microbes involved.
Based on field studies utilizing the fungicide benomyl, Carey et al. (1992) suggested that,


Table 2-1. Summary of nutrient uptake kinetic studies.
Fungus
Host
Nutrient
concentration
OiM)
Km
(pM)
^max
c
^min
(pM)
Reference
Glomus fasciculatum
Lycopersicon esculentum
(root segments)
1-100 kh2po4
1.61-0.35
0.1-0.32*
n.dc
(Cress et al., 1986)
none
w
w
3.9-42
0.10-0.25 Ia
n.d
Glomus mosseae
Glycine max
(whole plant)
30 KH2P04
20
58b
n.d
(Karunaratne et al.,
1986)
none
w
3.5
19b
n.d.
Pisolithus tinctorius
Pinus caribea
(whole plant)
20 Na2HP04
3.89
0.30a
0.32
(Pacheco and
Cambraia, 1992)
none
w
ft
16.44
0.23a
11.98
Glomus
macrocarpon
Zea mays
(root segments)
1.5-1070 Zn
5.3-0.38
0.08-0.47"
n.d.
(Sharma et al., 1992)
none
M

4.5-0.95
0.03-0.55"
n.d.
# imol g fresh weight'1 h'1
b nmol m'2 s'1
c n.d. = not determined


11
in another, could survive in the same environment due to niche separation. Mycorrhizas
may add flexibility to a plants physiological strategy by allowing it to profit from a
broader range of nutrient uptake mechanisms. Furthermore, mycorrhizas may add an
element of efficiency to soil nutrient exploitation by roots. Campbell et al. (1991)
proposed that plant species primarily acquire resources either by efficiently exploiting a
given resource (precision foraging) or by extensive development of roots and occupation
of a high resource site (scale foraging). Although untested, mycorrhizas may give an
advantage to the plant group with a tendency to efficiently exploit a given soil volume
by accessing nutrients outside of the roots nutrient depletion zone.
Exudates and secretions
Root and hyphal secretions and exudates modify nutrient availability in the soil
(Darrah, 1993; Duff et al., 1994). Several different mechanisms are involved and include
release of enzymes, chelating agents such as organic anions and siderophores, and
changes in rhizosphere pH by C02 from respiration and H+ excretion during uptake of
cations.
Nitrogen and phosphorus are often present in organic forms (Stevenson, 1986),
which are less available than the organic forms. Ectomycorrhizal and ericoid fungi permit
plant utilization of organic N from proteins and peptides (Abuzinadah and Read, 1986;
Abuzinadah and Read, 1989; Bajwa et al., 1985; Bajwa and Read, 1985), which are
otherwise unavailable N sources to plants (Abuzinadah and Read, 1986). Arbuscular-
mycorrhizal fungi do not appear capable of utilizing complex organic-N sources (Frey


LIST OF FIGURES
Figure
page
2-1 Length of external hyphae spreading from mycorrhizal roots of Trifolium
subterraneum after (a) 28 days and (b) 47 days. Bars represent standard
error of the mean [with permission from (Jakobsen et al., 1992)] 7
3-1 Assessment of arbuscular activity in Panicum chamaelonche roots from
the field site in 1991. (A) Percentage of root length with arbuscules in
benomyl-treated and nontreated plots, (B) Percentage root length with
metabolically active arbuscules in benomyl-treated and nontreated plots
and (C) Precipitation. Each symbol represents the mean of three replicates
SE 42
3-2 Total dry weight of mycorrhizal (M) and nonmycorrhizal (C) plants, (A)
Pirns elliottii and (B) corn in response to 0, 20, 60 or 150 kg benomyl ha'
1 in the greenhouse. Each symbol represents the mean of seven replicates
SE 45
3-3 Soil hyphal length (total) and activity (active) of mycorrhizal (A) Pinus
elliottii and (B) corn plants in response to 0, 20, 60 or 150 kg benomyl
ha'1 in the greenhouse. Each symbol represents the mean of seven
replicates SE 46
3-4 Mycorrhizal colonization of (A) slash pine and (B) corn grown in the
greenhouse in response to 0, 20, 60 or 150 kg benomyl ha'1. Each symbol
represents the mean of seven replicates SE 47
4-1 Pinus elliottii (A) shoot-phosphorus concentration, (B) shoot-phosphorus
content and (C) total dry weight in response to different competition
treatments and grown at either 0.32, 3.23 or 32.26 /xM P for 18 wk. Each
symbol represents the mean of six replicates SE. Inoculated grass was
not colonized at the end of the experiment and therefore was not included
in the analysis
vm
62


