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Alternative tomato farming system practices and their influence on the persistence and infectivity of Arbuscular Mycorrh...

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PAGE 1

ALTERNATIVE TOMATO FARMING SYSTEM PRACTICES AND THEIR INFLUENCE ON THE PERSISTENCE AND INFECTIVITY OF ARBUSCULAR MYCORRHIZAL FUNGI AND OTHER FUNGAL ROOT ENDOPHYTES By CHRISTOPHER RASMANN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Christopher Rasmann

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To Nita.

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iv ACKNOWLEDGMENTS I appreciate the opportunity my committee co-chairs, Drs. Jim Graham and Dan Chellemi, have provided in allowing me to pur sue this research proj ect. Their support and patience were important to me and instrume ntal in my ability to complete this work. Additionally, I am grateful to have been the recipient of a Hunt Brothers endowment in support of my research. I wa nt to thank Dr. Lawrence Dat noff for opening his lab for me to use as a home base and providing me with guidance and with many of the tools needed to accomplish my research. I would also lik e to express my gratitude to Dr. David Mitchell for his guidance and in sight throughout my time at the University of Florida and for the editorial input he provided w ith my research proposal and thesis. I am grateful for the support and assistance I received from many individuals, all of whom contributed to this project. I would lik e to thank Diane Bright, Dr. Jeff Buyer, Dr. Michel Cavigelli, Gloria Darlington, Dr. James Kimbrough, Dr. John Larsen, Dr. Ramon Littell, Gary Marlow, Dr. Joe Morton, Patty Rayside, Dr. Fabricio Rodrigues, Dr. Jeff Rollins, Mauricio Rubio, Brenda Rutherford and Dr. Carol Stiles.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... ..x CHAPTER 1 INTRODUCTION........................................................................................................1 Arbuscular Mycorrhizal and Other Funga l Root Endophytes in Agroecosystems: Context and Importance............................................................................................1 Study Site..................................................................................................................... .3 Conventional Production.......................................................................................5 Organic Production................................................................................................5 Disk Fallow...........................................................................................................6 Bahia Pasture.........................................................................................................6 Weed Fallow..........................................................................................................6 Reasoning and Objectives.............................................................................................7 2 LITERATURE REVIEW...........................................................................................10 Research on Arbuscular Mycorrhizal Fungi...............................................................10 Studying Arbuscular Mycorrhizae..............................................................................11 Non-Molecular Tools..........................................................................................13 Quantification of fungal coloniza tion in roots using microscopy................13 Spore extraction for identif ication and enumeration....................................18 Molecular Tools and Studying the Species Concept and Genetic Condition of Arbuscular Mycorrhizal Fungi.........................................................................22 Fatty Acid Methyl Ester Analysis.......................................................................29 Other Tools..........................................................................................................31 Arbuscular Mycorrhizal Fungi in Agroecosystems....................................................32 Phosphorus Fertilization......................................................................................32 Soil Disturbance..................................................................................................34 Pesticides.............................................................................................................35 Flooding...............................................................................................................36

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vi Organic, Conventional, and Grassla nd Farming Management Strategies...........37 Other Fungal Root Endophytes Known to Occur in Herbaceous Plant Roots...........40 3 MATERIALS AND METHODS...............................................................................43 4 RESULTS AND DISCUSSION.................................................................................52 Agricultural and Environmental Influen ces Impacting Edaphic Conditions at the Header Canal Study Site.........................................................................................52 Colonization of Roots of Tomato and Cover Crops by Arbuscular Mycorrhizal Fungi and Other Fungal Endophytes.......................................................................56 Arbuscular Mycorrhizal Fungi in Tomato and Cover Crop Roots......................56 Tomato Root Colonization by N on-Arbuscular Mycorrhizal Fungal Endophytes.......................................................................................................63 Microdochium bolleyi in Tomato Roots from the Organic Treatment................67 Infection Potential of Field Soil on Greenhouse-Grown Maize.................................73 Arbuscular Mycorrhizal Fungal Infection Potential............................................73 Non-Arbuscular Mycorrhizal Fungal R oot Endophyte Infection Potential........80 Spores: Enumeration and Identificati on of Arbuscular Mycorrhizal Fungal Morphotypes...........................................................................................................83 Spore Enumeration of Year Five Trap Culture Soil............................................83 Spore Morphotype Identification........................................................................85 5 CONCLUSIONS........................................................................................................99 LIST OF REFERENCES.................................................................................................105 BIOGRAPHICAL SKETCH...........................................................................................115

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vii LIST OF TABLES Table page 4-1 Results of soil analyses from year four and five of a 5-year cropping systems study at the Header Canal experimental farm site in Fort Pierce, FL......................53 4-2 Field tomato root colonization by arbu scular mycorrhizal fungi and other fungal root endophytes from year four and five of a 5-year cropping systems study at the Header Canal experimental fa rm site in Fort Pierce, FL....................................57 4-3 Field soil infection potential of ar buscular mycorrhizal fungi and other fungal root endophytes on maize from year four and five of a 5-year cropping systems study at the Header Canal experimental farm site in Fort Pierce, FL......................62 4-4 Diversity of arbuscular mycorrhizal f ungal morphotypes in year four and five of a 5-year cropping systems study at the Head er Canal experimental farm site in Fort Pierce, FL..........................................................................................................89

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viii LIST OF FIGURES Figure page 4-1 Header Canal experimental farm si te plots after the hur ricanes of 2004. Photo taken prior to final tomato transplanta tion in year five of a 5-year cropping systems study in Fort Pierce, FL. ............................................................................55 4-2 Field tomato root colonization by arbusc ular mycorrhizal fungi in years four and five of a 5-year cropping systems study at the Header Canal experimental farm site in Fort Pierce, FL...............................................................................................58 4-3 Correlation with least squares regression of the fatty acid 16:1 5 content in tomato roots to the observed proportion of colonization of tomato roots in field plots by arbuscular mycorrhizal (AM) fungi............................................................59 4-4 Bahia pasture plots with rows of tomato plants.......................................................61 4-5 Field tomato root colonization by all non-arbuscular myco rrhizal fungal root endophytes in year five of a 5-year cr opping systems study at the Header Canal experimental farm site in Fort Pierce, FL.................................................................64 4-6 Mass of enlarged fungal cells produced by Phialophora spp. within the cortex of tomato roots; blue color is a re sult of staining with trypan blue..............................65 4-7 Mass of enlarged funga l cells and hyphae produced by Phialophora spp. within the cortex of maize roots; blue color is a result of staining with trypan blue..........66 4-8 Clusters of resting spores (cystosori) formed by Polymyxa spp. within the cortex of a tomato root; blue color is a re sult of staining w ith trypan blue.........................68 4-9 Pigmented cells formed by Microdochium bolleyi within tomato roots..................70 4-10 Close-up of pigmented cells formed by Microdochium bolleyi within tomato roots. ........................................................................................................................ 71 4-11 Close-up of pigmented and trypan blue-stained cells formed by Microdochium bolleyi within tomato roots.......................................................................................72 4-12 Infection potential of treatment soil sa mpled in year four of a 5-year cropping systems study at the Header Canal experimental farm site in Fort Pierce, FL........74

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ix 4-13 Infection potential of treatment soil sa mpled in year five of a 5-year cropping systems study at the Header Canal experimental farm site in Fort Pierce, FL........75 4-14 Infection potential of disk fallow treatm ent soil sampled in year four and five of a 5-year cropping systems study at the Head er Canal experimental farm site in Fort Pierce, FL..........................................................................................................76 4-15 Results from Fatty Acid Methyl Ester (F AME) analysis of soil from year five of a 5-year cropping systems study at the Head er Canal experimental farm site in Fort Pierce, FL..........................................................................................................79 4-16 Maize roots with co lonization of a chytrid Olpidium spp. stained with trypan blue. Fungal structures are res ting spores and zoospore cysts.................................82 4-17 Mean arbuscular mycorrhizal spore numbers extracted / cm3 of undiluted treatment soil from year five of a 5-y ear cropping systems study at the Header Canal experimental farm s ite in Fort Pierce, FL......................................................84 4-18 Arbuscular mycorrhizal fungal spore w ith substantial parasitism evident through dark internal swirling; no morphot ypic designation could be assigned...................86 4-19 Damaged arbuscular mycorrhizal fu ngal spore with evidence of a defense response initiated by parasitism...............................................................................87 4-20 Surface ornamentations on the spore wall of an Acaulospora sp............................91 4-21 Multiple spore walls continuous with the layers of the subtending hypha can be seen in this example of the arbus cular mycorrhizal fungal morphotype Glomus intraradices ..............................................................................................................93 4-22 Sporocarp of Glomus sinuosum covered by peridial hyphae...................................94 4-23 Portion of a Glomus ambisporum sporocarp with an associated dense mass of mycelium; individual subtending hyphae lead to each spore...................................95 4-24 Gigaspora sp. with its large spor ogenous cell still attached....................................96 4-25 Echinulate auxiliary cells diagnostic for a Gigaspora spp. These structures were only found produced on external hyphae a ssociated with maize roots in the infection potential study...........................................................................................97 4-26 Smooth surfaced auxiliary cell diagnostic for a Scutellospora spp.; these structures were only found produced on external hyphae associ ated with maize roots in the infection potential study........................................................................98

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x Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ALTERNATIVE TOMATO FARMING SYSTEM PRACTICES AND THEIR INFLUENCE ON THE PERSISTENCE AND INFECTIVITY OF ARBUSCULAR MYCORRHIZAL FUNGI AND OTHER FUNGAL ROOT ENDOPHYTES By Christopher Rasmann December, 2006 Chair: James H. Graham Cochair: Daniel O. Chellemi Major Department: Plant Pathology Farming practices are known to significan tly impact arbuscular mycorrhizal (AM) fungi, as well as other soil microbial commun ities in agroecosystems. The effects of highly divergent land management strategies on the incidence and infectivity of AM fungi and other fungal endophytes were evalua ted in a 5-year cropping systems study at the USDA ARS Header Canal farm site in Ft. Pierce, FL. Two treatments, the conventional and disk fallow systems, repres ented farming practices considered to be detrimental to AM fungal populations. The remaining three treatments, the organic, bahia pasture and weed fallow systems, used practices thought to be more conducive to the presence of AM fungi. In December of year four and five of the study, tomato rhizosphere soil and fibrous roots of tomato were collected. Roots were cleared, stained and examined for colonization by AM fungi and other fungal endophytes. Rhizosphere soil was assayed

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xi for infection potential of AM and other r oot endophytic fungi based on maize seedling colonization at the five-leaf stage. Tomato r oots and rhizosphere soil from year five were also analyzed for the AM fungal fatty acid biomarker 16:1 5 c Sudangrass trap cultures were set up to determine abundance and dive rsity of AM fungal spore morphotypes. Of all investigated agricu ltural practices, phosphorus (P) fertilization and soil disturbance had the greatest influences on AM fungal infect ivity and abundance. All plots had very high levels of available soil P, which resulted in low levels of colonization across treatments. The bahia pasture syst em was the only treatment without regular extensive soil mixing, and these plots had the highest level of fiel d root colonization by AM fungi. Field roots were more heavily colonized by fungal endophytes other than AM fungi in all treatments; tomato plants fr om organic plots, though, were unique in encouraging colonization by Microdochium bolleyi Populations of root-associated AM fungi and other fungal root endophytes we re positively correlated in all studies. Hurricane-associated flooding and a shorte ned growing season played a role in reducing the infection potential in all treatmen ts except for the organic plots in year five compared to year four. The levels of primar y inoculum were maintained in organic plots, apparently through improved soil quality which resulted in decreased resistance to root development. Areas under active disturbance in disk fallow plots had the lowest levels of primary inoculum, but recovery to levels comparable to less disturbed treatments occurred after a single s eason of host root growth. Diversity of AM fungal morphotypes was typica l of agricultural fi elds, with at least 11 species present across treatments. Glomus spp. were the dominant spore type recovered in all treatments.

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1 CHAPTER 1 INTRODUCTION Arbuscular Mycorrhizal and Other Fungal Root Endophytes in Agroecosystems: Context and Importance Arbuscular mycorrhizal (AM) fungi form sy mbioses with most plants, are present widely in terrestrial environments, and have been identified as performing a variety of functions in their associati ons within natural ecosystem s and agroecosystems (Douds & Millner 1999; Jeffries et al 2003). These mycorrhizal asso ciations are believed to be capable of making a significant positive contribu tion to plant health as well as positively affecting soil quality. Among the benefits arbuscular mycorrhizae may provide to plants include increasing nutrient upt ake, particularly of P and Zn; improving plant access to water; positively modifying rh izosphere populations; and incr easing both pest and soil borne disease resistance in plants (Jeffries et al 2003; Johansson et al 2004). Additional potential positive effects that have been described as a result of the symbiosis between mycorrhizal fungi and plants are the improveme nt and maintenance of soil structure and alleviation of plant stress in otherwise inhospitable envi ronments, such as recently disturbed or polluted soils (D ouds & Millner 1999; Jeffries et al 2003; Johansson et al 2004). The AM fungi have a central role in eco systems and experimental evidence shows that considerable functional diversit y occurs among taxa within the phylum Glomeromycota (Munkvold et al 2004; Gosling et al 2006). This diversity, however, does not necessarily constitute benefits provided to all plants in al l environments (Bever

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2 et al 2001; Jeffries et al 2003; Munkvold et al 2004). Based on experimental evidence, the ability of AM fungi to function and ma intain the mycorrhizal condition under some field conditions common in industrialized agriculture appears limited (Kabir et al. 1999; Oehl et al. 2004; Gosling et al. 2006), and while the symbiosis is generally perceived as mutualistic, there are “occasional commensal and parasitic excursions from this norm” (Johnson et al 1997, p. 583). Management practices such as high P fertilizer applications; tillage-mediated soil disturba nce; and the application of pesticides, including the use of soil fumiga nts are considered potentia lly detrimental to populations of AM fungi. In fact, when considering th e widespread use of and production benefits gained from practices that may be deleteri ous to AM fungal populations, as well as the carbon cost inherent to plants in interactions with these obligately biotrophic fungi, the question can be raised as to whether or not AM fungi shoul d be considered when making management decisions in many agricu ltural systems (Ryan & Graham 2002). The AM fungi are not the only organisms widely capable of occupying the root cortex; there are many other fungal endophytes, so me of which may also exist within the stele (Yu et al 2001; Barrow 2003). Several definiti ons for the term endophyte exist and different uses of the word can sometimes be points of controversy (Wennstrm 1994; Sieber 2002). When used in this work, a relatively narrow definiti on will be adopted. The endophytes referred to herein are those f ungi observed existing within the roots of plants without causing apparent symptoms of di sease. Recognizing that AM fungi fit this definition of an endophytic organism, when the term endophyte is used in this work it will be qualified to distinguish fungi not recognized as residing within the phylum Glomeromycota. The individuals that ma ke up this group of root-occupying fungi may

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3 function as obligate or facultative biotrophs and exist within a range of plant-microbe interactions from mutualism to, in some inst ances, parasitism or pa thogenicity, as in the case of some formae speciales of Fusarium oxysporum (Morton 1998; Sylvia & Chellemi 2001; Sieber 2002). These forms are pathoge nic on select taxa of plants, and are seemingly innocuous or beneficial when associated with most other plants. The data for this study were collected from the USDA, ARS cropping systems project at Header Canal in Fort Pierce, Florida. The projec t provides, among other objectives, an opportunity to examine the eff ects varied farming management practices have upon the components that define soil qualit y. Part of determining the status of soil quality and health involves monitoring biol ogical indicators; this process begins by determining the condition of re sident plant health, and evaluating components such as soil microbial biomass, activity, and diversity (van Bruggen & Semenov 2000; Hill et al. 2000; Chellemi & Porter 2001). As discusse d above, both the AM f ungi and other root endophytes occur globally, and it is probable that all plants form intimate associations with at least one taxa of f ungal endophyte, while in many cases plants may be more or less indiscriminate in their as sociations (Sieber 2002). The value of these organisms as biological indicators of soil quality and health may be highly significant, and their impact on ecosystem functioning needs to be better understood. An integral aspect of that understanding in the context of agroecosystems involves the el ucidation of th e capability of fungal root endophytes to persist and rema in infective under in tensive management practices. Study Site The 5-year cropping systems study at the H eader Canal Farm in Fort Pierce, FL, has five land-management systems that are highly divergent, yet all use management

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4 methods not uncommon in the region. The land which is being farmed in this project had been in conventional tomato production usi ng raised plastic-mulch ed beds fumigated with methyl bromide plus chloropicrin (MeB r + CP) annually for 10 years prior to being leased for research. The entire research site has soil that ha s been classified as a Pineda fine sand (loamy, siliceous, hyperthermic, Ar enic Glossaqualfs), with a sandy texture from the surface to a depth of 96 cm (Lewis et al 2003; Watts & Stankey 1980). The average pH among system plots over years f our and five ranged from 6.8 to 7.7. Soil phosphorus levels were extremely high due to the previous fertilization history and nutrient applications made subsequent to the initiation of the land management treatments. Mean Mehlich-1 extracted phosphorus ranged from 794 to 1831 kg ha-1 among systems. During the initi ation of tomato crop production in year five of the study, two hurricanes (Frances and Jeanne) struck the area of Florida where the experimental farm site was located, causing signifi cant damage to the farm and surrounding communities. Rainfall totals exceeded 50 cm during the month of September, resulting in widespread flooding. Tomato transplanta tion was delayed approximately a month due to the hurricanes and the resultant field conditions. Systems were initiated in the summer of 2000 and, with the exception of one treatment, as will be explained below, were allowed 3 years to establish prior to resumption of tomato production. The five tr eatments were established in a randomized complete block design, with each treatment repl icated six times for a total of 30 plots, occupying 0.16 ha individually (plots are 68.5 x 23 m). Tomatoes in all treatments, except the bahia pasture system, were grown in raised beds with plastic mulch. The five systems are described below.

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5 Conventional Production This system continued the management pr actices of the precedi ng farmer, with the exception being replacement of the original soil fumigant with Telone C-35, a 62:35 formulation of 1, 3-dichloroprope ne (1, 3-D) and chloropicrin (CP). Trifluralin (Treflan) and napropamide (Devrinol) were spread and in corporated for weed control. Tomatoes in this treatment received an initial fertilizer application during bed fo rmation (20% of total N and K), with subsequent fertilization pr ovided during the growi ng season via injection through drip irrigation. In this system the 3-year transition was not necessary, so fall tomato production continued throughout system plots. During the spring and summer of 2001 this treatment had a cover crop of Japanese Millet ( Echinochloa crusgalli var frumentace a); it was weed fallowed in 2002, and disk fallowed in 2003. Organic Production During the first 3 years no tomato crop was raised on the plots in this treatment. Broiler litter and urban plant debris were broadc ast and incorporated into the soil in July. In August, a cover crop of sunn hemp ( Crotalaria juncea ) was planted, while in the following spring, Japanese millet was grown. After the transition pe riod, starting in the fourth year, the south half of each plot was used to produce a tomato crop following approximately 6 weeks of soil solarization of the beds, while the other half remained under a sunn hemp cover crop. During the fifth year, the entire plot was put into tomato production using the techni ques just described. The production plan for these plots was not certified organic due to their close proximity to treatments th at use technology prohibited by certifying bodies. However, the practices used in this tr eatment are aligned with methods that are in favor with advocates for organic agriculture.

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6 Disk Fallow The entirety of the plots in this treatmen t were disked regularly year-round to keep them free of vegetation. Disking depth was about 25 cm. Tomatoes were not grown in years one through three. In A ugust of year four, the south half of these plots received fertilizer and herbicides identical to those applied to the conventional system. After the final harvest, both halves were again disked re gularly. In year five, the entire plot was put into production using the same methods to integrate the northern half as were used in the southern half the year before. Bahia Pasture A bahia grass ( Paspalum notatum var. Argentine bahiagrass) pasture was established in these plots and managed th rough mowing and spring fertilization for the first 3 years. In year four, the southern halves of the plots we re put into tomato production using a strip tillage production system (Chellemi et al. 1999). A 46-cm strip was tilled into the bahia grass for planting, wh ile the grass on either side of the row was managed through mowing and use of the herb icide sethoxydim (Poast). The fifth year involved cropping both halves of these pl ots using the strip-till technique. Weed Fallow These plots were left undisturbed for th e first 3 years of transition, and weed communities were allowed to establish. The s outhern halves of the treatment plots were put into tomato production beginning in year four after disking and using the same practices used in the conventi onal system. The entire plots were cultivated to tomato during the fifth year.

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7 Reasoning and Objectives The main priority of this study is to characterize the impacts that varied management practices have upon an important and highly influentia l group in the soil community, the AM fungi. Across one soil t ype, a field with a uniform history of intensive agricultural production using MeBr + CP was transitioned into five divergent management strategies. Two treatments repres ented farming practices considered to be highly detrimental to AM fungal populations, the conventional plots and the disk fallow plots. The remaining three treatments used practices that are thought to be more conducive to encouraging the presence of AM fungi. Organic plot management included an intensive cover-crop regimen and nutrient so urces that are more slowly available than in the other systems, while weed fallow plots were left undisturbed during the transitional period, allowing for the unrestricted devel opment of an endemic plant population, and were not exposed to the use of agrichemical s until the re-initiation of tomato production. In the bahia pasture treatment, a sod known to be highly mycotrophic was established during the transitional period, and was maintain ed after strip-tilled rows were generated for tomato transplants. Specifically of interest in this proj ect was whether systems encouraged or discouraged the relative abundance and inf ectivity of fungal r oot endophytes. In addition, while considerable literature exists detailing and speculat ing on the impacts of certain agricultural practices on AM fungi, there is little information regarding the ability and extent to which populations recover from adverse conditions once land management practices are changed. Little is known about the long-term repercussions, if any, that farming strategies such as repeated soil fumigation have on AM fungal presence and infectivity. The quantificati on of the AM fungal presence among the systems during year

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8 four and five is important in that it can pr ovide information about the effects management has upon these organisms with transition to production and on the differences in populations occurring among th e systems during production. While considerable interest is deve loping in enhancing understanding of the possible functional role of non-AM funga l root endophytes in agroecosystems, particularly those fungi that have been designated as dark septate endophytes (DSE), there is little information re garding their ability to persist under in tensive agricultural management. Gathering information regarding structures associated with the occurrence of non-AM fungal root endophytes as they exist in roots can provide a provisional identification or classification. Investigating the degree to wh ich field tomatoes and other host crops are colonized may help us dete rmine the AM fungal a nd other root endophyte abundance and aggressiveness within the various treatments and help us understand how these have been affected by management strategies. The infectivity of AM and other fungal ro ot endophytes was quantified in end-ofseason field tomato roots, and their infec tion potential in field soil was assayed in a greenhouse study. The non-AM fungal endophyte s found existing in field roots were identified when possible based on their morphological features within the root association. Fatty acid methyl ester (FAME) analysis was performed on year five tomato roots and field soil, providing an independent measure of biomass by quantification of the AM fungal fatty acid biomarker, 16:1 5 c. Spore extractions from a greenhouse study in which trap crops were grown in field so il were completed, achieving a provisional accounting of AM fungal morphotype s occurring in the field. In year five, spore numbers from the harvested trap cultures were enumerated.

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9 The above work was accomplished to eluc idate the impact of industrialized agricultural practices, as represented by the hist ory of the farm site at Header Canal and perpetuated in two of the five treatments, on the survival, infectivity and occurrence of AM and other fungal root endophytes. In a ddition, it was important to determine the degree to which farming strategies that ha ve been considered more amenable to encouraging the existence and functioning of AM fungi were able to affect these populations after long-term exposur e to intensive tomato production.

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10 CHAPTER 2 LITERATURE REVIEW Research on Arbuscular Mycorrhizal Fungi Our knowledge of the existence of AM f ungi is generally accepted to have begun with descriptions Ngeli made in 1842 (Koide & Mosse 2004). In the late 19th and early 20th century, detailed descriptions of the mor phological results of the symbiosis were in place, including both of the most important di agnostic structures: vesicles (Janse 1897) and arbuscules (Gallaud 1905). While the rela tionship between plants and AM fungi has been known to exist for well over 100 years, it s widespread occurance in ecosystems and intensity therein, although specu lated upon in some earlier litera ture, has been established much more recently (Read et al 1976). A partial list of ensuing work, occurring on a broad range of scales while spanning multiple disciplines, includes: attempting to develop a taxonomic system and understand the phyloge ny of the AM fungi; ascertaining and characterizing the effects of the mycorrhizal relationship on the symbionts and on the broader environment, as well as investiga ting the natural and an thropogenically derived edaphic and environmental conditions that impact the partnership; understanding the regulating factors in and the physiology of mycorrhi zae; and the attempt at development of AM fungi as an agricultural technology (K oide & Mosse 2004). Research that is of particular interest in the cont ext of this project in cludes studies that relate to the ecology of mycorrhizae, especially in managed systems.

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11 Studying Arbuscular Mycorrhizae Accurate methods to detect, quantify a nd identify AM fungi are paramount to developing an understanding of mycorrhizae in any environment and context. A variety of obstacles exist in studying arbuscular mycorrhizae, however, stemming mostly from their obligately biotrophic nature and complex, not ye t fully understood, genetic condition. Classical, biochemi cal and molecular tools have been developed and used with varying degrees of success by researchers. While tools and techniques exist both for quantification and identification of AM fungi as well as for measuring their metabolic activity, linking identi fied active taxa to possible ecological roles is more complicated (Millner & Wright 2002). There appears, based on experimental evidence, to be cons iderable interand even intra-phylo and morphotypic functional diversity in the AM fungi. Munkvold et al (2004) compared the hyphal growth of 24 isolates from four de scribed morphotypes and their ability to influence the growth and P-nutrition of Cucumis sativus and found considerable variability existing within mo rphotypes in their mycelial exte ntion and in the ability to promote growth. Another study compari ng the capabilities of 16 isolates of Glomus intraradices from a single field produced similar re sults, with five-fold differences in final hyphal lengths (Koch et al 2004). Further confounding understanding of the func tional diversity of AM fungi is that when taken out of the microcosm of a c ontrolled experiment, the fungal role in mycorrhizal functioning seems plastic, de pending largely on its environment (Johnson et al 1997). If functional richness abounds in th e AM fungal genotype, and is shown to not be dependent upon species diversity, but rather on isolates as influenced by their environment, it would represent a shift in a standard view of microbial ecology as it

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12 relates to this type of mycorrhiza. The same uniquely diverse genetic makeup of AM fungi that makes them difficult to characterize, and in some cases investigate, may serve to place the organism in position to ensure the perpetuity of the relationship between symbionts in the presence of adversity through functional redundancy in the larger population. This would be especially impor tant to understand in the context of agricultural or other distur bed environments where alte ration of habitat occurs. There remains substantial debate as to whether maintaining taxonomic diversity is important if functional diversity is preserve d and is active. Our understanding of these concepts with regards to AM fungi and the scale on which we judge them has changed as our tools for reseach have de veloped and improved. In the early stages of mycorrhizal research the AM fungi were considered functionally redunda nt; we now are beginning to better understand the influence that the edaphi c and climatic environment as well as the host plant imparts on the functi onal phenotype of the fungi. Li kewise, in natural systems, the phenotype of the fungal component in mycorrh izal associations can directly affect the structure of plant commun ities (van der Heijden et al 1998; Klironomos et al 2000; Graham & Miller 2005). Futu re research determining the extent to which functional diversity and resiliency exists among AM f ungal species and isolates as well as the degree to which it is present in given popul ations will help clarify the association between these factors (Kirk et al 2004). Furthermore, wh en considering ecological studies of the mycorrhizal relationship it is vi tal that, where possible, experimental results from controlled environments be investigat ed on the broadest scal e possible to verify their real world signific ance (Graham & Miller 2005).

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13 Prior to discussing research on AM fungi in ecosystems in greater depth, it is important to describe some of the more im portant and commonly used tools and methods available for the research of these organisms, including problems associated with their use. Non-Molecular Tools Quantification of fungal coloni zation in roots using microscopy Assessing the levels of fungal infection in host plants is performed in most studies involving AM fungi. Barring the use of bioc hemical or molecular techniques, both of which are still often correlated with classical methods, examination of roots takes place under a microscope. In order to best prep are samples for observation, harvested roots should be processed rapidly after taking any n ecessary measurements from the fresh root system followed by drying the roots to be exam ined as soon as possible to stop metabolic activity. To visualize intracortical fungi when using non-vital stains, the plant’s pigmentation and cellular const ituents need to be clarif ied prior to staining. Phillips and Hayman (1970) were among the fi rst to use a proce dure that is still commonly used today, due to its low cost, lo w technical requirements and consistent results; host cytoplasm is cleared thro ugh heating roots in 10% (w/v) potassium hydroxide (KOH) followed by rinsing, with ro ots undergoing subse quent staining using trypan blue. An exceptional alternative clea ring agent is concentr ated chloral hydrate, which renders the roots transpar ent, although its legal status ma kes it difficult to acquire. Several variations on the basic procedure have been developed. The length of time required to clear roots varies wi th the type and age of the root, the size of the root sample and the temperature at which clearing take s place. Calibration is essential when establishing clearing procedures, as too little clearing will reveal little in terms of fungal

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14 structures, while too much will destroy roots, stripping the cortex from the vascular tissue. The root samples to be processed ar e typically best kept to < 2 g, with segment lengths cut to 13 cm. Heating of samples in KOH generally is accomplished in an oven or hot water bath (typically 6095C), but is fastest when autoclaved. It can also be done at room temperature, if extendi ng the process to hours or days is not problematic. Further clearing with acidified bleach (1.7% NaOCl with 1% diluted HCL v/v in water) or alkaline hydrogen peroxide (0.5% NH4OH with 0.5% H2O2 v/v in water) at room temperature provides additional removal of pigments in root tissue that contain particularly recalcitrant obs curing compounds. This step is especially valuable when further clearing in KOH would lead to destruc tion of the sample, but, once again, in this procedure the sample should be observed frequen tly to achieve the best results for a given type of root. If bleaching is performed using alkaline H2O2, the sample should subsequently be acidified with diluted HCL to enhance the staining that follows. Roots should be thoroughly rinsed with water after each clearing and bleaching step. Non-vital stains are stil l the most commonly used technology for enhancing visualization of AM fungi in roots. These stains are valuable in that they provide a means to view the entirety of colonization at the time of plant harvest, and are therefore useful for observation of both viable and nonviable fungal tissue. Trypan blue (0.05% w/v) was used in the study that popularized the hot KOH clear ing technique and is still widely used today (Phillips & Hayman 1970). This, and similar stains such as 0.05% (w/v) cotton blue, 0.01% (w/v) acid fuschin and 0.03% (w/v) Chlorazol black E (CBE), are typically accomplished by placing the roots in dye that has been suspended in lactoglycerol (lactic acid – glycerol – water, 1:1:1 or 1:2:2) soluti on and heating. Stained

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15 roots can then be stored in l acto-glycerol or diluted glycerol without dye, a step that can also enhance visualization of fungal tissue by removing excess stain, especially important when using trypan blue or CBE. Stain c oncentrations and staining time should be assessed and adjusted according to the root type and the needs of a particular study. Higher concentrations and longe r staining times are more lik ely to be required with cotton blue and acid fuschin, while over-staining can often occur when CBE is used. Trypan blue is particularly useful when examining roots under a dissecting microscope for determination of colonization levels, whil e a higher contrast stain such as CBE is probably more appropriate for morphological studies. In addition to the destructive, non-vital staining techniques described above, nondestructive and vital staini ng methods have been devel oped. Technologies such as epifluorescence and confocal laser-sc anning microscopy (CLSM) allow for the examination of living tissue, albeit with inherent limitations in the use of both. Epifluroescence microscopy appears to only reveal collapsed a nd therefore inactive arbuscules, providing informati on of select value, while CL SM requires specialized and expensive equipment and supplies. Vital stai ning using nitro blue tetrazolium chloride (NBT), diaminobenzidine (DAB) and phosphatase assay kits can also be useful to visualize tissue that was metabo lically active at the time of pl ant harvest. These methods are considerably more expensive than non-vi tal stains, their use can be an involved process, and investigators may still require or benefit from the use of specialized equipment.

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16 An excellent general review of clearing and staining techniques was recently written by Vierheilig et al (2005). Brundrett et al. (1996) provides detailed guidance with regards to non-vital stai ning materials and procedures. There are two general appro aches to studying AM fungi colonization levels that have evolved and generally supplanted a multitude of other techniques, favored because they minimize compromises researchers need to make with regards to accuracy, ease of execution, and reproducibility. One approach was adapted from a procedure developed by Newman (1966) to improve th e ability of researchers to estimate total sample root lengths, which he called the “line intersec tion method,” now often called the grid-line intersect or just the line-intersect method. Th is procedure was devise d to provide another quantitative evaluation besides root weights for assessing th e amount of root tissue in a sample, and yields accurate, reproducible es timations. As commonly adapted for the study of AM fungi, this method was descri bed, as well as tested for accuracy and compared to several other techniques by Gi ovannetti and Mosse (1980). In the lineintersect method, previously cl eared and stained roots are spread on a Petri dish onto which a grid of straight lines are affixed, drawn or scored. The grid of lines is then used as a reference, where intersections between them and the root sample are tallied and observed for evidence of infection using a dissecting microscope. This will yield data representing both the total root intersections and the total infected intersections, from which the proportion of infected tissue can be de rived. An appropriate volume of roots is chosen, not so many that individual roots can no t be observed, but not so few that at least 100 intersections can not be tallied. This m eans that when fine roots are being observed, the dry weight of roots to be examined will rarely exceed 0.2 g.

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17 The grid of straight lines is usually laid out so that 1.27cm ( 0.5in.) squares are formed. The line-intersect method can be used to not only estimate the proportion of infected root, but also the tota l root length, using the formula N A R = 2 H where R = total root length, A = the area of the surface over which the roots are observed, N = the number of intersections between roots and lines, and H = the length of all straight lines. When 1.27cm squares are used, the tota l root length in cm = the number of rootline intersections, or R = N. Giovannetti a nd Mosse (1980) determined that increasing the number of observations from 100 to 200 improved the standard error of percent infection measurements from 4 to 3 when one set of observations was performed, while increasing the number to 300 yields a smaller improvement. This number can therefore be adjusted according to a researcher’s need for accuracy. McGonigle et al (1990) developed a method that not only allows the determination of colonization levels and se paration according to structures encountered, it additionally assists with problems encountered in distinguishing AM fungal structures from other cortical objects by virtue of its reliance on the use of a compound microscope to examine intersections. The use of di ssecting microscopes to study root – endophyte interactions is inherently limited due to the relatively lower magnifications available when compared to compound microscope. A r oot system, even when adequately cleared and stained, and with its cortex intact after proces sing, can be a confusing mess of colors and shapes due to root cell content staining as well as the coexistence of a diversity of microorganisms within the root. This problem can be multiplied when an investigator is

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18 studying field roots or has limited expe rience differentiating among the varied components of the root and its inhabitants. The technique described as the magni fied intersections method (McGonigle et al 1990) examines intersections between th e eyepiece crosshairs and roots at a recommended magnification of x 200, higher th an that capable of achievement using dissecting scopes. In using this procedure, between two to four s lides of roots from a cleared and stained subsample are observed. Th e field of view is moved so four, six or eight passes are made across a slide along its sh ort axis. Where the crosshair intersects with a root section, the field of view is adjusted so the ver tical line is parallel with the long axis of the root. Inte rsections are counted and clas sified according to their AM fungal structural content. Although the accuracy of individual inters ection assessment increases with this method, there are drawbacks. The authors th emselves noted that the preparation of sampling and subsequent observation is a much lengthier process us ing their technique when compared to the line-int ersect method. Further, it re mains unclear as to whether this represents an improvement in the assessm ent of the true colonization level of a root system. The additional information yielded in determination of the level of types of AM fungal structures present also seems to have limited value. Where these determinations are perceived to be important or useful, th e magnified intersections method is clearly a superior investigative process to the line-in tersect method; however, it requires more skill on the part of the microscopist. Spore extraction for identification and enumeration The separation of spores from soil or ot her plant growth medi a is required for classification, as AM fungal structures formed during the association with roots are not

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19 suitable for differentiation among morphotypes except at the higher taxonomic levels. Spore extraction is important not only for the determination of the presence of morphotypes using classical techniques, but is also currently necessary for molecular studies in identifying phylotypes. This secti on will detail the technique of spore removal and subsequent procedures that relate to morphological classifi cation and quantitative evaluations, while the subject of molecular stud ies will be dealt with subsequently. Spore extractions in ecological studies are typical ly accomplished for two reasons: 1) to quantify the level at which s porulation is occurring in th e field at different plant phenological stages, or to check for fluctuations in production related to seasonal or plant successional factors; and 2) for identificati on purposes, either from field soil, or from greenhouse pot cultures. The processes of spore extraction that are most commonly used are adaptations of a method derived to extract nematodes, first modified by Gerdemann (1955). The method varies among researchers, but the general process involves homoge nization of a soil or root-soil complex in water followed by si eving, centrifugation and decanting. Some investigators additionally incl ude steps involving separation in sucrose density gradients with additional centrif ugation (Brundrett et al 1996). The specific method used in this study will be detailed in the Materials and Methods section. Enumeration of spores is generally associ ated with directly or recently sampled field soil, although investigators in some studies count spores concurrently or exclusively from trap cultures (Liu & Wang 2003). Spor e enumeration data is best observed with reservation and understood with qualification in ecological research. It does not necessarily represent an accura te indication of AM fungal pr esence, since the degree to

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20 which spores are present in field soil or in pot cultures depends on many variables. Individual isolates are known to sporulate more heavily than others, with some isolates rarely forming spores under any conditions. Sporulation generally appears to occur in predisposed isolates after reaching a threshol d of colonization within a plant and can be affected by a multitude of environmental c onditions. Additionally, spores are not the most important infective propagule in many systems (Clapp et al 1995; Klironomos & Hart 2002), and are only a small fraction of the total AM fungal biomass in any system. Identification based on spores directly ha rvested from the field can be a difficult ordeal, owing to the large pr oportion of parasitized spor es and associated organic material that is generally present, co mplicating both morphological and molecular identification. This problem is commonl y resolved by segregating healthy-looking morphotypes from field soil extractions, th en inoculating greenhouse or potted field grown host crops with the individual morphot ypes. Growth media in pot cultures is usually sterilized sandy or sandy loam soil fe rtilized during crop gr owth using a weak Hoagland’s solution without phosphorus. The host crop is then harves ted at a particular developmental stage or at senescence, a nd a second extraction is performed, yielding spores more suited for identification. There are currently two systems of taxonomi c classification broadly recognized for AM fungi, based primarily on morphologi cal and genetic variation among groups, respectively. The revisions to taxonomy have been continuous duri ng the long history of research of these organisms, so it is with great relief that most researchers note that considerable homology has occurred, and th at effort has been made to maintain congruence where possible thus far between the systems (Koide & Mosse 2004).