6
in improved nutrient uptake by mycorrhizal plants include the amount of absorbing
surface area, fungal growth rates, nutrient uptake kinetics and hyphal distribution.
Hyphae can extend far beyond the nutrient depletion zone (primarily P) of roots. Using
an exclusion screen technique, Li et al. (1991a) located hyphae of Glomus mosseae up
to a maximum measured distance of 11.7 cm from roots of Trifolium repens L. after 49
d. Ectomycorrhizal rhizomorphs are likely to extend substantially further.
In a separate comparative study on arbuscular-mycorrhizal (AM) fungi and P
uptake, Acaulospora laevis, Glomus sp. and Scutellospora calospora developed hyphae
up to 11 cm from the roots of the host plant, Trifolium subterraneum L., after 47 d (Fig.
1). However, hyphal densities with increasing distance from the mycorrhizal roots were
not the same for all fungi. Acaulospora laevis had a constant hyphal density up to 11 cm,
while for Glomus sp. it decreased after 3 cm, and for S. calospora the highest hyphal
density was observed closest to the root and declined exponentially thereafter. The hyphal
P uptake rates for the three fungi (calculated average for 28-47 d) were 2.8, 0.8 and 0.6
fmol P m1 s'1, respectively, with considerably higher rates for the initial 28-day period.
The consequence of these differences was a substantial contrast in plant P content among
the mycorrhizal treatments. The previously listed characteristics of absorbing surface
area, fungal growth rates, nutrient uptake kinetics and hyphal distribution indicative of
improved nutrient uptake were all favorable in the A. laevis treatment, which was also
associated with the highest plant P content.
Depending on the mycorrhiza and the initial soil nutrient concentration, the
contribution by hyphae to total plant nutrient uptake (Marschner and Dell, 1994;


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
INFLUENCE OF MYCORRHIZAS ON PLANT COMPETITION FOR
PHOSPHORUS BETWEEN SLASH PINE AND GRASS
By
Christian Thomas Pedersen
December 1995
Chairperson: Dr. D. Sylvia
Major Department: Soil and Water Science
Individual plants benefit from the mycorrhizal condition primarily by improved
nutrient uptake, especially of phosphorus (P), resulting in enhanced plant survival and
growth in resource-limited conditions. On a broader scale, mycorrhizas have the potential
to mediate plant competition and subsequently may be important at the community level.
In the southeastern United States, slash pine (Pinus elliottii Engelm. var. elliottii)
is grown in plantations, where it competes for nutrients with grasses and other
herbaceous vegetation. One objective of my research was to assess mycorrhizal
contribution to intra- and interspecific plant competition for P in the greenhouse between
pine and Panicum chamaelonche Trin., a dominant grass species at a local plantation site.
The grass was inoculated or noninoculated with the arbuscular mycorrhizal (AM) fungus,
Glomus sp. (INVAM FL329, formerly INVAM FL906), and pine was inoculated or
xi


60
Twenty milliliters of concentrated HC1 were added to the sample and also evaporated to
dryness. Phosphorus was determined colorimetrically by a slightly modified procedure
of Murphy and Riley (1962). Reagent, quantitatively diluted with deionized water, was
added directly to the samples. Since some solutions were at the detection limit, less
reagent was added to later samples in order to concentrate them. The resulting P-
depletiOn curve was fit using a curve-fitting procedure (SigmaPlot; Jandell Scientific, San
Rafael, California), and uptake calculations were made based upon this idealized curve.
Total surface area of roots and hyphae were determined using image analysis software
(Mocha; Jandell Scientific, San Rafael, California) or gridline-intersect methods
(Giovannetti and Mosse, 1980). The maximum uptake rate, 1^, was calculated based on
the root surface area and the quantity of P absorbed from the nutrient solution during the
first 45 min. The minimum solution concentration from which a nutrient can be
absorbed, C^, was considered the asymptotic value where the solution P concentration
no longer decreased.
Results
Greenhouse Competition Study
No colonization was found in the inoculated grass plants at the end of the
experiment. Therefore these treatments were excluded from further analyses. Also, the
ubiquitous EM fungus, Thelephora terrestris (Ehrh.) Fr., was found growing on the
noninoculated pine (pine ) treatments, but not in the inoculated pine (pine+) treatments.
The mean soil solution pH was 4.0.


To Karen,
for her love, support, patience
and her sense of humor that carried
us both through this adventure


112
Koide R.T., L.F. Huenneke, S.P. Hamburg, and H.A. Mooney. 1988. Effects of
applications of fungicide, phosphorus and nitrogen on the structure and productivity of
an annual serpentine plant community. Funct. Ecol. 2:335-344.
Koide, R., T. and X. Lu. 1992. Mycorrhizal infection of wild oats: Parental effects on
offspring nutrient dynamics, growth and reproduction, p. 55-58. In D.J. Read, D.H.
Lewis, A.H. Fitter and I.J. Alexander (ed.) Mycorrhizas in Ecosystems. CAB
International, Wallingford, United Kingdom.
Koslowsky S.D. and R.E.J. Boerner. 1989. Interactive effects of aluminum, phosphorus
and mycorrhizae on growth and nutrient uptake of Panicum virgatum L. (Poaceae).
Environ. Poll. 61:107-125.
Kothari S.K., H. Marschner, and V. Romheld. 1991. Contribution of the VA
mycorrhizal hyphae in acquisition of phosphorus and zinc by maize grown in calcareous
soil. Plant Soil 131:177-186.
Kough J.L., V. Gianinazzi-Pearson, and S. Gianinazzi. 1987. Depressed metabolic
activity of vesicular-arbusuclar mycorrhizal fungi after fungicide application. New
Phytol. 106:707-715.
Krishna K.R., K.G. Shetty, P.J. Dart, and D.J. Andrews. 1985. Genotype dependent
variation in mycorrhizal colonization and response to inoculation of pearl millet. Plant
Soil 86:113-125.
Krishna K.R., H.M. Suresh, J. Syamsunder, and D.J. Bagyaraj. 1981. Changes in the
leaves of finger millet due to VA mycorrhizal infection. New Phytol. 87:717-722.
Kucey R.M.N. and E.A. Paul. 1982. Carbon flow, photosynthesis, and N2 fixation in
mycorrhizal and nodulated Faba beans (Vicia faba L.). Soil Biol. Biochem. 14:407-412.
Kuiters A.T. and H.M. Sarink. 1986. Leaching of phenolic compounds from leaf and
needle litter of several deciduous and coniferous trees. Soil Biol. Biochem. 18:475-480.
Larsen, J., I. Jakobsen, I. Thingstrup, and S. Rosendahl. 1994. Benomyl inhibits hyphal
P transport but not fungal alkaline phosphatases in a cucumber-G/o/nwj caledonium
symbiosis. -155. In Fourth European Symposium on Mycorrhizas. Granada, Spain, July
11-14, 1994.
Leake J.R., G. Shaw, and D.J. Read. 1989. The role of ericoid mycorrhizas in the
ecology of ericaceous plants. Agrie., Ecosys. Environ. 29:237-250.


Table Al-1. Tests for single degree of freedom contrasts for root-length density, plant biomass, plant
P content and percent colonization. Each parameter was analyzed separately for grass and pine.
Root-Length Density
(m cm"3 of soil)
Plant
Biomass
(g)
Plant P
Content
(mg P g1)
Colonization
(%)
Uptake Rate
(fmol P cm'2 s'1)
Contrasts for grass:
Grass+ / grass'
.06
n.s.
.02
.001
.05
(Grass+ / grass') over all pine
.06
n.s.
.03
.02
n.s.
(Pine+ / pine ) over all grass
.04
n.s.
n.s.
n.s.
n.s.
Intra- / interspecific
n.s.
n.s.
n.s.
.05
n.s.
Contrasts for pine:
Pine+ / pine'
<.001
.001
.01
.006
.03
(Pine+ / pine ) over all grass
<.001
<.001
.06
.01
n.s.
(Grass+ / grass') over all pine
n.s.
n.s.
n.s.
n.s.
n.s.
Intra- / interspecific
n.s.
n.s.
.07
n.s.
n.s.
oo
4^