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21 The taxonomic system built on the foundation of the morphological variation of spores exists in descriptions associated w ith the International Culture Collection of VA Mycorrhizal Fungi (INVAM), currently held at West Virginia University and maintained by Joe Morton. The system is not isolated fr om advances in molecular research, and has accounted for the elevation of the AM fungi to the level of division Glomeromycota that occurred based based on SSU rDNA sequence analysis by Schler et al (2001). Additionally, results of SSU rD NA analysis helped elucidat e the existence of the two most recently recognized families within th is taxonomic system: the Paraglomaceae and Archaeosporaceae (Morton & Redecker 2001). The INVAM collection currently recognizes one order, Glomales, within the phylum Glomeromycota. This order is divide d into two suborders: the Glomineae, which contains four families and five genera; and the Gigasporineae, with one family and two genera (INVAM 2006a). There are, ind eed, many divergent anatomical and morphological characteristics independent of spores that have been identified among these groups, some of which have aided in th e separation of taxa. Examples include the formation of auxiliary cells associated w ith the Gigasporineae, the interand intracellular vesicles formed by the Glomineae, as well as differences in the shape, size and staining intensity of hyphae and arbuscules in the cortex. The ability to use these characteristics for diagnostic purposes, t hough, is extremely limited and therefore they are generally only noted in morphotype descript ions from pure culture. They can, in certain circumstances, provide evidence of the existence of taxa that are otherwise not evident through the primar y means of morphotype id entification: spore wall characterization. The use of alternative characteristics as a means of identifying AM

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22 fungal taxa would only be important when pr imary evidence was lacking, for example, when spores are not extracted but colonized roots are observed. Id entification with the use of spores is preferred as morphotype de scriptions are formed based on a variety of spore attributes including the number of walls external and internal anatomical feature structure, size and staining ch aracteristics, and spore ontogeny. The system of classification based on SSU ribosomal gene analysis does not refer in its descriptions to the other morphologica l, biochemical, phenological or other sources of genetic variability that o ccur among and within identified taxa. This classification groups the AM fungi to th e species level (Schler et al 2001; Schler 2006), and has rearranged the taxonomic organization creating multiple orders, as well as adding and shifting family-level cla ssifications. Phylotypes cu rrently recognized by this classification include four orders: Glom erales, now divided into two groups; Diversisporales, a grouping that includes the Acaulosporaceae, the Gigasporaceae, as well as two new families, Diversisporaceae a nd Pacisporaceae; the Archaeosporales with Archaeosporaceae and the newly formed Ge osiphonaceae; and the Paraglomerales containing the Paraglomeraceae. Some of the problems with speciating the AM fungi and associated difficulties with molecular anal yses are discussed in the next section. The separation of AM fungi from Zycomycota in to their own phylum, the Glomeromycota, is widely accepted; finer taxonomic levels, however, will ideally ultimately depend on a more comprehensive knowledge of mo rphological and molecular variability. Molecular Tools and Studying the Species Concept and Genetic Condition of Arbuscular Mycorrhizal Fungi While the knowledge of the existence of mycorrhizae is over a century old, and interest in studying these organisms has long b een high due to their ubiquitous nature and

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23 perceived potential as an agricultural techno logy, the concept of species has yet to be firmly established with consensus. As noted previousl y, spore morphology has historically been the most important factor in AM fungal species determination, but there are a number of problems associ ated with relying solely on spores for identification in ecological studies. Some practical difficultie s include rapid sample degradation, presence of associated, but non-sporula ting morphotypes, the lack of significant distinguishing characteristics among some taxa, and the ex treme laboriousness involved in processing and examining numerous samples (Redecker et al 2003). The use of molecular tools has emerged and developed as an option for id entification, but ther e are a number of associated problems using molecular met hods in trying to identify AM fungi. Heterogeneous nuclear material exists with in individual AM fungi, and the reasons for this state remain yet to be definitively confirmed. Additionally, polymorphism occurs in typical marker genes adding to the complications of studying an organism that is already difficult to classify (Redecker et al 2003). Two important questions were raised by Landis et al (2004) and also discussed by Sanders (2004): what is the best way to quantify AM fungal richness in a given ecosystem, the classical method that categ orizes by spore morphotypes, a molecular method, or some combination of both? Also, is there a most relevant level on which to measure diversity when studying an organi sm which up to this point has had a continuously transitioning species concept? Sa tisfactory answers to these questions still elude us and will require a great deal of additional work. Two published studies cited by Sanders ( 2004) highlight the problems associated with using spore morphotype as the sole method for speciation. The first involved

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24 research investigating AM fungal diversity in understory vegetation under forest cover in England, and found that PCR amplification detected a Glomus spp. phylotype strongly, yet it was not found sporulating during long-term soil and plant sampling (Clapp et al 1995). Spore morphology as a measure of dive rsity was also found lacking in a study by Rosendahl and Stukenbrock (2004), in which rDNA analysis of AM fungi on sand-duneoccupying grasses in Denmark was performed. Phylogenetic analysis revealed 11 groups of distinguishable AM fungi, but only four correlated with specie s identified by spore morphotype. In addition, those identified cl ades were only “patchily distributed in the grassland,” suggesting that a significant presence and gr eater diversity could exist undetected in the form of the non-sporul ating population (Ros endahl & Stukenbrock 2004, p. 3184). Sanders, in his review, specula tes that using spore morphotype counts only give us the barest beginnings of the ecological story (2004). While the complications related to mo rphotype characterization might seem daunting, they are at least a concrete st andard providing guidance, irrespective of ecological significance. Molecular methods, de spite years of attempts at development, are still in the germinal stage in terms of adequately answering key ecological questions conclusively. The main problem lies in the previously mentioned genetic variation which occurs in the phylum and even within indi vidual spores (Pawlowska & Taylor 2004; Sanders 2004). Before delving into th e problems associated with molecular characterization, I will discuss some of th e published evidence regarding the nuclear condition and life history of AM fungi. The vast majority of fungi exist vegetative ly as haploids, and it has recently been advanced that at leas t one of the AM fungi, Glomus intraradices is also haploid; the

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25 ploidy of the larger group of AM fungi rema ins to be positively determined however, and may even be variable (Hijri & Sanders 2003). The DNA content of nuclei has been estimated for 11 AM fungi other than G. intraradices and ranged from approximately 127 Mb to 1058 Mb (Hosny et al 1998). Interestingly, both extremes came from morphotypes of the same genus, Scutellospora In contrast, G. intraradices the species used most extensively in molecular studi es involving AM fungi, has a genome size estimated to be about 16.5 Mb (Hijri & Sanders 2003). Al so, spores and hyphae have been determined to be multinucleate, with the number of nuclei in each spore ranging from 800 to 50,000 (Sanders 2002). When Pawlowska and Taylor (2004) looked at the ploidy status of isolates of Glomus etunicatum starkly different conclusions we re derived compared to the study describing G. intraradices as an organism with a haploid genome. Their methods involved investigation of the transmission of polymorphic markers as well as examining the rDNA variant distribution among spore nucle i using PCR to clone the ITS1 region. That study looked at the variation within four field isolates of G. etunicatum the results of which supported the case for the homokar yotic condition, and therefore the conclusion that high variability is most likely a produc t of polyploidy. One of the most intriguing aspects of this study is their additional investigation of a G. intraradices strain, the same morphotype described in the 2003 work of H ijri and Sanders as haploid, and using the same PCR amplification techniqu e predicted it was polyploidic. The AM fungi have defied almost every attempt at their culturing due to their obligately symbiotic nature, although de velopments in monoxenic culture using transformed root tissue have been successful (de la Providencia et al 2004). Studies

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26 involving fungal persistence in soil subsequent to spore ge rmination generated results showing only an ephemeral existence in the so il in the absence of a host (Sanders 2002). There has been evidence, however, for longe r-term survival in soil after initial germination, though with arrested development (Logi et al 1998). Experimental evidence revealed that is olates of the morphotype Glomus caledonium germinated without a host, stopped growth after 15 days, and subsequently retracted its protoplasm from hyphal tips, and then isolated empt y hyphae by forming septations. These AM fungal isolates retained infectivity for exte nded periods, with some remaining viable 6 months after initial germination (Logi et al 1998). The AM fungi have been assumed to exist as asexual organisms, although this has not yet been definitively proven, but is instead a result of the lack of any evidence for the occurrence of sexual processes. The origin of the high levels of genetic variation within individuals remains an object of speculation in the absence of sexual recombination, with anastomosis and subsequent nuclear excha nge among different isolates remaining under investigation as a possibility. Anastomosi s has been known to occur in AM fungi for almost 50 years (Godfrey 1957), and has been periodically observed and quantified since that time as occurring extensiv ely in various AM fungi. Th e process of anastomosis has not been seen to occur between different morphot ypes, nor to this date in isolates of the same morphotype from different geographica l locations. This has led to speculation upon the existence, and nature of unidentified rec ognition factors that seem to exist, during in vitro studies, on a finer level than any curre nt taxonomic system describes (de la Providencia et al 2004; Sanders 2002). Bo th de la Providencia et al (2004) and Giovannetti et al (2001) have looked at the nature and levels of anastomosis in AM fungi

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27 in isolates from familes of both described s uborders. They produced contradictory data with regards to the number of anastomose s formed per unit length of hyphae; however, they agreed that Glomeraceae strains formed anastomoses between individual hyphae. In their work, de la Providencia et al. (2004) found Gigasporaceae strains formed bridges only upon the same hyphal strand. These studies, and another by Giovannetti et al (2004), provided important information th rough the visualization of cytoplasmic movement and nuclear exchange between anastomosing hyphae in both symbiotic and presymbiotic growth (de la Providencia et al 2004; Sanders 2002). This suggests a mechanism whereby resource and genetic shar ing might exist, alt hough not yet detected on a level higher than intra-isolate. There are two widely accepted yet con ceptually divergent schools of thought regarding the nuclear condition of AM fungi that can explain detected polymorphism in single spore rDNA and the genetic diversity in these apparently asexual multinucleate fungi. The first model involves a collecti on of coexisting genetically diverse nuclei existing within each fungal individual that is passed on to successive generations. Not every spore in the next generation would rece ive the same complement of nuclei with the same frequency as the parent generation. An astomosis, in this heterokaryotic model, would result in the transfer of intact nucl ei from any donating fungi. Karyogamy would not have to occur to change a phenotype, as the hundreds to thousands of nuclei present in AM fungal cells would gradually accumula te mutations during replication (Sanders 2002; Pawlowska & Taylor 2004). The other m odel argues for a genome that contains more variability within each nuclei, the hom okaryotic model. This would involve an assemblage of similar nuclei with variance manifested through the presence of multiple

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28 gene copies either on individual or, in th e case of polyploidy, multiple chromosomes. Genetic diversity could arise from either errors in mitotic recombination and accumulation of other mutations, or thr ough as yet undescribed genetic exchanges involving sexual recombinat ion (Sanders 2002; Pawlowska & Taylor 2004). Taking into account the complexity and our lack of understa nding regarding the AM fungal genome, the use of molecular tech niques for ecological studies of AM fungi can be perceived as tools under development. Identification in such research almost exclusively targets the SSU rDNA, therefore re vealing an inherently limited picture of diversity. Considering the hi gh degree of polymorphism of rDNA that exists in these fungi, and the possibility that considerable va riation may even occur within individuals, interpretation of results can prove problem atic. Reliance on a single genetic locus for identification may prove less popul ar as our understanding of th e source of the incredible diversity within this organism increases. Quantification using molecular technique s in ecological stud ies of arbuscular mycorrrhizae is no less difficult than identi fication. While quantitative techniques like Real-Time PCR (RT-PCR) have proved invalu able in the study of other organisms, it probably has the most potential for the study of AM fungi when the presence of only one isolate is being measured. So far, quantita tive measures with this technology have been limited to root-associated AM fungi, wher e it shows some promise for specialized studies. Alkan et al. (2004) describe the use of RT-PCR to achieve quantitative results in detecting Glomus intraradices in tomato and alfalfa. Their RT-PCR data was calibrated with microscopic observations, thus enabling th em to subsequently screen large numbers

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29 of samples with reduced labor. They sugge sted that a similar application of using calibration curves to allow for high-throughput colonization assays using RT-PCR. This method appears likely to be useful for rapid identification and quantif ication of individual fungi for which reliable, estab lished primers exist, though less useful for quantification in field plants, where a complex of diverse AM fungi are present Ishii and Loynachan (2004) described two methods us ing nested-PCR, in which root segments infected with AM fungi previ ously stained by trypan bl ue were used in the extraction of DNA. The four greenhouse-grow n plants inoculated with an isolate of Glomus mosseae acquired from INVAM were maize ( Zea mays ), alfalfa ( Medicago sativa ), soybean ( Glycine max ) and sudangrass ( Sorghum sudanense ). Both methods used bead beating to expedite sample proces sing. The use of this technique would allow correlation with microscopic observations of the same samples, and could confirm colonization levels if phylum-specific prim ers were available and quantitative PCR was used. Further results from the use of molecula r and other investigative tools will be presented in a subsequent section wher e ecological research is discussed. Fatty Acid Methyl Ester Analysis Fatty acids occur at a reasonably constant proportion of cell biomass as they exist largely as components of membrane lipids. Si gnature fatty acids are biomarkers that can differentiate groups of organisms with in studied communities and can provide quantitative estimates of their microbial bi omass. These compounds are represented in literature by the number of carbon atoms with the degree of their unsaturation separated from the carbon chain length by a colon. The position of a double bond is indicated next, following the symbol indicating the number of car bon atoms from the terminal methyl

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30 end of the molecule to the first occurren ce of unsaturation (Zelles 1999). The suffixes c and t stand for the cis and trans isomers of the unsatu ration, respectively. Fatty acid analysis using gas chromatogr aphy (GC) has proven to be a useful tool for structural analysis of soil and plant-a ssociated microbial communities. Fatty acid methyl ester (FAME) analysis has been de veloped into an automated process by MIDI Inc. (Newark, Del.), whereby whole cell fatt y acids are extracted, then analyzed using appropriate software. Analysis of whol e cell fatty acids to study broad microbial communities that occur within varying agricu ltural management strategies has been well established (Cavigelli et al 1995; Buyer & Drinkwater 1997). The use of FAME extraction analyzed using the MIDI system is widespread due to the ease of sample processing; however, careful interpretation of the results is necessary, since both cellular and extracellular lipids are det ected with this technique. Extracellular lipids can be derived predominantly from organic matter as opposed to the ac tive fraction of the targeted organism, and therefore, data shoul d be considered accordingly (Zelles 1999). The fatty acid 16:1 5 c has been confirmed as a biomarker for AM fungi in studies investigating colonization of roots (Graham et al 1995), in estimations of fungal biomass in the soil (Olsson et al 1995), and in work that requires quantification of the AM fungal presence associated with both substrates (Thygesen et al 2004). The AM morphotypes within the Glomineae are reported to have higher concentrations of 16:1 5 c compared with some members of the Gigasporineae, within which it appears to negligible. Therefore, the use of this particular fatty acid will not detect comprehensively at the phylum level; however, it is capable of being used for detection and quantification of the AM fungal morphotypes that are considered the most important in agricultural

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31 ecosystems. As a result, the use of 16:1 5 c has good potential for use in studies to reasonably quantify AM fungal abundance of members of the Glominaeae in roots and soil when finer levels of identification are not deemed necessary; eith er in lieu of using labor-intensive root, spore and hyphal quantif ication, or as a means of complementing classical techniques. The fatty acids 18:1 9c and 20:1 9c have been identified as possible biomarkers for members of the suborder Gigasporineae, yielding useful results in studies on plant roots (Graham et al 1995) and from soil (Madan et al 2002), although th eir extensive use has yet to be undertaken. Perhaps compli cating the matter of th e use of fatty acids that appear to be produced in abundance other than 16:1 5 c is the evidence that fatty acids seem to be differentially produced in the various AM fungal structures (Graham et al 1995; Sakamoto et al 2004). For example, some evidence shows that the external hyphae of Gigaspora rosea seems to lack 20:1 9c, while it has been shown to be abundant within spores (Sakamoto et al 2004). Other Tools There are additional techniques for the char acterization of AM fungi in soil and roots, including variations of the methods listed previously. Those described above, however, are the most commonly found in re search currently. Undoubtedly, as our understanding of these organisms and the technology used to study them advances, a more distinct picture of the ecological ro le of mycorrhizae will similarly develop. A good introductory overview of so me of the other techniques used in AM fungal research, such as the quantification of extraradical hyphae, assays for functional activity, and identification using immunological methods is given in Millner and Wright (2002), and references contained therein.