93
Uptake rates for both pine+ and grass+, based on the combined surface area of
roots and hyphae, were lower than for the respective noninoculated treatments (Fig. Al-
5, Table Al-1). Uptake rate based on root surface area alone increased the difference
(data not shown). There were no differences in uptake rates for either pine or grass
between their respective intra- and interspecific competition treatments.
Soil-P content for grass+ was higher than grass', whereas pine+ had slightly less
P in the soil than pine' (Table Al-3).
Discussion
The higher plant biomass and P content of pine+, compared to pine', were
associated with increased root-length density in the pine+ treatments. The mycorrhizal
fungus apparently contributed to increased growth of pine+ which is supported by the
lower soil-P content in the pine+ compartments. Although pine' was found to have higher
levels of colonization than pine+, the EM fungus, P. tinctorius, was more effective than
T. terrestris at increasing plant growth. It was not possible to determine the additional
quantity of P contributed by mycorrhizal fungi since the nonmycorrhizal control was lost
when it was colonied by T. terrestris. The lower uptake rate in pine+ plants with a higher
surface area may have occurred for two reasons: (i) P depletion zones overlapped due
to a high density of absorbing surface area or (ii) not all of the surface area used in the
calculation was involved in nutrient absorption.
Sample calculations with hyphae alone demonstrate that hyphal depletion zones
overlapped even at the lowest hyphal density measured. This would result in less P


20
must be some measure of improved fitness derived from mycorrhiza; otherwise the
symbiosis would have been selected against. The alternative is that mycorrhizal fungi are
parasites with maximum adaptability to plant resistance strategies. This, however, is
unlikely considering the exchange of nutrients between the two organisms, which is
characteristic of a true mutualism.
The effects of the mutualism on plant growth and survival influence interactions
beyond the single plant level (Brundrett, 1991; Francis and Read, 1994). Plants rarely
grow alone, except in extreme or anthropogenic environments, and consequently end up
competing for similar resources, especially inorganic nutrients, water and light. Under
conditions limiting growth, mycorrhizal plants have distinct competitive advantages.
Thus, from a holistic and functional perspective, mycorrhizal research reaches its full
value when applied to natural or managed ecosystems where interactions occur. Current
issues pertain to the involvement of mycorrhizas in plant community development,
stabilization and diversity, as well as to questions of environmental sustainability and the
economics of agricultural production systems. A relevant question, then, is to what extent
is the force of this symbiosis manifested in plant communities?
Plant Interactions
During competition, plants utilize several different strategies for optimal resource
capture with many of them overlapping those found in the mycorrhizal symbiosis. The
choice of strategy depends primarily on a sites resources and the amount of disturbance
(Grime, 1979; Tilman, 1982). Literature summarized in the first part of this chapter has


41
benomyl ha"1 equivalent. Plants were grown from March to May 1993 under a mean
photosynthetic photon flux density of 608 /mol m"2 s'1 and 18/35C (min./max.)
temperature regime.
The plants were sampled before, and then 2, 4 and 6 wk after benomyl
application. The harvest procedures were the same as for pine, with the exception of
estimation of root colonization. Washed root segments (1 to 2 cm) were cleared with
10% KOH for 30 min, rinsed several times with tap water, acidified for 30 min in 5%
HC1 and stained overnight in 0.05% aniline blue in lactoglycerol. Colonization was
determined using a gridline-intersect method (Giovannetti and Mosse, 1980). Although
fungi other than AM existed in this particular system, the differentiation of saprophytic
from characteristic AM fungal hyphae was based on gross morphological differences.
Arbuscular mycorrhizal fungi generally had a somewhat larger hyphal diameter (4
compared to <2 /m), stained darker with aniline blue, were not dematiacious, lacked
septation or clamp connections and demonstrated a less angular growth pattern compared
to other fungi present. Prior to statistical analysis, percentage colonization was
transformed using the arcsine, square root transformation.
Results
Field Study
Initial AM colonization of P. chamaelonche in the field was high, indicating that
root growth and mycorrhizal development commenced earlier than the first fungicide