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32 Arbuscular Mycorrhizal Fungi in Agroecosystems There has been an abundance of work published focusing on the existence, aggressiveness and state of functioning of AM fungi within ecosystems, including a substantial number of reviews and even meta-ana lyses of other researchers’ results. In a review of the diversity of AM fungi in a large variety of managed and natural ecosystems, the morphotype numbers found among the various studies ranged from 3 to 46 per site (Douds & Millner, 1999). Other stud ies that were cited in that work have shown, as was discussed in the first chapter, that the choice of crop rotation (or lack thereof), tillage and fertiliz ation strategies all can prof oundly affect populations of AM fungi. A meta-analysis of data from independently published field studies was accomplished by Treseder (2004), looking at the impacts that fertilization and CO2 enrichment have on the mycorrhizal state. The author found that across studies, mycorrhizal abundance was reduced by 15% due to N fertilization, 32% by P fertilization and increased 47% as a result of elevated CO2 levels. While ectomycorrhizal fungi were included in the meta-analysis, 18 of 20 studies compared for impact of P fertilization focused on the AM fungi. In the following sections, I will undertake a brief review of literature, and will concentrate on research that focuses on anth ropogenically derived alterations to the environment under agricultural production and their impact on AM fungal biomass and infectivity. In addition, an emphasis will be placed on field conditions that were important in this study. Phosphorus Fertilization. If such a thing as common knowledge exists for AM fungi, it is related to their role in plant P nutrition and, to a lesser degree, the impact hi gh P levels can have on the

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33 organism. This does not mean accurate ch aracterizations of th ese fungi occur in discourse as most, if not all, public awarene ss of mycorrhizae is due to the marketing of these fungi as an agricultural cure-all. Within th e agricultural scienc es it is widely accepted that they are capable of providi ng P to plants, especially, and perhaps exclusively, when soil availabi lity of the nutrient is m oderate to low (Schubert & Hayman 1986; Thingstrup et al 1998), and that high levels of available soil P contribute to depressed colonization of roots by AM fungi (Daft & Nicholson 1969; Thingstrup et al 1998). The mechanism for increased absorption of soil P by mycorrhizal roots is likely twofold. The primary mechanism seems to be due to the increased so il exploration that roots with the associ ation are capable of undertaking (Sanders & Tinker 1971; Koide 1991). There is also evidence that AM fungal hyphae may be able to excrete compounds increasing the solubility of the inorganic P (Joner et al 2000; Tawaraya et al 2006). The decrease in levels of the a ssociation when high levels of available soil P are present is apparently more a result of th e P concentration in the plant than it is related to actual soil levels (Koide & Li 1990; Koide 1991), although the two are obviously connected. Additionally, sporulation has been shown to be related to a threshold of AM fungal root biomass and possibly plant P concentrat ion (Douds & Schenk 1990), providing an explanation for why spore numbers are drama tically reduced when available soil P is high. This suppression of the mycorrhizal condition has been perceived as a selfregulatory process, whereby the plant reduces C allocation to the fungi when it is more P self-sufficient (Graham et al 1981). The mechanisms through which plants inhibit infection by AM fungi are probably quite va ried, considering the length of time the

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34 organisms have had to coevolve and the dive rsity of the many plant and fungal genotypes involved. There is evidence, though, that both biochemical means and anatomical structures can be involved (B rundrett & Kendrick 1990; Fortin et al 2002). The continuance of the mycorrhizal relationship when soil P is readily available can lead to a reduction in crop yield, through the misapplie d C investment wit hout a commensurate benefit in terms of plant P ac quisition (Graham 2000; Kahiluoto et al 2001; Ryan & Graham, 2002). Soil Disturbance The external hyphal network of AM fungi in soil is an important inoculum source for plant roots, so it comes as no surprise that disruption of soil through ag ricultural practices such as primary til lage and cultivation can reduc e the overall biomass and root colonization levels. This has been shown in many studies with similar results; only a few will be mentioned. A study by Drijber et al (2000) considered changes in the broad soil microbial community in moldboard plowe d, reduced tillage, no-tilled an d sod plots. Using FAME analysis, AM fungal biomass was determined to have decreased significantly in the more heavily disturbed treatments compared to the no-till and s od plots. Jasper et al (1991) investigated the infection poten tial of annual pasture, heathl and and forest soil sown with clover plants after disturbance. There wa s a significant decrea se in the AM fungal infection potential in heathland and forest soils after soil mixi ng, which led them to speculate that disturbance is probably most important wher e the initial propagule number is already low, a condition that was overcome in the pasture soil. More recently, an ongoing tillage-rotation experi ment in its sixth year in Chile compared different levels of soil disturbance on a variety of AM fungal-related parameters (Borie et al 2006). The

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35 less disturbed treatments had higher spor e numbers and greater levels of root colonization, as well as higher total cont ent of the AM fungal-derived glycoprotein, glomalin, when compared the more highly disturbed treatments. Pesticides One might anticipate that the use of pestic ides, particularly broad range biocides and fungicides, would have a negative effect on mycorrhizal survival and functioning. While this is generally the case, the impact of many of these agrichemicals on AM fungal communities is often slight and usually appear s to be only ephemeral in nature, and it can be difficult to predict the response to an indi vidual pesticide. The range of responses has varied from deleterious to negligible to s timulatory (Koide & Mosse 2004). It has, in fact, proven difficult to control AM fungi with chemical methods for research purposes, a situation that has made the development of non-mycorrhizal mutants all the more welcome. One of the fungicides considered most effective, benomyl, gives only partial control and requires frequent reapplication. In a study looking at th e P nutrition of the perennial common bluebell ( Hyacinthoides non-scripta ), individual colonies were grown in 28-L cubic blocks of soil (Merryweathe r & Fitter, 1996). The colonies receiving fungicide were immersed monthly in a soluti on of benomyl (50% w.p.) for control of AM fungi. While a significant re duction occurred, col onization was still measurable, reaching approximately 40% at the final assessment. In a recent work, six soil fumigants were investigated for their effects on the structure and functionality of the microbi al community, including AM fungi, using FAME profile analysis (Klose et al 2006). The sandy loam soil used in the microcosm study was fumigated for 24 hours, and then s ubjected to FAME extr action periodically

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36 over the next 90 days. Four of the six fumiga nts had a significant depressive effect on AM fungi biomass for the duration of the e xperiment, while the ot her two did not differ significantly post-fumigation from control soil; neither methyl bromide with chloropicrin, nor methyl isothiocyanate had a significant impact. Although the authors imply a lack of community recovery in the case of the four e ffective fumigants, the absence of change in the AM fungal biomarker during the course of this study is not su rprising, considering that no host was available for co lonization in the microcosm. A succinct review of the impact that some pesticides have on AM fungal populations is contained in a sec tion from a publication by Gosling et al (2006), while a more comprehensive, albeit dated, tr eatment is undertaken in Trappe et al (1984). Flooding There has been less information published regarding the mycorrhizal status of agricultural crops during flooded conditions than there has of wetland plants. It has been demonstrated repeatedly that AM fungal asso ciations can persist and benefit rice in wetland production (Solaiman & Hirata 1997; Solaiman & Hirata 1998), and it is well established that AM fungi colonize many we tland plants. While they are known to be aerobic organisms, there is an apparent means through which at least some taxa of these fungi are capable of surviving and functioning in flooded conditions. However, in native wetland plants, colonization levels have been reported to slow dur ing periods of flooding, with substantial increases in mycorrhizal development during any extended periods between flooding (Miller 2000; Ray & Inouye 2006). Even when soils have been submerged for long stretches, resulting in li ttle development of AM fungal colonization within roots, levels rebound in field plants and infection potential studies after drawdown of water levels (Miller 2000; Ray & Inouye 2006).

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37 In a study of post flood syndrome in the Midwest and Great Plains, Ellis (1998) examined maize and soybean plants that were grown in fields that had been flooded for long periods the previous year. Plants we re sampled three times during the growing season. Additionally, a greenhouse experiment was undertaken in which maize plants were grown in sterilized soil that was subsequently inoculated with Glomus intraradices spores and then flooded for either 0, 1 or 3 we eks. While flooded fields initially had low infection potentials the following year, colonization levels steadily increased as the season progressed. There was no significant di fference among the levels of colonization in the greenhouse study, and overall levels were actually highest in the two flooded treatments. The author attributed the low le vels of infection in fields during seasons subsequent to flooding episodes not to the extended periods of saturated soil, but rather to the absence of host tissue after the near complete crop death. These and other (Sah et al 2006) studies indicate that flooding conditions do not preclude the presence of a functional populat ion of AM fungi. Mechanisms for coping with both long-term saturate d conditions in wetlands and short-term flooding in agricultural fields and othe r land exist, and infective propagules persist after wet conditions subside. The reduced colonizati on during times of flooding is likely due to severely stressed plant root systems, which is relieved when drier conditio ns prevail and normal root growth can proceed. Organic, Conventional, and Grassla nd Farming Management Strategies In distinguishing among studies that focus on individual agricultural practices, it is easy to neglect research of a broader sc ope that compares pertinent management strategies. Of particular inte rest here is research that fo cuses on organic and grassland (or pasture) agriculture. Both of these represent types of farmin g that have been considered

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38 to be relatively amenable to the encourag ement of AM fungal popul ations in terms of biomass, functioning and diversity. Organic agriculture is not a static management style and farmers incorporate a tremendous diversity of practices with yield goals, type of crop, certifying body, soil type and geographic location among some of the more important vari ables involved in deciding practices. Farming strategies in orga nic agriculture that ar e thought to have the potential to minimize disrup tion of AM fungal populations include crop rotations and cover cropping, the absence of highly soluble ch emical fertilizers, and reduced reliance on pesticides with use restricted to botani cally derived or mined sources. While not exclusive of either organic or conventional ma nagement, but rather an integral part of both in many instances, grassland or forage agriculture differs from agronomic or horticultural row crop production. Agroecosystem s dedicated to or integrating long-term stands of native grasses or forage crops are generally distinguis hed by a lower relative agrichemical input, reduced traffic and soil disturbance and increased levels of plantderived organic compounds in the soil when co mpared to row crops. It is important to bear in mind that there can be consid erable overlap among farming management strategies and that what differe ntiates one from the other, in terms of what specifically influences AM fungal populations in a gi ven work, may not be readily deduced. Oehl et al (2004) found 35 morphotypes of AM fungi in one field site in Switzerland where the systems trial had been in place for longer than 22 years. This large number of identified taxa was a product of their trapping techniques, in which cultures were kept for extended periods of time under different tr ap plant species. Morphotype richness, spore abundance and inf ection potential were all higher in two

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39 organically managed systems when compared to two conventional systems. They attributed these differences, as well as the significantly higher colonization levels in biodynamic plots and the development of differe nt morphotype populations in the organic treatments, to qualitative and quant itative variation in fertilizers. Thirty-seven morphot ypes of AM fungi were collect ed from a single one-hectare field under mixed grasses and fo rbs in North Carolina (Bever et al 2001). The method used to extract so many species from a single field was a rigorous variation of trapping techniques; long, successive trapping periods multiple trap plant species and other changes in environmental conditions were used in order to simulate the year-round field conditions from which the samples were take n. The authors speculated that when a mycorrhizal complex exists, as opposed to indi vidual strains associating with one plant species, the host specific ity of AM fungi is high with re spect to fungal growth. That is, on certain plants, host specificity is manifest ed through more or less dominant AM fungal types developing. Similarly, the study lent cr edence to the idea that AM fungi exert an influence on plant community structure, but on ly in particular plant-fungal combinations. A central philosophy of organic farming is that management prac tices should strive to maximize biological functioning and diversity in order to maintain the health of the broader ecosystem, and AM fungi are perceive d as key players in these systems (Oehl et al 2004; Harrier & Watson 2004). Most stud ies comparing organic and conventional management systems have shown higher relative colonization levels (Douds et al 1993; Ryan et al 2000), as well as spore number and morphotype diversity (Douds et al 1993; Oehl et al 2004) in organic farming. This is t ypically interpreted as a product of the lower levels of slowly available soil P that co mes from the use of fertilizers acceptable in

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40 organic agriculture (Ryan & Gr aham 2002). Where nutrient app lications are in excess of crop needs and build to high levels in the soil and when tillage is a frequent occurrence for seedbed preparation and cultivation purpos es, this ideal of enhancing mycorrhizal relationships may be no more likely than in other high-input systems. Communities of AM fungi in the extensive root network in grassland and forage ag riculture, on the other hand, can be expected to develop a consider able external hyphal network, undisturbed by tillage (except during periods of establis hment) and with reduced occurrence of inhibition due to excessive nutrient levels. Other Fungal Root Endophytes Known to Occur in Herbaceous Plant Roots As non-AM fungal root endophytes represen t a diverse range of fungal taxa, and probably exist within most plant species ar ound the world, it is speculated that they contribute substantially to plant health and soil ecos ystem functioning (Sylvia & Chellemi 2001; Sieber 2002). However, as me ntioned in the introdu ction, the ecological roles of these endophytes can not be ascribed as being clear cut or uniform. In order to begin to understand them, a great deal of further work needs to be undertaken to ascertain both the phylogenetic status of many of these organisms and their host ranges (Jumpponen & Trappe 1998; Sieber 2002). Some of the non-AM fungal endophytes identified as commonly occurring in herbaceous plant roots include Philophora spp. and their teleomorphs, as well as species of Fusarium Microdochium Trichoderma and the loosely described group of anamorphic f ungi currently known as DSE’s (Sieber et al 1988; Skipp & Christensen 1989; Jumpponen 2001; Sylvia & Chellemi 2001; Sieber 2002). Phialophora spp. are some of the few endophytes commonly inhabiting herbaceous plants that have been identifie d and classified as belonging in that group.

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41 The DSE are an informal grouping that comprises Melin’s (Melin 1922; Wang & Wilcox 1985; Jumpponen & Trappe 1998) form taxon Mycelium radicis atrovirens (MRA) and other root-occupying fungi that meet a morphological standard and are apparently non-deleterious to the host plant. Melin desc ribed a fungus isolated from Pinus sylvestris and Picea abies roots that did not sporulate, which he believed was nonmycorrhizal and possibly harmful to the plant. Further, it grew on media, forming “grey air mycelium of tenuous hyphae” and produced a “dark olive-green pigment” when cultured (Melin 1922, p. 256; Wang & Wilcox 1985). The term MRA has continued to be used by some researchers for sterile fungi with darkly pigmented, septate hyphae found both growing in root cells and isolated from the soil (Wang & Wilcox 1985; Sieber et al 1988; Jumpponen & Trappe 1998). In a study examining mycorrhizal coloniza tion of plant communities at different altitudes in Austria, Read and Haselwandter (1981) not ed the existence of these endophytic fungi throughout the range of thei r survey and referred to the group as DS fungi. Thereafter, the term DSE has come into favor fo r dark septate endophytes and is used most frequently, although the exact mean ing and appropriateness of this term is sometimes questioned. A commonly used de scription for DSE includes those rootoccupying, biotrophic, mitosporic fungi with septate, melanized hyphae that typically also produce melanized microscler otia within the root (Treu et al 1996; Jumpponen & Trappe 1998; Sieber 2002). The search for a universally accepted name, or perhaps the dissolution of the need for a group, should come as taxonomic affinities are determined. It is interesting to take note of Barrow’ s (2003) suggested refining of the designation DSE to systemic endophytic fungi (SEF). His a nd others’ work take note of the difficulty

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42 in quantifying colonization by these endophytes due to their exte nsive network of nonstaining, hyaline hyphae (Yu et al 2001; Barrow 2003). In the present study a great deal of hyaline hyphae was encountered while obser ving samples; the abundance of hyaline hyphae with lesser amounts of da rk, septate hyphae occurring, as well as the existence of other poorly defined structures made grouping into classes difficult. Therefore, these non-AM fungal endophytes were not otherwise designated in this study, except where species or other taxonomic units could be a ssigned. It is unclear whether changing the designation to SEF would truly be a refinement or if there is instead an overlap of two or more groups of endophytes. Additionally, it would seem such a change would likely open the door to greatly expanding the numbe r of fungi included, potentially leading to more confusion.

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43 CHAPTER 3 MATERIALS AND METHODS Root and soil sampling. Soil samples were collected twice for use in infection potential and trap culture studies. Soil a nd root sampling for this study took place in years four and five of the Header Cana l cropping systems project, and occurred in December of 2003 and 2004. While plant and soil sampling were completed on the same day in both years, plots in year five were planted 36 days late r than in year four (October 7, 2004 compared to September 2, 2003) due to the severe weather. Four beds of tomato plants were in each half plot under production in 2003, but the hurricanes of 2004 destroyed a substantial portion of the beds th at had been formed in September; only two beds were established in each plot half wh en tomatoes were planted in October. To approximate the rhizosphere of the plan t, for each sample that was taken, the probe entered at the base of a plant and wa s angled slightly to capture a substantial portion of the root system along with the soil. Soil cores were taken from beneath plants in the middle beds and from within the middl e 100 plants in each row. Thirty cores from each plot were taken to a depth of about 20 cm, with 15 samples collected walking East to West in one center row, and the second 15 samples gathered walking West to East in the other center row. The cores from each plot were combined and homogenized in plastic bags, with the composite sample volume being approximately 2 L. Each of the five tomato production treatments was repl icated six times, producing a total of 30 samples from the portion of the plots then us ed for tomato production in year four, and 60 samples in year five.

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44 Soil sampling was also conducted within the transition areas in the organic, bahia pasture, weed fallow and disk fallow treatments in year four. Thirty cores were taken in each plot from the area parallel to the por tion of the plots from which the tomato production soil samples were taken. Cores we re taken from beneath the cover crop sunn hemp plants in the organic system, and from the bahia sod in the bahia system. Ragweed ( Ambrosia artemisiifolia ) was one of the most abundant and consistently present weeds across the weed fallow system plots, therefor e, soil samples were taken to approximate this plant’s rhizosphere in that treatment. Cores were also taken from the disk fallow plots in the areas being activ ely disked. There were 24 to tal composite samples taken from these transitional portions of the syst ems (four treatments x six replications). In year five, all previously transitioni ng portions of the plots were put into production. However, in the bahia pasture a nd disk fallow treatments, a considerable portion of the plots were under sod or being actively disked, respect ively. The section between each half plot remained free of tomato production throughout the experiment, and in these two systems this area approxi mated the continuance of the rotation. Therefore, soil samples were taken from th is middle portion of th e plots in these two systems using the same method described for sampling the transitional areas from the previous year, generating a total of 12 composite samples. Root samples were taken from all plots with tomato plants and treatment plots with cover crops and were used to assess co lonization by AM fungi and other fungal endophytes. Four tomato plants from each pl ot were dug from the center 100 plants of the middle beds. Loose soil was removed by ge ntly shaking roots pr ior to cutting the entire root system from the stem. A total of 120 tomato plants were removed in year four

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45 (four plants x 30 plots), and 240 pl ants were collected in year five (both halves of plots, eight plants x 30 plots). In y ear four, root samples were al so taken from cover crops from the plots with sunn hemp (organic), ragweed (weed fallow) and bahia plants. The bahia root samples were extracted from the paths between the tomato rows and not the tomato sod. The root systems in both year four and fi ve were placed in pa per bags and dried at 70 C. Fine roots were subsampled for clearing and staining, r oots were dried and subsample weights were between 0.05 and 0.07 g. Infection potential study sample preparation. Infection potential studies provide a method to evaluate the relative amounts of viable inoculum by growing a mycotrophic crop that forms abundant roots quickly in ei ther diluted (with sterilized sand), or undiluted soil (as in this study). Quite di fferent from full-seas on studies, infection potential results are typically timed to provide data regarding levels of primary infection on host crop roots. Harvested roots are exam ined for either the percentage colonization by AM fungi or total mycorrhizal root length as described in the section on quantification of fungal colonization in root s using microscopy. In prep aration for this and other evaluations, each of the composite soil samp les from individual plots was sieved to remove structural roots and to further homogeni ze soil. Fibrous roots were cut into small pieces (< 2 cm) and mixed with the soil. A portion of soil from each plot was removed for fertility and other analyses. The remaini ng soil and roots from i ndividual plots were divided in half, with one half designated for the spore trap culture described below, and the rest used for the infection potential study. For the infection poten tial test, soil was distributed at a volume of 125 mL among five to seven 150-mL conetainers based upon the amount of soil in the initial composite sample. Two germinated maize seeds were

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46 planted and watered into each tube, then late r thinned, selecting the healthiest looking plant. Infection potential plants were not fertilized, but were watered as necessary. Trays holding the conetainers were ra ndomly rearranged weekly. Ma ize plants were harvested approximately 30 to 40 days after planting, at the five-leaf stage of development. Soil was gently washed from the roots prior to their separation from the plant, and root systems were then each placed in paper ba gs and dried at 70 C. Dried roots were subsampled for clearing and staining, with subsample weight typically ranging from 0.03 to 0.05 g. Trap crop sample preparation. Acquisition of fresh s pores for morphotype identification through culturing diluted field soil or previously extracted spores using live plant roots is highly desirable, if not necessary, as the rapid de gradation in quality of field collected spores can render them usel ess (Millner & Wright 2002; Redecker et al 2003). The portion of soil not used in the infection potentia l study was used to grow trap plants for subsequent isolation of AM fungal spores. The soil designated for the trap culturing in each of the composite samples was divided between two 450-mL tubes. Five sudangrass ( Sorghum sudanense ) seeds were planted and then watered into the soil in each tube, later thinned to three plants. Trays holding the conetainers were randomly rearranged biweekly through the first 6 months of growth, a nd were fertilized weekly using 35 mL of a Hoagland solution with a ve ry low concentration of P (0.25 mM) after nutrient deficiency symptoms first develope d. The plants were allowed to naturally senesce, and were harvested approx imately seven months after planting. Trap culture processing, spore extraction and observation The method used to extract spores from trap cultures was an adaptation of the process mentioned in the

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47 previous chapter (Gerdemann 1955; Brundrett et al 1996). After the sudangrass plants had senesced, the aboveground biomass was removed and the soil within each conetainer was allowed to air dry. After removal from the tube, the soil and root system from each pot was broken down, with roots cut into smal l (< 2 cm) fragments. A representative 100 cm3 of soil and root mixture were suspended in 500 mL of DI wate r for 5 minutes with agitation. The soil-root suspension was then po ured over a set of three sieves. The sieve openings, top to bottom were 2 mm, 1 mm, a nd 38 m. The material on the top sieve was discarded, while the contents in the 1mm sieve were gently wa shed over the bottom sieve; the material remaining after rinsing wa s thrown away. The material in the bottom sieve was then distributed equally among six 50-mL centrif ugation vials followed by the addition of equal parts DI wate r and 50% sucrose (w/v). Th e sucrose was injected into the soil and associated material at the bo ttom of each tube after adding water, using a syringe with a piece of tubing attached, creating two layers with a clear interface. The six tubes were put in an International mode l HN centrifuge and spun for 5 minutes at approximately 771 x g. After centrifugation, th e more dense soil was pelleted at the bottom of each tube, while the remaining r oot tissue and other organic debris became suspended at the top. From each of the si x tubes, the 20-mL of solution surrounding the interface between sucrose and DI water was re moved individually using a syringe, taking equal proportions from above and below the interface to maximize spore recovery. This collected solution was drained from the spor es in suspension using vacuum filtration. The filtration system consisted of 55-mm circles of Whatman 50 hardened filter paper within a Bchner funnel and flask attached to a KNF Laboport vacuum pump. During filtration, after spores from all six tubes we re extracted, the syringe carrying the solution

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48 with spores was rinsed several times by load ing and ejecting DI wa ter from the syringe onto the filter paper. This served seve ral purposes; it was a method of clearing the contents of the syringe for the next sample, it ensured that spores remaining in the syringe after extraction were recovered, and it washed the sucrose from the spores and the filter paper, which improved the subsequent handling of spores. The filter paper was then moved to a Petri dish and observed under an Olympus SZH10 stereo microscope for enumerati on and identification. Enumeration was undertaken using year five trap cultures and was based on the method used by INVAM (2006b). The area of the ocul ar field at 4x magnification was determined to be 23.8 mm2, and the area of the 55-mm diameter filter paper was 2375.8 mm2, providing 100 possible fields of view for each spore extraction. The average number of spores counted in 40 randomly chosen fields was determined, yiel ding an approximation of the spores / cm3 of soil (average number of spores f ound x total possible fields / 100 cm3 of soil from which spores were extracted). Spore morphotypes were segregated after e numeration using the sharpened end of a thin wooden dowel. Spores were transferred to a slide, and observed after covering them with the several drops of a semi-permanent mounting agent consisting of polyvinyl-lactoglycerol (PVLG) with Melzers reagent (1:1 v/v). Spore morphotypes were observed and identified to the extent possible using an Olympus BX51 compound microscope with brightfield and differential inte rference contrast (DIC) optics. Joe Morton, the curator of INVAM at West Virginia University, provi ded assistance with morphotype delineations, and the INVAM collection descripti ons were used for classification.

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49 Clearing and staining roots. Roots were cleared and st ained for examination of colonization levels both in the IP study and for field roots. The procedure used for clearing and staining was a modified version of that described by Brundett et al (1996). Fibrous roots were subsampled and placed into scintillation vials. Roots were covered with 10% KOH (w/v) and placed in an oven at 95 C until cellular contents and pigments were largely removed, and before significant da mage occurred to the root system. This time period varied among the di fferent plant roots. Maize roots from the IP study were heated for 15 minutes, while roots of tomat o, sunn hemp, and rag weed were cleared for 25 minutes, and bahia roots were re moved after 30 minutes of heating. After rinsing several times with tap water, all roots, with the exception of those from maize plants, received an additional bleaching step. An acidified bleach solution was prepared using diluted Ultra bleach ( 1.7 % NaOCl) and 1 mL of diluted HCL per 100 mL of bleach. The bleach was added until th e sample was covered, and then the sample was swirled in the solution for up to 30 seconds. Total bleaching time varied among samples with additional applications initiate d until the roots became noticeably lighter in color; however, bleaching never exceeded 90 se conds. Roots were rinsed repeatedly after bleaching, then soaked in water for 5 minutes before staining. Staining was with 0.05% (w/v) trypan blue in lacto-gly cerol (1:2:2 lactic acid glycerol water). Maize and tomato samples were covered with stain and placed in a 95 C oven for 10 minutes, while the rest of the r oot samples were heated with stain for 12 minutes. Stained roots were ri nsed and then destained in l acto-glycerol for at least 24 hours before examination.

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50 Determination of % colonized root-length. Colonization levels were determined using the line-intersect met hod described in the section on quantification of fungal colonization in roots using microscopy, and derived from Giovannetti and Mosse (1980). Roots from both the infection potential study and field plants were observed using this method. Examination was with an Olympus SZ H10 stereo microscope using dark-field illumination. Intersections were scored for presence of AM fungi and other endophytes. When structures of questionable origin were encountered, root sections were removed from the Petri dish, mounted on a slide, and observed under an Olympus BX51 compound microscope using brightfield and differential interfer ence contrast (DIC) optics. Soil fertility and fatty acid analysis. Soil from each plot was sent to Waters Agricultural Laboratories in Camille, GA for fert ility analysis in both year four and five. The Mehlich-1 extraction procedur e was used to determine levels of available P, as well as other nutrients. Subsamples from composite soil and tomato roots were sent to John Larsen at the Danish Institute of Agricultural Sciences, fo r FAME analysis in his lab. Approximately 0.2 g of tomato root tissue from each plot, a po rtion of fine roots from each of the four tomato plants sampled, was stored in 2% (v /v) acetic acid, and a 10-g subsample of soil from each plot composite was subjected to lyophilization for preservation during transport to Denmark. Whole cell fatty acids were extracted and methyl esters separated using gas chromatography and subsequently identified using the automated procedure developed by MIDI Inc. (Newark, DE).

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51 Statistical analysis. One-way analysis of variance was us ed to test for differences among treatments with regards to colonization levels. Means determined to be significantly different ( P < 0.05) were separated using Tukey' s HSD test. Data were transformed where necessary to achieve normal distributi on. Pearson’s correlations were used to examine relationships between FAME results from soil and roots and colonization levels in the IP and field plants, respectively, as well as interactions among endophytes and between endophytes and edaphic factors. Stat istical analyses were performed using SAS release 8.02 for Windows.

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52 CHAPTER 4 RESULTS AND DISCUSSION Agricultural and Environmental Influences Impacting Edaphic Conditions at the Header Canal Study Site Over the production history of this field, including the period during this research project, multiple applications of fertilizer ha ve resulted in extremely high P levels (Table 4-1). The relatively higher levels of this nutrient that occur in the organic treatment are due to the annual addition of br oiler litter in these plots, at the rate of approximately 22,400 kg ha-1, which should result in about 800 kg of applied P2O5 ha-1, or 350 kg of applied P ha-1. The increase of soil P levels by 600 kg ha-1 between years four and five in the organic plots as revealed by soil testing indicate higher P concentration in the manure, greater relative mineralizati on leading up to the time of sampling, or a combination of both of these factors. The over-application of P compared to plant need when using broiler litter as a nutrient source is a common problem, resulting from the low N:P ratio of the material, because fertilizer rates ar e often determined based on the N needs of a crop. The plots in other treatments, duri ng seasons of tomato production, received a comparatively conservative 56 kg ha-1 of applied P2O5. As discussed in the literature review, high levels of available P in th e soil result in plant P sufficiency with accompanying higher tissue concentrations and correlative lower levels of plantassociated AM fungal biomass and spore produc tion. All treatments had very high levels of soil P, and this was exacerbated by applica tions that exceeded plant uptake, with the most extreme example occurring in the organic plots.

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53 Table 4-1. Results of soil analyses from year four and five of a 5-year cropping systems study at the Header Canal experimental farm site in Fort Pierce, FL. Year Treatment %OM P (kg ha-1) Bulk density (g cm-3) a pH S (kg ha-1) Mn (kg ha-1) Bahia pasture 0.75 ( 0.04)795 ( 55)Not m easured 7.14 ( 0.09)77.90 ( 7.86)23.15 ( 1.18) Conventional 0.60 ( 0.04) 821 ( 51) Not measured 7.60 ( 0.16)67.95 ( 18.35) 23.52 ( 2.09) Disk fallow 0.53 ( 0.03) 819 ( 58) Not measured 7.70 ( 0.07)44.20 ( 5.58) 25.59 ( 1.08) Organic 1.70 ( 0.14) 1220 ( 90) Not measured 6.88 ( 0.07)78.03 ( 11.40) 37.43 ( 2.61) 4 Weed fallow 0.82 ( 0.02) 884 ( 51) Not measured 7.40 ( 0.06)58.24 ( 6.12) 26.76 ( 1.43) Bahia pasture 0.73 ( 0.05)794 ( 36)1.43 ( 0.02)7.04 ( 0.10)24.22 ( 7.19)39.48 ( 2.28) Conventional 0.55 ( 0.03) 849 ( 35) 1.55 ( 0.02) 7.47 ( 0.10) 42.28 ( 9.02) 11.29 ( 2.96) Disk fallow 0.44 ( 0.02) 899 ( 31) 1.55 ( 0.02) 7.65 ( 0.04) 27.60 ( 4.83) 40.25 ( 1.22) Organic 2.36 ( 0.25) 1831 ( 80) 1.24 ( 0.02) 7.10 ( 0.03) 13.64 ( 10.28) 76.26 ( 2.68) 5 Weed fallow 0.69 ( 0.02) 859 ( 52) 1.47 ( 0.02) 7.34 ( 0.07) 17.45 ( 8.65) 47.88 ( 2.79) Values are treatment means SE. a Bulk density was not determined in year four plots.

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54 Organic matter levels were highest in th e organic plots (Table 4-1), due to the broadcast of urban plant debris at an approximate rate of 70,000 kg ha-1, spread concurrently with the broiler litter. The bahia pasture and weed fallow treatments both had long-term stands of vegetation, resulti ng in substantial car bon input through rootderived deposition, and theref ore had higher organic matter levels than either the conventional or disk fallow plots. As woul d be expected on a field with a homogeneous soil type, the addition of ample organic matte r had the corresponding effect of decreasing soil bulk density in individual treatments, as was determined through soil testing in year five. While bulk densities were high for a plow layer, they were not uncharacteristically so for sandy soils. Additions of organic matter are known to have a number of positive effects on soil structure. Water-stable aggregates are formed as organic matter is added from external inputs or directly from plant and mi crobial deposition and decomposition. Many physical, chemical and biological factors inter act in forming soil aggregates, including a substantial contribution derive d from the activity of AM fu ngi (Rillig & Mummey 2006). The benefits that organic matter lend to the development and maintenance of soil structure were evident after tw o hurricanes swept over the experi mental farm site in 2004. While most treatment plots had their raised beds destroyed (Figur e 4-1A), many of the organic plots largely retained their bed st ructure (Figure 4-1B), despite substantial flooding. Soil fertility test results additionally indicated that flooding conditions persisted long enough to have a substantia l impact on nutrient av ailability, revealin g the extent of

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55 Figure 4-1. Header Canal experimental farm si te plots after the hur ricanes of 2004. Photo taken prior to final tomato transplanta tion in year five of a 5-year cropping systems study in Fort Pierce, FL. A) Remnants of raised beds for tomato production in conventional system. B) Ra ised beds still la rgely intact in organic plots.

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56 waterlogging. Manganese (Mn) is extremel y soluble at the low redox potentials that occur in soil with flooding (Tisdale et al 1993a); this effect is ev ident at the farm site when comparing extractable Mn2+ between year four, a season with normal rainfall, and year five, a season with abnormally high rainfall. The status of sulfur (S) was also altered in the field between the two seasons. The form of S absorbed by roots is SO4 2-, and is easily leached from soils. Additionally, when soil moisture is high, mineralization from organic matter and oxidation of i norganic S is decreased (Tisdale et al 1993b). Reduced amounts of S were extracted in treatments during year five as a result of these processes. Colonization of Roots of Tomato and Cover Crops by Arbuscular Mycorrhizal Fungi and Other Fungal Endophytes Arbuscular Mycorrhizal Fungi in Tomato and Cover Crop Roots Tomato root colonization by AM fungi wa s very low to moderately low across treatments, with significantly higher levels o ccurring in the bahia pa sture plot in both years (Table 4-2; Figure 4-2). The organi c system had significantly lower levels of colonization compared to all other treatment s in year five. The colonization levels observed in year five tomato roots were co rroborated by FAME analysis (Figure 4-3). There was a strong correlation betw een colonization levels and 16:1 5 c content measured from root samples on the plot level. The overall pattern and levels of colonizat ion in tomatoes among treatments did not vary much between years (Table 4-2). Ther e are two main distinguishing characteristics that separate the bahia treatment, which had the highest levels of colonization, from the others: the bahia pasture plot s had neither extensive soil mixing nor raised bed formation prior to tomato production. Instead, main tenance of a highly mycotrophic sod was preserved throughout the 5-year experiment, except where narr ow strips were tilled for

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57 Table 4-2. Field tomato root colonization by arbuscular mycorrhizal (AM) fungi and other fungal root endophytes from year four and five of a 5-year cropping systems study at the Header Canal experimental farm site in Fort Pierce, FL. Year Orientation Treatment AM fungal % colonizationa Other fungal root endophyte % colonizationab M. bolleyi % colonization acd 4 South Bahia pasture Conventional Disk fallow Organic Weed fallow 24.1 ( 3.5) 8.2 ( 3.4) 8.8 ( 1.8) 8.2 ( 2.3) 14.4 ( 3.5) Was not measured Was not measured P values Treatment 0.0039 North Bahia pasture Conventional Disk fallow Organic Weed fallow 32.6 ( 4.6) 9.2 (1.6) 9.9 (2.5) 6.7 (1.7) 12.7 ( 1.8) 52.1 ( 2.0) 39.6 ( 1.1) 47.9 (5.7) 44 ( 1.4) 37.9 ( 5.0) 0.6 ( 0.4) 0.1 (0.1) ND 12.8 ( 2.2) ND 5 South Bahia pasture Conventional Disk fallow Organic Weed fallow 30.3 ( 2.6) 15.2 ( 2.4) 11.7 ( 1.4) 4.3 ( 0.9) 13.0 ( 2.0) 58.4 ( 4.8) 47.8 (5.4) 48.8 ( 6.3) 37.5 (1.6) 36.2 (3.3) 0.5 ( 0.4) ND ND 13.0 (3.1) ND P values Treatment Orientation Interaction <0.0001 0.4011 0.1541 0.0083 0.5263 0.2828 <0.0001 0.7934 0.9856 aProportion data were transformed to the squa re root of acrsine prior to performing ANOVA; values in table are actual mean propor tions of root colonization of treatments SE. b Colonization levels of non –arbuscular myco rrhizal fungal root endophytes were not measured in year four tomato roots. c Colonization levels of Microdochium bolleyi were not measured in year four roots. d ND indicates treatments in year five where Microdochium bolleyi was not detected.