ACKNOWLEDGMENTS
I thank Dr. David Sylvia for giving my wife and me a warm welcome to Florida
and for his continued support throughout my studies. I also thank the members of my
supervisory committee, Drs. Nicholas Comerford, James Graham, David Mitchell and
Donn Shilling, for their inputs. I would like to especially thank Drs. David Mitchell,
David Hubbell and Suresh Rao for their major contributions to the philosophical aspects
of the Doctorate in Philosophy. Appreciation is extended to the National Science
Foundation for partially funding this research (Grant No. BSR-9019788). The opportunity
to use the laboratory facilities of Drs. Rao and Comerford is gratefully acknowledged.
Mary McLeod, Dongping Dai, Drs. Amiel Jarstfer and Linda Lee deserve thanks for
their invaluable technical guidance and patience throughout the process. Thanks also go
to the many laboratory assistants that have stood by my side and who probably do not
realize how much they have contributed. I appreciate Mike Allens technical support with
my Benlate field work. To the many graduate students who have passed through and the
few that still remain: The interactions we had were valuable to me. Specifically, I would
like to thank Dr. Pauline Grierson, Len Scinto and Dr. Philip Smethurst for their
supportive conversations that put things in perspective. Many thanks also to David
Farmer and Steve Trabue for the late night reality checks on the second floor of McCarty
Hall. Lastly, I am grateful for my familys love and support, which, even though they
were far removed from my research activities, was of great importance.
in


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94
uptake on a unit surface area basis, which was expressed by the uptake rate calculations.
Pine uptake rate was substantially lower then grass which was due to the much higher
pine absorbing surface area attributable to external hyphae of mycorrhizal fungi. Since
the observed differences are not completely explained by this, it is likely that not all
hyphal surface area was active in uptake.
Even though AM fungi of grass, had access to the additional P in the hyphal
compartment, grass+ growth was not stimulated. In fact, grass+ had a lower P content
than grass' when competing with pine, inoculated or not. Colonization of grass+ and AM
hyphal lengths were lower when competing with pine. It is not clear why this reduction
in AM growth occurred; however, it is possible that EM fungi may have exhibited some
form of antibiosis. Due to the distance between plants it is less likely that the effect was
induced by pine.
A total P budget based on soil and plant P content was not calculated since the
pine" plants became colonized and the AM fungi did not contribute to increased P uptake
from the hyphal compartment.
In conclusion, inoculation of pine with P. tinctorius enhanced pine P uptake over
the pine' treatment, which was colonized by T. terrestris. Competition between pine and
grass could not be addressed adequately due to the lack of AM contribution to grass
uptake.


115
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40:161-170.
Newman E.I. 1988. Mycorrhizal links between plants: Their functioning and ecological
significance. Adv. Ecol. Res. 18:243-270.
Newman E.I. and W.R. Eason. 1993. Rates of phosphorus transfer within and between
ryegrass (Lolium perenne) plants. Funct. Ecol. 7:242-248.
Newman E.I., W.R. Eason, D.M. Eissenstat, and M.I.R.F. Ramos. 1992. Interactions
between plants: the role of mycorrhizae. Mycorrhiza 1:47-53.
NewshamK.K., A.H. Fitter, and A.R. Watkinson. 1994. Root pathogenic and arbuscular
mycorrhizal fungi determine fecundity of asymptomatic plants in the field. J. Ecol.
82:805-814.
Newsham K.K., A.R. Watkinson, H.M. West, and A.H. Fitter. 1995. Symbiotic fungi
determine plant community structure: Changes in a lichen-rich community induced by
fungicide application. Funct. Ecol. 9:442-447.
Nilsson M.-C. 1994. Separation of allelopathy and resource competition by the boreal
dwarf shrub Empetrum hermaphroditum Hagerup. Oecologia 98:1-7.