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58 Figure 4-2. Field tomato root colonization by arbuscular mycorrhiza (AM) fungi in years four and five of a 5-year cropping systems study at the Header Canal experimental farm site in Fort Pierce, FL. The treatments in the study were bahia pasture (B), conventional (C), di sk fallow (DF), organic (O), and weed fallow (WF).

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59 Figure 4-3. Correlation with least square s regression of the fatty acid 16:1 5 content in tomato roots to the observed proportion of colonization of tomato roots in field plots by arbuscular mycorrhizal (A M) fungi. Results are from year five of a 5-year cropping study at the Header Canal farm site in Fort Pierce, FL. Data were normalized by transformation to square roots.

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60 tomato transplantation (Chellemi et al. 1999) (Figure 4-4). The minimal levels of disturbance creating conditions to sustain preservation of the external hyphal network associated with bahia roots provided transp lants with a directly infective, thoroughly distributed and effectiv e source of inoculum. The other treatments all had soil mixing to different degrees prior to tomato transplantation, and, with the ex ception of organic plot s in year five, this resulted in full season colonization levels that were not st atistically distinguis hable from each other (Table 4-2). The significantly lower levels of colonization in year five organic tomatoes may be attributed to soil-related factors, including conditions that both enhance root growth and reduce dependency on mycorrhizal f ungi. It has already b een established that the P status in the field was well in excess of plant need; levels in the organic plots were so high that it is surprising th at AM fungi were able to co nsistently gain ingress. Additionally, the lower bulk density and im proved aggregate formation that develops from organic matter additions in these plots would improve the physical environment for root growth by reducing resi stance for root extension and improving drainage after waterlogging. Reduced relativ e colonization levels in th ese conditions may not be a result of less inoculum or decreased infectivit y of the inoculum present, but rather may be derived from conditions that allowed roots to gr ow in the organic treatment at a rate that outpaced the ability of AM fungi to develop; a faster rate than occurred in the other treatments with regularly homogenized soil. This possibility is supported by the results of the infection potential study that show that the levels of primary infection were not comparatively depressed in the organic treatments (Table 4-3).

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61 Figure 4-4. Bahia pasture plots wi th rows of tomato plants. In this treatment, the only soil disturbance after establishment of th e sod occurred in years four and five of the project, when 46-cm rows were tilled for tomato transplants. Picture taken during year five of a 5-year cr opping study at the Header Canal farm site in Fort Pierce, FL.

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62 Table 4-3. Field soil infection potential of arbuscular mycorr hizal fungi and other fungal root endophytes on maize from year four and five of a 5-year cropping systems study at the Header Canal experimental farm site in Fort Pierce, FL. Year Orientation Treatment AM fungal % colonizationa Non-AM fungal root endophyte % colonizationa North Bahia pasture Disk fallow Organic Weed fallow 23.4 (1.7) 4.3 ( 0.8) 14.8 (2.3) 21.7 ( 2.5) 52.2 ( 1.2) 29.6 ( 3.8) 46.6 ( 3.3) 53.1 ( 3.8) 4 South Bahia pasture Conventional Disk fallow Organic Weed fallow 26.5 ( 2.3) 24.0 ( 1.5) 25.4 ( 5.7) 18.0 ( 2.0) 21.2 ( 3.5) 54.9 ( 3.3) 40.0 ( 3.1) 34.4 ( 4.5) 28.4 ( 3.0) 45.4 ( 6.5) P values Treatment Orientation Interaction 0.0040 0.0018 0.0010 0.0001 0.1668 0.0739 North Bahia pasture Conventional Disk fallow Organic Weed fallow 16.9 ( 2.5) 8.3 ( 1.8) 8.8 ( 0.7) 17.1 ( 1.7) 8.8 ( 1.2) 48.7 ( 2.9) 33.3 ( 6.9) 29.8 ( 3.1) 19.7 ( 2.5) 34.2 ( 3.0) South Bahia pasture Conventional Disk fallow Organic Weed fallow 18.1 ( 4.4) 9.0 ( 1.5) 9.7 ( 1.4) 22.7 ( 4.9) 7.6 ( 1.3) 47.6 ( 4.7) 28.7 ( 6.1) 30.7 ( 3.1) 17.6 ( 1.8) 38.9 ( 3.0) 5 Middle Bahia pasture Disk fallow 13.4 ( 1.5) 3.7 ( 1.6) 28.8 ( 2.9) 19.2 ( 1.4) P values Treatment Orientation Interaction < 0.0007 0.8685 0.1571 < 0.0001 0.8300 0.7773 aProportion data were transformed to the squa re root of acrsine prior to performing ANOVA; values in table are actual mean propor tions of root colonization of treatments SE.

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63 Colonization levels of AM fungi were also determined for cove r crop roots in the northern half of year-four plots, the por tion that remained out of production until the following year. Mean root colonization le vels were approximately 17.2 2.5 and 21.8 3.3 %, respectively, for bahia roots from the bahia pasture and ragweed roots from the weed fallow plots. Sunn hemp was colonized to a much lesser extent, with average levels among organic plots only reaching 3.4 0.6 %. Again, it is likely th at a lack of soil homogenization up to that point in the w eed fallow plots probably allowed for the development of an extensive mycelial network as in the bahia pasture treatment. This consequently maximized the spatial distribut ion of AM fungal inoculum. Tillage was performed in the organic plots to establis h cover crops, however, and soil P had been steadily building due to manure applications. Additionally, the mycorrhizal status of sunn hemp under conditions of P suffici ency may simply be negligible. Tomato Root Colonization by Non-Arbuscular Mycorrhizal Fungal Endophytes The extensive occurrence of other endophy tic fungi and fungal-like organisms was noted in year-four tomato roots, and quantifie d in the year-f ive roots (Table 4-2; Figure 4-5). In addition, quantification of this group of organisms was determined through the infection potential study using soil from treatment s in both year four and five (Table 4-3). When considered as a discrete group, the levels of infection of tomato roots in year five by these other fungal endophytes was considerably greater than that of the AM fungi in the same year. The majority of non-AM fungal endophyt es identifiable th rough the direct observation of tomato and other field roots, appeared to be Phialophora spp., as determined by the presence of distinctive, en larged fungal cells w ithin corti cal tissue (Figures 4-6 and 4-7). These structures are often called microsclerot ia in studies where

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64 Figure 4-5. Field tomato root colonization by all non-arbuscular myco rrhizal fungal root endophytes in year five of a 5-year cropping systems study at the Header Canal experimental farm site in Fo rt Pierce, FL. The method used to determine colonization levels was id entical to and concurrent with assessments for the presence of arbuscu lar mycorrhizal fungi. The treatments in the study were bahia pasture (B), conventional (C), disk fallow (DF), organic (O), and weed fallow (WF).

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65 Figure 4-6. Mass of enlarged fungal cells produced by Phialophora spp. within the cortex of tomato roots; blue color is a result of staining with trypan blue.

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66 Figure 4-7. Mass of enlarged f ungal cells and hyphae produced by Phialophora spp. within the cortex of maize roots; blue color is a result of staining with trypan blue.

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67 the fungi are referred to as DSE. The hyphae as sociated with these structures were only occasionally pigmented, although in individual roots the extent of naturally pigmented hyphae could be considerable. These hyphae we re often stained by procedures used to detect AM fungi. Polymyxa spp. are endophytes clas sified in the order Plasmodiophorales, and were found on severa l occasions in tomato roots; these organisms were easily recognized by the cluste rs of resting spores formed within the cortex (Figure 4-8). There was a diversity of other structures that were only occasionally observed in field roots, a lthough identification wa s not always achieved. Unidentified structural features were often not positiv ely related to taxa due to the lack of distinguishing characteristics (an example be ing intracortical hyphae without distinctive associated features), or the absence of conf irming descriptions. Identifying taxa was not a priority when designating colonization levels of non-AM fungal root endophytes, although tissue had to clearly be fungal, or from fungal-like organisms, such as the plasmodiophorids, in order to be positively designated during colonization assessments. It is important to note that none of these observed organisms were determined to be deleterious to the health of their host plants in any way, nor was there any obvious antagonistic relationship among st udied organisms. In the season that both groups of fungi were quantified from field roots, thei r concurrence was positively correlated (r = 0.472, P < 0.0005). Similar observations were ma de in the infecti on potential study and will be discussed below. Microdochium bolleyi in Tomato Roots from the Organic Treatment The distinctive, corn-cob-like structures produced by M. bolleyi within tomato roots in this study were the stimulus for exam ining the occurrence of a broader range of

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68 Figure 4-8. Clusters of resting spores (cystosori) formed by Polymyxa spp. within the cortex of a tomato root; bl ue color is a result of staining with trypan blue.

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69 fungal endophytes than just the AM fungi (Fi gure 4-9 and 4-10). After clearing tomato roots for observation, these masses were the most clearly distinguishable structures where they occurred, irrespective of staining. These masses of fungal tissue did occasionally absorb trypan blue and stain blue (Figure 411), but this did not enhance visualization compared to naturally pigmented tissue. To mato roots in the organic treatment were unique in that they were the only roots in this study in which significant levels of this organism were measured (Figure 4-5), a phenomena that occurred in both years, although it was only measured in year five. The diagnostic structures for this organism as it develops within the plant cortex have been called alternatively, chlamydospores dark cells, or microsclerotia, and are known to preferentially occupy the inner and outer cortex (Murray & Gadd 1981; Kirk & Deacon 1987; Sieber 2002). While these struct ures were obvious within roots when the organism produced them, they appeared to be a small portion of the total biomass produced by M. bolleyi as extensive colonization o ccurred through hyaline hyphae that did not stain with trypan bl ue (Figure 4-10). In fact, extension of the transparent intracortical hyphae appeared to terminate w ith these pigmented structures, making their origins difficult to discern. The ability of M. bolleyi to flourish in the organic pl ots was not due to the tomato plant host crop in which the organism was dete cted, as this was the production crop in all systems and the same variety was used across the field. However, the use of cover crops and organic matter applications in the organi c treatment may provide the answer to its selectively prolific nature. Microdochium bolleyi is a saprophytic fungus that is known to occur widely within the roots of small grains, in which it ha s been reported to be a

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70 Figure 4-9. Pigmented cells formed by Microdochium bolleyi within tomato roots.

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71 Figure 4-10. Close-up of pigmented cells formed by Microdochium bolleyi within tomato roots. Transparent cells and hyphae can be seen associated with the structure.

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72 Figure 4-11. Close-up of pigmented and trypan blue-stained cells formed by Microdochium bolleyi within tomato roots.

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73 weak parasite with a possible antagonistic interaction with pathoge nic fungi and thus a potential biocontrol agent (Murray & Ga dd 1981; Kirk & Deacon 1987). The organic treatment, with a spring crop of Japanese millet directly preceding tomato production, was the only system with a cover crop similar to the small grains. Extensive sampling of this cover crop did not occur, a lthough an informal sampling found M. bolleyi to be very common within its roots. Infection Potential of Field Soil on Greenhouse-Grown Maize Arbuscular Mycorrhizal Fungal Infection Potential Whereas determining the mycorrhizal stat us of end-of-season roots provides a measure of the ability of AM fungi to deve lop associations in the field over time, the infection potential bioa ssay is used to provide an indi cation of the infective inoculum available to create primary inf ections. In order to estimate this potential, it is crucial that host plants are harvested in a timely manner. It has been determined previously that secondary infection in maize roots can be mi nimized when plants are harvested at the five-leaf stage of developmen t (approximately 30 days after seed germination) (Moorman & Reeves 1979). The results from the study on the infec tion potential of AM fungi revealed relationships between these organisms and ma nagement tactics that were not evident from the full season tomato plants that we re investigated (Tab le 4-3; Figures 4-12 through 4-14). Soil sampled from both ha lves of system plots produced generally uniform levels of colonization on maize roots in year four, with the exception being the northern halves of disk fallow plots which di ffered significantly from all other treatments regardless of management strategy (Figure 4-12 ). The northern portion of the disk fallow

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74 Figure 4-12. Infection potentia l of treatment soil sampled in year four of a 5-year cropping systems study at the Header Cana l experimental farm site in Fort Pierce, FL; infection potential was de termined by colonization levels of arbuscular mycorrhizal (AM) fungi on gr eenhouse grown maize plant roots at the five-leaf stage. Columns with the same letter above SE bars indicate no significant difference (means separated at = 0.05 using Tukey’s HSD). The treatments in the study were bahia past ure (B), conventional (C), disk fallow (DF), organic (O), and weed fallow (W F).Treatment codes are followed by an N or S, designating the plot half samp led (N = north, S = south). Conventional plot soil was not sampled in the northern half in year four.

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75 Figure 4-13. Infection potentia l of treatment soil sampled in year five of a 5-year cropping systems study at the Header Cana l experimental farm site in Fort Pierce, FL; infection potential was de termined by colonization levels of arbuscular mycorrhizal (AM) fungi on gr eenhouse grown maize plant roots at the five-leaf stage. The treatments in the study were bahia pasture (B), conventional (C), disk fallow (DF) organic (O), and weed fallow (WF).Treatment codes are followed by an N or S, designating the plot half sampled (N = north, S = south).

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76 Figure 4-14. Infection potential of disk fallow treatment soil sampled in year four and five of a 5-year cropping systems st udy at the Header Canal experimental farm site in Fort Pierce, FL; infection potential was determined by colonization levels of arbuscular mycorrhizal (AM) fungi on greenhouse grown maize plant roots at the five-leaf stage. During this study, the southern half of disk fallow plots produced thei r first tomato crop in year four and finished a second year of production at th e end of year five. Northern plot halves only produced one tomato crop, ha rvested at the end of year five. Actively disked areas were located in th e northern half of plots in year four, and in between plot halves in year five.

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77 treatment was still actively disked at the time of sampling and had been managed accordingly to eliminate plant growth for the f our previous years. All other treatments were statistically similar, with the exception of slightly lower levels of colonization in the northern half of organic plots at the end of their first year of tomato production when compared to bahia pasture and conventional sy stem plots. This difference in organic plots might be attributable to the impact of the high P levels in th at treatment, although the results do not carry over to the following year. While the extremely low levels of AM co lonization occurring on infection potential hosts grown in actively disked soil is not su rprising, especially considering the generally adverse conditions due to other agricultural ma nagement practices in the field, a direct comparison between the northern and southern halves of plots revealed an unexpected response (Figures 4-12 and 4-13). After a si ngle full season of host growth, such as occurred in the southern half of disk fallow plots, the infection poten tial recovered to the extent that it was indistinguishable from all other treatments. The implication is that AM fungal inoculum density and infectivity fo llowing 14 years of in tensive agricultural management, including three consecutive ye ars of continuous disturbance, was not significantly impacted compared to the othe r farming practices i nvestigated after host roots were allowed to persist for several months. This indicates that inoculum recovers from periods of intense disturbance in the absence of host tissue more rapidly than previously suspected (Gosling et al 2006). The intense flooding and reduction in exposure to host roots in year five appears to have altered treatment differences compared to the previous year (Table 4-3; Figure 413). Both halves of the disk fallow plots ha d been put into producti on and at the end-of-

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78 season sampling time they both had similar le vels of colonization, albeit reduced from colonization levels that occurred in disk fallow production plots from the year before. However, the infection potential of that por tion of plots where disking continued, in the stretch of land between the northern and southe rn plot halves, colonization levels were almost identical to actively disked soil from the year before (Figure 4-14). This suggests a possible baseline level of viable inoculum in this field regardless of management, from which recovery to more extensive levels o ccur after host roots prime the soil. Because tomato roots were in the soil for 36 fewer days in year five compared to year four, it is possible to surmise the reduced recovery of inf ection potential in year five was partially a product of the shorter priming period. The infection potential of all treatment s was reduced in year five, with the exception of organic plots (Table 4-3). Determination of the 16:1 5 content of field soil using FAME analysis helped confirm the inf ection potential study results in year five (Figure 4-15), and correlation at the treatment level was verified (r = 0.9001, P = 0.037). When comparing the colonization levels of full season field tomatoes to infection potential maize plants in the organic treatment the discrepancy mentioned in the previous chapter is clear; despite low levels of coloni zation occurring on tomato plants, the organic plot had considerable active inoculum as evidenced by s ubstantial primary infection on maize in infection potential assays. The likely reasons for the low levels of field tomato root colonization were discusse d in the previous chapter, an d are related to the clearest explanations for higher infec tion potentials in the organic treatment. The improved soil structure in these plots allowed for more ra pid drainage from saturated conditions and also reduced resistance to root growth when compared to the other tr eatments with higher

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79 Figure 4-15. Results from Fatty Acid Methyl Ester (FAME) analysis of soil from year five of a 5-year cropping systems st udy at the Header Canal experimental farm site in Fort Pierce, FL. Results show the levels of the arbuscular mycorrhizal (AM) fungal biomarker 16:1 5 within treatments. Content of treatment soil is relative to a fatty acid standard (19:0). The treatments in the study were bahia pasture (B), conventiona l (C), disk fallow (DF), organic (O), and weed fallow (WF).Treatment codes are followed by an N or S, designating the plot half samp led (N = north, S = south).

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80 bulk densities. This would lead to a quicker recovery of to mato plant root growth from stressful conditions, and a generally more favorab le habitat for roots in the organic plots. Concurrent with increased rela tive root growth is a greate r extension of an active AM fungal mycelial soil network, and thus increase d generation of inoculum. Where the pace of root growth exceeds the ability of the AM fungi to develop and advance in the cortex, lower overall colonization levels can be e xpected to occur. Additionally, extensive development within plant tissue is more likely to be retarded in plants that have access to abundant P, while the primary infection of newer fine roots may proceed unabated. The bahia pasture plots had infection potential levels that were not clearly elevated compared to other systems as occurred in fi eld roots, although colonization levels were significantly higher than conven tional, disk fallow and weed fallow plots in year five (Figure 4-13). The moderation of bahia past ure infection potentials compared to full season field root colonization ca n be attributed to sample preparation for the greenhouse study. Tomato transplant colonization levels in bahia pasture plots benefited from a largely undisturbed, spatially dense mycelium in the soil; but after cores were sampled at the end of each growing season, soil sample s from each plot were bulked and thoroughly homogenized. While coloniza tion levels in infection potential maize plants were decreased compared to field plants, the bahia pasture plots consisten tly generated levels of inoculum that overcame the short-term di sturbance with moderate levels of primary infection. Non-Arbuscular Mycorrhizal Fungal Root Endophyte Infection Potential As in the field roots, no negative inte raction among these groups of fungi was detected. Instead, positive correlatio ns occurred in year four (r = 0.441, P = 0.002) and year five (r = 0.201, P = 0.117) with respect to coloniza tion levels of both groups of

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81 endophytes within maize roots from the infect ion potential study. Levels of colonization by non-AM fungal endophytes in the field were comparable to th e results of the infection potential study (Tables 4-2 and 4-3). Bahia pasture soils had the highest overall infection potential for non-AM endophytes, while organic plots had significantly lower levels of colonization in year five compared to othe r systems. In addition, there was a general decline in the colonization levels in the infection potential study in year five compared to the previous year, probably because of the widespread waterlogging of soils. There was no evidence of M. bolleyi in maize roots, although this doe s not preclude the presence of this endophyte. The maize roots were quite young at the tim e of harvest and M. bolleyi is known to preferentially occupy naturally se nescing root cells, generally forming the darkly pigmented diagnostic structures wher e the plant forms papillae (Kirk & Deacon 1987), while hyphae are hyaline and do not stain. The morphological st ructures of fungal root endophytes observed in maize were simila r to those seen in tomato roots, although extensive occurrence of the chytrid Olpidium spp. was observed. (Figure 4-16). Making assessments on the status of su ch a broad group of organisms based on their ability to initiate pr imary infection would be difficult and probably be of limited value. However, it is clear from these data in addition to that from full-season tomato roots, that considerable root colonization by a diverse set of fungal taxa occurred in this field and that colonization can develop quickly.

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82 Figure 4-16.Maize roots with colonization of a chytrid Olpidium spp. stained with trypan blue. Fungal structures are resti ng spores and zoospore cysts.

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83 Spores: Enumeration and Identification of Arbuscular Mycorrhizal Fungal Morphotypes Spore Enumeration of Year Five Trap Culture Soil Sudangrass cultures were harvested and spores extracted from the root-soil complex for each of the replicate pots that we re established. Spores were then counted and a mean derived for each replicate pot to develop an approximation of the number of spores occurring in the rhizosphere of host pl ants in each treatment. Spore counts were performed in both years of tomato producti on and in both halves of all plots. Spores extracted and observed from year five soil were scarce (Figure 4-17). While significant differences existed among treatmen ts, with the bahia pasture and conventional plots having more spores than the other tr eatments, and organic and disk fallow plots having the fewest, spore number s in all treatments were very low. Spore numbers may have been slightly to moderately underesti mated in both years, however, as a result of adapted harvesting and enumeration procedures that were used to improve spore retrieval for mounting. Also, the final step of spore ex traction used vacuum filtration to drain and wash away sucrose solution from spores. When on wet filter paper, spores were remarkably easier to grasp and manipulate for mounting when compared to spores free floating in water within a Petri dish. When observed associated with white filter paper, though, juvenile and hyaline spores are much harder to see and therefore, were undercounted While year five sudangrass cultures were grown in undiluted field soil, year four cultures were grown in soil diluted with ve ry low P sand in order to stimulate spore generation. Early in the study, it seemed questionable whether sporulation (or colonization of plant roots in the infection pote ntial test) would occur at all at the extreme

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84 Figure 4-17. Mean arbuscular mycorrhizal fungal spore numbers extracted / cm3 of undiluted treatment soil from year five of a 5-year cropping systems study at the Header Canal experimental farm s ite in Fort Pierce, FL. Spores were extracted from field soil used to grow a sudangrass trap culture in a greenhouse study. Columns with the same letter above SE bars indicate no significant difference (means separated at = 0.05 using Tukey’s HSD). The treatments in the study were bahia past ure (B), conventional (C), disk fallow (DF), organic (O), and weed fallow (WF).

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85 P contents that exist in the Header Canal fi eld soil; therefore, the initial cultures were diluted in half with low-P sand. Since re sults from trap culture spore enumerations already have diminished value when compared to counts made directly from the field, the spore numbers from the year four extraction using diluted soil, which further modified the edaphic environment, are not included. Sporulation was approximately three to nine times higher in the dilu ted year four soil than in undiluted year five soil. It is difficult to ascribe the detected difference in sporulation simply to the reduced P leve ls derived through soil dilution, however, because the field conditions in year five we re significantly altered by climatological conditions. Considerable parasi tism existed in spores extr acted both years (Figures 4-18 and 4-19) and the extremely wet field conditions in the final year of tomato production may have provided optimal conditions for the many organisms that prey on AM fungal spores. Sporulation also may have been a ffected by phenological di fferences in tomato plants that occurred at time of sampling between years, as a result of the later planting date in year five due to the severe weat her that was experienced in the end of 2004. Spore Morphotype Identification Identification of AM fungal spore morphot ypes can be a challenging prospect in the best of circumstances, such as when ha rvested in abundance from pure trap cultures grown in sterilized soil. Differentiating among the more than 150 morphotypes described in the INVAM collection is a product of the ability of the investigator to capture examples and visualize incremen tal variability within groups cl assified as genera. Even inter-familial differences can be hard to asce rtain, as crucial diagnostic structures or developmental stages may be absent or otherwise missed from culture.

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86 Figure 4-18. Arbuscular mycorrhizal fungal s pore with substantial parasitism evident through dark internal swirling; no mo rphotypic designation could be assigned.

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87 Figure 4-19. Damaged arbuscular mycorrhizal fungal spore with evidence of a defense response initiated by parasitism.

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88 Cultures generated directly from field so il, as in this study, can be even more problematic. In addition to the issues related to parasitism mentioned above, considerable associated debris can obscur e field soil cultures and competition among root inhabiting fungi can cause alterations in AM fungal community spore production and representation. Because of this, in addition to the a ssociated sampling technique limitations, the field diversity of AM funga l spore morphotypes detected in this study must be considered as incomplete and ma y considerably under-represent the richness present at the Header Canal site during the 2 years that sampling and identification took place. At least 11 individual morphotypes were id entified as having sporulated between the two sampling dates; it is likely that more existed, but were not captured or identified. Joe Morton, the curator of the INVAM collect ion, identified two of the spore types present, Archaeospora trappei and Glomus etunicatum and additionally assisted with multiple confirmations of other types. Morphotypes identified from the various treatments are listed in Table 4-4, but the dive rsity present in the field or even that which was encountered in this study is not definitivel y revealed. For example, the vast majority of spores fell into one of the last four categories on the table, distinguished as a Glomus spp. morphotype. In reality, si nce substantial variation was observed in each group, these categories are constructs for spores that could represent considerable morphotypic diversity. This variation, however, could not be confirmed to represent different individual morphotypes. Additionally, it is important to understand that many of the observed differences might be attributed to individual spore morphot ypes encountered at

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89 Table 4-4. Diversity of arbuscular mycorrhizal fungal morphotypes in year four and five of a 5-year cropping systems study at th e Header Canal experimental farm site in Fort Pierce, FL. Morphotypic diversity was determined by extracting spores from sudangrass trap cu ltures grown in field soil. Morphotypes extracted Treatments where morphotype recovered Acaulospora spp.a Bahia, Conventional, Disk Fallow, Organic Archaeospora trappei Bahia, Conventional, Disk Fallow Gigasporaceaeb Bahia, Disk Fallow, Weed Fallow Glomus ambisporum Bahia Glomus clarum Conventional, Disk Fallow Glomus etunicatum Bahia, Weed Fallow Glomus geosporum all Glomus intraradices Bahia, Conventional, Disk Fallow, Weed Fallow Glomus mosseae all Glomus sinuosum all Glomus spp. (orange)c all Glomus spp. (small hyaline)c all Glomus spp. (small pale yellow)c all Glomus spp. (yellow)c all aHigh quality spores of these morphotypes we re not acquired, and sporiferous saccules were not retained after extraction; th is grouping may therefore include multiple morphotypes. bFiner taxonomic designations were not possible with the spore types extracted. cThese groupings are constructs that could be identified as Glomus spp., but could not be classified on a finer taxonomic level; multiple morphotypes may exist within each category.

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90 different developmental stages or affected by the environment to somehow alter morphological characteristics. The diversity accounted for is not unreas onable for a field with a history of intensive agriculture and constitutes more morphotypes than have been reported as existing in some surveys of natural and disturbed environments (Douds & Millner 1999). Considering this, and magnified by the likely und er-representation of spore types in this study, it is possible to speculate that substa ntial diversity of AM fungal morphotypes has been retained in this field across treatme nts relative to descriptions from other agricultural environments. Based on the methods used in this study, the organic plots had the fewest positively identified spore types, while bahia pasture contained the most. This is not unexpected considering the abundance of available P in th e organic treatment, a condition previously detailed as being known to contribute to reduc tions in both colonizat ion and sporulation. Glomus spp. spore types were the most co mmon and most frequently identified morphotypes by a large margin in all systems. Acaulospora spp. spore types were recognized as small to moderately sized spores without a pore connecting to the subtending hyph ae. Instead, their spore walls are entire, typically have no associated hyphae and have nothing remaining of their sporiferous saccule after the extraction process. The inte rnal germinal walls diagnostic for this group after staining with Melzer’s reagent could only occasionally be visualized, while the ornamentations that are on so me of the spore walls of Acaulospora spp. provided a more frequent means for identificati on (Figure 4-20). Spores of Glomus spp. are similar in size

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91 Figure 4-20. Surface ornamentations can be seen on the spore wall of this Acaulospora sp.

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92 to the Acaulosporaceae, but of ten have subtending hyphae, of which some or all of the layers are continuous with the spore walls, wh ich constitutes the pore diagnostic for this spore type (Figure 4-21). No internal ge rminal walls are formed. Some types of Glomus spp. develop aggregates with numerous associ ated individual spor es, producing masses known as sporocarps (Figures 4-22 and 4-23) Spores of members of the family Gigasporaceae are considerably larger than other groups when mature and have an associated, bulbous sporogenous cell that form s during spore development (Figure 4-24). The two groups within this family can be distinguished between each other in a number of ways, including the formation of germination shields in Scutellospora spp. that are absent in Gigaspora spp and the number and associated characteristics of the spore walls. Although large spores with spor ogenous cells were found, confirming that Gigasporaceae were being obser ved, none of the other spore-derived features of this group were available for discrimination in this study. Instead, the presence of both groups was confirmed by structures attached to external hyphae in th e infection potential study. Both the echinulate auxiliary cells formed by Gigaspora spp. (Figure 4-25) and the smooth but knobby auxiliary cells generated by Scutellospora spp. were discovered (Figure 4-26).

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93 Figure 4-21. Multiple spore walls continuous w ith the layers of the subtending hypha can be seen in this example of the ar buscular mycorrhizal fungal morphotype Glomus intraradices

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94 Figure 4-22. Sporocarp of Glomus sinuosum covered by peridial hyphae.

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95 Figure 4-23. Portion of a Glomus ambisporum sporocarp; this aggregate can be seen associated with a dense mass of myce lium with individual subtending hyphae leading to each spore.

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96 Figure 4-24. Gigaspora sp. with its large spor ogenous cell still attached.

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97 Figure 4-25. Echinulate auxili ary cells diagnostic for a Gigaspora spp. These structures were only found produced on external hyph ae associated with maize roots in the infection potential study.

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98 Figure 4-26. Smooth surfaced auxiliary cell diagnostic for a Scutellospora spp.; these structures were only found produced on external hyphae a ssociated with maize roots in the in fection potential study.

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99 CHAPTER 5 CONCLUSIONS The experimental farm at the Header Ca nal site represented a multiplicity of potential adversities to the existence a nd functioning of AM fungal and other soil microbial communities. The diversity of farming strategies among the treatments occurred across a single so il taxonomic unit, effectively eliminating the substantial influence that soil type and texture would have on microbial communities. Resilient populations of AM and other fungal root endophytes were found to reside within all systems and no permanent impacts related to anthropogenic or environmental alterations could be confirmed. However, field-wide suppression of th e mycorrhizal association and short-term changes in AM fungal activity re lated to management practices and flooding were detected. Of all investigated agri cultural practices, phosphorus fertilization and soil disturbance had the greatest influences on AM fungal infectivity and abundance; however, their impacts were manifested in dist inct ways and persiste d over different time scales. Historical fertilization practices led to an enormous P bank in all treatments, with the most extreme levels developing in organi c plots where P applications were well in excess of annual crop need. The abundant available P supply within the soil in all treatments led to a general repression of AM fungal colonization and probably resulted in a low incidence of sporulation. Tomato ro ots in the bahia pasture treatment had the highest average levels of my corrhizal colonization of any roots observed in this study, reaching a maximum of 32 % of root tissue co lonized, a degree of association that, in

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100 most environments, would be considered to be at the low end of moderate. This relatively larger amount of colonization in bahia pasture treatments was not due to reduced available P, but to the lack of significant soil disturba nce occurring in the plots of that treatment. This P effect represents a long-term obstacle to more extensive AM fungal development in plants and spore production. Mitigation of the soil nutrient content would require long-term crop production and harvest without significant future P additions. Tillage mediated soil disturbance did not present a long-term suppression of AM fungal infectivity, alt hough primary infection was dramati cally decreased in soil actively undergoing intensive tillage. Subsequent to the cessation of disking and following a full season of crop growth, field crop colonization le vels and infection po tential of soil were indistinguishable from other, less distur bed production systems. The ability of populations to quickly recover infectivity af ter years of host root absence to levels identical to those in a minimally disturbed system in the course of a single season represents an intriguing finding of this research. There was no clear evidence that either th e history of fumigati on at the farm site with MeBr + CP or the continued use of a lternative fumigants in the conventional plots had a lasting effect on the ability of AM or other fungal endophyte communities to colonize field roots, or to initiate primary infection on maize roots in a greenhouse study. Analysis of AM fungal signature FAME’s to measure biomass within soil did indicate very low habitation in conventi onal plots in year five; however this method detected that a similar response occurred in disk fallow pl ots, suggesting these differences may have been largely a consequence of saturated field conditions prevalent in late 2004.

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101 Repression of these fungal populations would be expected to have taken place directly after fumigation, while results from th ese studies only included end of season colonization and soil infection potentials, apparently allowing substantial time for recovery. Widespread waterlogging and a shortened growing season in year five, caused by two consecutive hurri canes, may have reduced the amount and infectivity of all fungal root endophytic propagules as measured by the infection potential study, but colonization levels of full season tomato roots were largely unaffected. This seeming dichotomy can be explained. The low incidence of field root colonization by AM fungi in the systems with regularly mixed soil may represent a mi nimum level of occurrence, as determined mainly by the P status of the host plants a nd the periodic disturbance of the soil. The levels of field root coloniza tion were relatively higher in the bahia pasture plots as a result of the spatially dense web of infectiv e hyphae associated with the sod roots. With the exception of AM fungi in the organic tr eatment, all fungal root endophyte infection potentials within soil were reduced in year fi ve compared to year four. A field-wide alteration of the degree of primary infection that these varied popul ations are able to generate is most likely due to reduced root growth as a result of a relatively shorter growing season (105 days in year four and 69 days in year five) in combination with highly stressful field conditions, with a conc urrent reduction of root-associated fungal inoculum. Diversity of AM fungal morphotypes at th e experimental site was typical of agricultural fields, as dete rmined through spore extraction from greenhouse grown host crops grown in undiluted field soil. Fe wer identifiable morphotypes were found in

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102 organic plots than in other treatments. Additional diversity is likely to exist, as a high proportion of extracted spores were parasitized, and pale spores were not readily detected using the methods undertaken in this study. Spore morphotype diversity could also be expected to be enhanced thr ough extractions occurring directly from field soil, especially if assessed at different points within a season and in association with others of the various plants that were sown or that establis hed naturally in plot s during this study. Non-AM fungal root endophytes constitute an extensive group of coexisting organisms that can be difficult and confusi ng to distinguish among using microscopy to visualize within roots. This creates c onditions that discourag e investigators from assessing levels of colonizati on. While some fungal structures stain using non-vital dyes and are therefore mistaken for AM fungal de velopment in some studies, others have no pigmentation and do not stain us ing conventional dyes; this ca n lead to overestimation of AM fungal colonization as we ll as inaccurate evaluations of the extent of other endophytic development. The presence of uns tained, hyaline hyphae within roots in this study makes it likely that non-AM fungal root endophyte colonization levels were underestimated; Microdochium bolleyi a saprophytic endophyte that was predominant only in tomato roots harvested from organic plots, undoubtedly suffered from underestimation for this reason. With the exception of the excl usive occurrence of M. bolleyi in organic plots, there were no dramatic differences in the types or levels of nonAM root endophyte colonization in end-of-season field plants or infection potential host roots that could be confirme d among treatments. In addi tion, there was no antagonism detected between AM and other endophytic root fungi with regards to their ability to colonize plant roots, as root co-occupancy was positively correlated.