Plant Biomass (g)
86
Figure Al-1. Plant biomass for (A) slash pine and (B) grass grown in the growth
chamber for 62 d. Each bar represents the mean of a minimum of six replicates SE.


LIST OF TABLES
Table Page
2-1 Summary of nutrient uptake kinetic studies 9
3-1 Test for homogeneity of slopes for the effect of Benlate 50 DF applied
in the field on percent Panicum chamaelonche roots with arbuscules and
their activity over time 43
4-1 Pirns elliottii (pine) and Panicum chamaelonche (grass) treatment
combinations planted in the competition study. Two plants were planted
per pot. The superscripts + and signify an inoculated or
noninoculated plant respectively. Pine was inoculated with Pisolithus
tinctorius and the grass was inoculated with Glomus sp. (INVAM FL329,
formerly FL906) 56
4-2 Ergosterol concentration (/xg g1) of Pinus elliottii roots inoculated with
Pisolithus tinctorius (pine+) or noninoculated (pine'), and grown in
combination with Pinus elliottii (pine) or Panicum chamaelonche (grass)
at 0.32, 3.23 or 32.26 /xM P for 18 wk. Each value represents the mean
of six replicates SE 64
4-3 Initial phosphorus uptake rate, I^ (/xmol P cm'2 s'1) and (/xM P), the
minimum solution concentration from which a nutrient can be absorbed,
for Pinus elliottii and Panicum chamaelonche grown in a hydroponic
solution containing 0.32 /xM P. Each value represents the mean of three
replicates SE 68
Al-1 Tests for single degree of freedom contrasts for root-length density, plant
biomass, plant P content and percent colonization. Each parameter was
analyzed separately for grass and pine 84
vi


43
Table 3-1. Test for homogeneity of slopes for the effect of Benlate 50 DF applied in the
field on percent Panicum chamaelonche roots with arbuscules and their activity over
time.
Slope over time
Roots with arbuscules
(%)
Roots with active arbuscules
(%)
Control
-0.104 **
-0.164 **
Benlate
-0.012
-0.010
indicates slope value is significantly different from 0 at P < 0.01


Plant Biomass (g) Plant-P Content (mg P)
100
Figure A3-1. Nonmycorrhizal Panicum chamaelonche (A) plant biomass and (B) plant
phosphorus content in response to 0.001, 0.003 0.010, 0.032, 0.100, 0.316, 0.1000,
3.162 or 10.000 mg P L1. Each symbol represents the mean of seven replicates SE.


49
dependent response by the mycorrhizal grass in the greenhouse. The range of
concentrations was based on previous values published in the literature (Trappe et al.,
1984). The sand used in the greenhouse minimized possible adsorption phenomena that
normally occur in field soils. Consequently, all the concentrations tested were above the
threshold required to obtain a maximum inhibition of mycorrhizal development. Not only
was all of the fungicide readily available, but it was also well above the manufacturers
recommended application rate, which together presumably caused a decrease in plant
biomass unrelated to the plants mycorrhizal status. In agreement with previous literature
(Trappe et al., 1984), the effect of benomyl on mycorrhizal pine was neutral although
sometimes an increase in growth has been observed (De la Bastide and Kendrick, 1990;
Pawuk and Barnett, 1981).
Arbuscules were counted in the field, since they are a distinguishing characteristic
of the mycorrhizal fungus, and, more importantly, they represent the site where active
exchange of nutrients between the symbionts occurs. The initial decrease in arbuscule
activity in the field following benomyl application has been documented in a greenhouse
study as well (Sukarno et al., 1993). Since the response to 5 kg benomyl ha'1 was minor
compared to total colonization, the benomyl application rate was increased. At the last
sampling as the plants started to senesce, the increase in arbuscule number and activity
in roots of plants treated with benomyl may be due to a reduction in the impact of
nonmycorrhizal fungi on plant growth and subsequent mycorrhizal functioning. Low
colonization and arbuscule numbers in the greenhouse study made it difficult to obtain
a reliable measure of arbuscule abundance to compare to the results in the field. The lack


56
Table 4-1. Pirns elliottii (pine) and Panicum chamaelonche (grass) treatment
combinations planted in the competition study. Two plants were planted per pot. The
superscripts + and signify an inoculated or noninoculated plant respectively. Pine
was inoculated with Pisolithus tinctorius and the grass was inoculated with Glomus sp.
(INVAM FL329, formerly FL906).
Plant Competition Treatments
Intraspecific
Interspecific
pine+ x pine+
pine+ x grass+
pine' x pine
pine+ x grass'
grass+ x grass+
pine' x grass+
grass' x grass'
pine' x grass


121
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of mycorrhizae of the submersed macrophyte, Vallisneria americana. Estuaries
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Wilson A.D. and D.J. Shure. 1993. Plant competition and nutrient limitation during early
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30
and West (1994) found that compared to fungicide-treated plants, mycorrhizal plants had
lower soluble neutral sugars, starch, total N, and amino acids (alanine and
tyrosine/valine) and a higher concentration of the anti-feedant chemicals, aucubin and
catalpol. In their study, chewing insects were negatively impacted when feeding on
mycorrhizal plants; however, sucking insects developed better on mycorrhizal plants. The
authors hypothesized that a higher C/N ratio in the mycorrhizal plants allowed more C
to be allocated to plant defense mechanisms, such as secondary plant metabolite
production. Localization of the secondary metabolites may partly account for the
differential response between insect types. Viewed in terms of plant competition, plants
able to efficiently modify their C balance to simultaneously reduce insect pests and still
maintain the mycorrhizal association may have a competitive advantage in the long run.
Plant Succession and Community Structure
Limited information is available on the ecological relevance of mycorrhizas in
plant competition. Plant competition can be viewed in terms of single plant interactions,
but its importance lies at the population and community levels. The interactions occurring
at the ecosystem level are obviously complex and many have been set aside for the sake
of simplicity. As has been suggested by various authors (Brundrett, 1991; Francis and
Read, 1994; Newman, 1988) mycorrhizas are likely involved in plant community
structuring, but the magnitude of their effect is unknown. Increasing the competitive
ability of individuals within a population enhances their ability to capture resources and
improves their fitness. One of the major components determining early succession is


106
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2,4-dichlorophenoxyacetic acid by mycorrhizal fungi at three nitrogen concentrations in
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Duchesne, L.C. 1994. Role of ectomycorrhizal fungi in biocontrol, p. 27-45. In F.L.
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Dueck T.A., P. Visser, W.H.O. Ernst, and H. Schat. 1986. Vesicular-arbuscular
mycorrhizae decrease zinc-toxicity to grasses growing in zinc-polluted soil. Soil Biol.
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ex. Laws. New Phytol. 127:719-724.


82
massflow of nutrients from the hyphal to the plant compartment, water used to bring the
boxes to their original weight was added only to the plant compartment. Plants were
grown in a growth chamber with mean temperatures of 23/29C (dark/light cycle,
respectively) and a mean photosynthetic photon flux density of 1000 mol m'2 s'1 at plant
height. Plant shading was minimized by the distance between plants. Boxes were
randomized each time plants were watered in the growth chamber.
Plants were harvested after 62 d. The surface sand layer containing a crusted algal
mat approximately 5-mm thick was removed and treated separately. Sand was removed
from roots first by shaking and then by rinsing the roots with water. All root pieces were
collected. A sample of subsurface sand was retained for further analysis. Plant tissue-P
status, colonization and root and hyphal surface areas were measured as in the Materials
and Methods of Chapter 4. Root and hyphal surface areas were measured separately for
the plant and hyphal compartments. For absorbing surface area calculations involving
intraspecifically competing mycorrhizal plants, half of the total hyphal surface area from
the hyphal compartment was added to the surface area of the plant on each side. Plant
P-uptake rate was calculated as the change in plant P content during the time of the
experiment based on combined root and hyphal surface area. The mean total hyphal
density for each compartment and treatment was calculated.
Sand for P analysis was thoroughly mixed prior to analysis. A 4.5- to 5.5-g
sample in 20-ml, borosilicate vials was treated overnight with 5 ml of concentrated HC1.
This was evaporated and 10 ml of 0.1 N HC1 were added. Phosphorus was analyzed after
24 h following the procedure of Murphy and Riley (1960). Surface and subsurface soil