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103 While many of the management practices associated with intensive agricultural production have long been considered detrimenta l to the ability of AM fungal populations to form associations with pl ants and even to persist in the soil, little research has examined the degree to and rate at which recovery is likely to occur upon cessation or alteration of farming strategies. In addition, relatively little work has been accomplished regarding the impact farming strategies ha ve on the diverse group of non-AM fungal root endophytes that are known to be widespread in agricultural fiel ds; nor has the extent that these organisms occur together in the cortex with their mycorrhizal counterparts been thoroughly investigated. This study has shown that general repre ssion of field root colonization by AM fungi occurs in this field when abundant ava ilable P and periodic so il disturbance takes place, although persistent infective propagul es remain. Continuous tillage has the clearest negative impact on the ability of AM fungi to initiate prim ary infection, but this effect is lost after a single season in th e presence of host substrate. Field-wide waterlogging in year five seems to have contributed to reduci ng the production of infective propagules of all root endophytic fungi except for the AM fungi in the organic treatment. The mechanism through which fl ooding reduced the ability of endophytes to cause primary infection was likely related to reduced root growth and propagule formation in the soil of treatments with highe r bulk density, as well as slower rates of water drainage. Morphotypes of Glomus spp. were the most common spore type extracted from host crops and no reduction in morphotype diversity c ould be verified in any treatment. Non-AM fungal root endophytes were more abundant and infective than AM fungi in all treatments; Phialophora spp. were among the most commonly identified

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104 fungi within field roots, while Olpidium spp. colonized maize roots heavily in the infection potential study. Coexistence between groups of endophytic fungi was general and positively correlated. The fungal endophyte M. bolleyi was unique to organic tomato roots, probably due to significant organic ma tter inputs and the production of a favorable cover crop directly preceding tomato transplantation.

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105 LIST OF REFERENCES Alkan, N., Gadkar, V., Coburn, J., Yarden, O. & Kapulnik, Y. (2004) Quantification of the arbuscular mycorrhizal fungus Glomus intraradices in host tissu e using realtime polymerase chain reaction. New Phytologist 161 877-885. Barrow, J.R. (2003) Atypical morphology of dark septate fungal root endophytes of Bouteloua in arid southwestern USA rangelands. Mycorrhiza 13 239-247. Bever, J.D., Schultz, P.A., Pringle, A. & Morton, J.B. (2001) Arbuscular mycorrhizal fungi: More diverse than meets the ey e, and the ecological tale of why. BioScience 51 923-930. Borie, F., Rubio, R., Rouanet, J.L., Morales, A., Borie, G. & Rojas, C. (2006) Effects of tillage systems on soil characteristics, gl omalin, and mycorrhizal propagules in a Chilean Ultisol. Soil & Tillage Research 88 253-261. Brundrett, M., Bougher, N., Dell, B., Grove T. & Malajczuk, N. (1996) Separating spores from soil. Working with myco rrhizas in forestry and agriculture (eds M. Brundrett, N. Bougher, B. Dell, T. Grove & N. Malajczuk), pp. 155-162. ACIAR Monograph Series Number 32, Canberra. Brundrett, M., Bougher, N., Dell, B., Grove T. & Malajczuk, N. (1996) Clearing and staining mycorrhizal roots. Working with mycorrhizas in forestry and agriculture (eds M. Brundrett, N. Bougher, B. De ll, T. Grove & N. Malajczuk), pp. 179-182. ACIAR Monograph Series Number 32, Canberra. Brundrett, M., Bougher, N., Dell, B., Grove, T. & Malajczuk, N. (1996) Measuring root colonization by mycorrhizal fungi. Working with mycorrhizas in forestry and agriculture (eds M. Brundrett, N. Bougher, B. Dell, T. Grove & N. Malajczuk), pp. 184-189. ACIAR Monograph Seri es Number 32, Canberra. Brundrett, M. & Kendrick, B. (1990) The r oots and mycorrhizas of herbaceous woodland plants. II. Structural aspects of morphology. New Phytologist 114 469-480. Buyer, J.S. & Drinkwater, L.E. (1997) Compar ison of substrate utiliz ation assay and fatty acid analysis of soil microbial communities. Journal of Microbiological Methods 30 3-11. Cavigelli, M.A., Robertson, R.P. & Klug, M. J. (1995) Fatty acid methyl ester (FAME) profiles as measures of soil microbial community structure. Plant Soil 170 99-113.

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106 Chellemi, D.O. & Porter, I.J. (2001) The ro le of plant pathology in understanding soil health and its applicatio n to production agriculture. Australasian Plant Pathology 30 103-109. Chellemi, D.O., Rhoads, F.M., Olson, S.M., Rich, J.R., Murray, D., Murray, G. & Sylvia, D.M. (1999) An alternative, low-inpu t production system for fresh market tomatoes. American Journal of Alternative Agriculture 14 59-68. Clapp, J.P., Young, J.P.W., Merryweather, J.W. & Fitter, A.H. (1995) Diversity of fungal symbionts in arbuscular mycorrhizas from a natural community. New Phytologist 130 259-265. Daft, M.J. & Nicolson, T.H. (1969) Effect of Endogone mycorrhiza on plant growth. II. Influence of soluble phosphate on endophyte and host in maize. New Phytologist 68 945-952 de la Providencia, I.E., de Souza, F.A., Fe rnandez, F., Delmas, N.S. & Declerk, S. (2004) Arbuscular mycorrhizal fungi reveal distinct patterns of anastomosis formation and hyphal healing mechanisms between different phylogenic groups. New Phytologist 165 261-71. Douds, D.D., Janke, R.R. & Peters, S.E. (1993) VAM fungus spore populations and colonization of roots of maize and soybean under conventional and low-input sustainable agriculture. Agriculture, Ecosystems and Environment 43 325-335. Douds, D.D. & Millner, P.D. (1999) Biodiversity of arbuscular mycorrhizal fungi in agroecosystems. Agriculture, Ecosystems and Environment 74 77-93. Douds, D.D. & Schenck, N.C. (1990) Relatio nship of colonization and sporulation by VA mycorrhizal fungi to plant nut rient and carbohydrate contents. New Phytologist 116 621-627. Drijber, R.A., Doran, J.W., Parkhurst, A.M. & Lyon, D.J. (2000) Changes in soil microbial community structure with tillage under long-term wheat-fallow management. Soil Biology & Biochemistry 32 1419-1430. Ellis, J.R. (1998) Post flood syndrome and vesicular-arbuscular mycorrhizal fungi. Journal of Production Agriculture 11 200-204. Fortin, J.A., Bcard, G., Declerck, S., Dalp, Y., St-Arnaud, M., Coughlan, A.P. & Pich, Y. (2002) Arbuscular mycorrhi za on root-organ cultures. Canadian Journal of Botany 80 1-20. Gallaud, J. (1905) tudes sur le s mycorrhizes endotrophes. Revue General de Botanique 171, 5-500. Gerdemann, J.W. (1955) Relation of a la rge soil-borne spore to phycomycetous mycorrhizal infections. Mycologia 47 619-632.

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107 Giovannetti, M., Fortuna, P., Citernesi, A. S., Morini, S. & Nuti, M.P. (2001) The occurrence of anastomosis formation and nuc lear exchange in intact arbuscular mycorrhizal networks. New Phytologist 151 717-724. Giovannetti, M. & Mosse, B. (1980) An eval uation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytologist 84 489-500. Giovannetti, M., Sbrana, C., Avio, L. & Stra ni, P. (2004) Patterns of below-ground plant interconnections established by means of arbuscular mycorrhizal networks. New Phytologist 164 175-181. Godfrey, M.R. (1957) Studies on British studied of Endogone. III. Germination of spores. Transactions of the British Mycological Society 40 203-210. Gosling, P., Hodge, A., Goodlass, G. & Be nding, G.D. (2006) Arbuscular mycorrhizal fungi and organic farming. Agriculture, Ecosystems and Enivronment 113 17-35. Graham, J.H. (2000) Assessing costs of arbuscular mycorrhizal symbiosis in agroecosystems. Current advances in mycorrhizae research (eds G.K. Podila & D.D. Douds), pp. 127-140. APS Press, St. Paul, MN Graham, J.H. (2001) What do root pathogens see in mycorrhizas? New Phytologist 149 357-359. Graham, J.H., Hodge, N.C. & Morton, J.B. (1995) Fatty acid methyl ester profiles for characterization of glomalean f ungi and their endomycorrhizae. Applied and Environmental Microbiology 61 58-64. Graham, J.H., Leonard, R.T. & Menge, J.A. ( 1981) Membrane-mediated decrease in root exudation responsible for phosphorus inhibi tion of vesicular arbuscular mycorrhiza formation. Plant Physiology 68 548-552. Graham, J.H. & Miller, R.M. (2005) Mycorrhizas: Gene to function. Plant and Soil 274 79-100. Harrier, L.A. & Watson, C.A. (2004) The potenti al role of arbuscular mycorrhizal (AM) fungi in the bioprotect ion of plants agains t soil-borne pathogens in organic and/or other sustainable farming systems. Pest Management Science 60 149-157. Hijri, M. & Sanders, I.R. (2003) The arbuscular mycorrhizal fungus Glomus intraradices is haploid and has a small genome in the lower limit of eukaryotes. Fungal Genetics and Biology 41 253-261. Hill, G.T., Mitkowski, N.A., Aldrich-Wolfe, L., Emele, L.R., Jurkonie, D.D., Ficke, A., Maldonado-Ramirez, S., Lynch, S.T. & Ne lson, E.B. (2000) Methods for assessing the composition and diversity of soil microbial communities. Applied Soil Ecology 15 25-36.

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108 Hosny, M., Gianinazzi-Pearson, V. & Dulieu, H. (1998) Nuclear DNA content of 11 fungal species in Glomales. Genome 41 422-428. INVAM (International Culture Collecti on of VA Mycorrhizal Fungi) (2006a) Classification of Glomeromycota http://invam.caf.wvu.edu/fungi/taxonomy/classification.htm Accessed 8/27/2006. INVAM (International Culture Collecti on of VA Mycorrhizal Fungi ) (2006b) Enumeration of spores http://invam.caf.wvu.edu/methods/spores/enumeration.htm Accessed 8/27/2006. Ishii, S. & Loynachan, T.E. (2004) Rapid and reliable DNA extraction techniques from trypan-blue-stained mycorrhizal r oots: comparison of two methods. Mycorrhiza 14 271-275. Janse, J.M. (1897) Les endophytes radi caux de quelques plantes Javanaises. Annals du Jardin Botanique Buitenzord 14 53-201. Jasper, D.A., Abbott, L.K. & Robson, A.D. (1991) The effect of soil disturbance on vesicular-arbuscular mycorrhizal fungi in soils from different vegetation types. New Phytologist 118 471-476. Jeffries, P., Gianinazzi, S., Perotto, S., Turn au, K. & Barea, J.M. (2003) The contribution of arbuscular mycorrhizal fungi in sustaina ble maintenance of plant health and soil fertility. Biology and Fertility of Soils 37 1-16. Johansson, J.F., Paul, L.R. & Finlay, R.D. (2004) Microbial interactions in the mycorrhizosphere and their significa nce for sustainable agriculture. FEMS Microbiology Ecology 48 1-13. Johnson, N.C., Graham, J.H. & Smith, F. A. (1997) Functioning of mycorrhizal associations along the mutual ism – parasitism continuum. New Phytologist 135 575-585. Joner, E.J., Ravnskov, S. & Jakobsen, I. (2000) Arbuscular mycorrhizal phosphate transport under monoxenic conditions usi ng radiolabeled inorganic and organic phosphate. Biotechnology Letters 22 1705-1708. Jumpponen, A. (2001) Dark septate endophytes – are they mycorrhizal? Mycorrhiza 11 207-211. Jumpponen, A. & Trappe, J.M. (1998) Dark septate endophytes: A re view of facultative biotrophic root-colonizing fungi. New Phytologist 140 295-310. Kabir, Z., O’Halloran, I.P. & Hamel, C. (1999) Combined effects of soil disturbance and fallowing on plant and fungal comp onents of mycorrhizal corn ( Zea mays L.). Soil Biology and Biochemistry 31 307-314.

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109 Kahiluoto, H., Ketoja, E., Vestberg, M. & Saar ela, I. (2001) Promotion of AM utilization through reduced P fertilization. 2. Field studies. Plant Soil 231 65-79. Kirk, J.L., Beaudette, L.A., Hart, M., Moutoglis P., Klironomos, J.N., Lee, H. & Trevors, J.T. (2004) Methods of studyi ng soil microbial diversity. Journal of Microbiological Methods 58 169-188. Kirk, J.J. & Deacon, J.W. (1987) Control of the take-all fungus by Microdochium bolleyi and interactions involving M. bolleyi, Phialophora graminicola and Periconia macrospinosa on cereal roots. Plant and Soil 98 231-237. Kirk, J.J. & Deacon, J.W. (1987) Invasion of na turally senescing root cortices of cereal and grass seedlings by Microdochium bolleyi Plant and Soil 98 239-246. Klironomos, J.N. & Hart, M.M. (2002) Col onization of roots by ar buscular mycorrhizal fungi using different sources of inoculum. Mycorrhiza 12 181-184. Klironomos, J.N., McCune, J., Hart, M. & Nevi lle, J. (2000) The influence of arbuscular mycorrhizae on the relationship between plant diversity and productivity. Ecology Letters, 3 137-141. Klose, S., Acosta-Martnez, V. & Ajwa, H. A. (2006) Microbial community composition and enzyme activities in a sandy loam soil after fumigation with methyl bromide or alternative biocides. Soil Biology & Biochemistry 38 1243-1254. Koch, A.M., Kuhn, G., Fontanillas, P., Fumagalli, L., Goudet, I. & Sanders, I.R. (2004) High genetic variability and low local diversity in a populat ion of arbuscular mycorrhizal fungi. Proceedings of the Nati onal Academy of Science 101 23692374. Koide, R. (1991) Nutrient supply, nutrient demand and plant-response to mycorrhizal infection. New Phytologist 117 365-386. Koide, R.T. & Li, M. (1990) On host regula tion of the vesiculararbuscular mycorrhizal symbiosis. New Phytologist 114 59-74 Koide, R.T. & Mosse, B. (2004) A history of research on arbuscular mycorrhiza. Mycorrhiza 14 145-163. Landis, F.C., Gargas, A. & Givnish, T.J. (2004) Relationships among arbuscular mycorrhizal fungi, vascular plants and environmental c onditions in oak savannas. New Phytologist 164 493-504. Lewis, D., Liudahl, K., Nobl e, C. & Carter, L. (2003) Soil survey of Okeechobee County, Florida USDA, NRCS in cooperation with Univ ersity of Florida, IFAS and AES, Soil and Water Science Department and Fl orida Department of Agriculture and Consumer Services.

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110 Liu, R. & Wang, F. (2003) Selection of appropria te host plants used in trap culture of arbuscular mycorrhizal fungi. Mycorrhiza 13 123-127. Logi, C., Sbrana, C. & Giovannetti, M. (1998) Cellular events involved in survival of individual arbuscular mycorrhizal symbiont s growing in the absence of the host. Applied and Environmental Microbiology 64 3473-3479. Madan, R., Pankhurst, C., Hawke, B. & Smith, S. (2002) Use of fatty acids for identification of AM fungi and estimation of the biomass of AM spores in soil. Soil Biology & Biochemistry 34 125-128. Melin, E. (1922) On the mycorrhizas of Pinus silvestris L. and Picea abies Karst. A preliminary note. The Journal of Ecology 9 254-257. Merryweather, J. & Fitter, A. (1996) Phosphor us nutrition of an obligately mycorrhizal plant treated with the fungicide benomyl in the field. New Phytologist 132 307311. McGonigle, T.P., Miller, M.H., Evans, D.G ., Fairchild, G.L. & Swan, J.A. (1990) A new method which gives an objective measure of colonization of roots by vesiculararbuscular mycorrizal fungi. New Phytologist 115 495-501. Miller, S.P. (2000) Arbuscular mycorrhizal colonization of semi-a quatic grasses along a wide hydrologic gradient. New Phytologist 145 145-155. Millner, P.D. & Wright, S.F. (2002) Tool s for support of ecological research on arbuscular mycorrhizal fungi. Symbiosis 33 101-123. Moorman, T. & Reeves, F.B. (1979) The ro le of endomycorrhizae in revegetation practices in the semi-arid west. II. A bi oassay to determine the effect of land disturbance on endomycorrhizal populations American Journal of Botany 66 1418. Morton, J.B. (1998) Fungi. Principles and applications of soil microbiology (eds D.M. Sylvia, J.J. Fuhrmann, P.G. Hartel & D. A. Zuberer), pp. 72-93. Prentice Hall, Upper Saddle River, NJ. Morton, J.B. & Redecker, D. (2001) Two new families of Glomales, Archaeosporaceae and Paraglomaceae, with two new genera Archaeospora and Paraglomus, based on concordant molecular and morphological characters. Mycologia 93 181-195. Munkvold, L., Rasmus, K., Vestburg, M., Rose ndahl, S. & Jakobsen, I. (2004) High functional diversity within species of arbuscular mycorrhizal fungi. New Phytologist 164 357-364. Murray, D.I.L. & Gadd, G.M. (1981) Preliminary studies on Microdochium bolleyi with special reference to colonization of barley. Transactions of the British Mycological Society 76 397-403.

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112 Sah, S., Reed, S., Jayachandran, K., Dunn, C. & Fisher, J.B. (2006) The effect of repeated short-term flooding on mycorrhiz al survival in snap bean roots. HortScience 41 598-602. Sakamoto, K., Iijima, T. & Higuchi, R. ( 2004) Use of specific phospholipid fatty acids for identifying mycorrhizal fungus Gigaspora rosea. Soil Biology & Biochemistry 36 1827-1834. Sanders, I.R. (2002) Ecology and evolution of multigenomic arbuscular mycorrhizal fungi. The American Naturalist 160 S128-S141. Sanders, I.R. (2004) Plant and arbuscular fungal diversity – are we looking at the relevant levels of diversity and are we using the right techniques? New phytologist 164 415-418. Sanders, F.E. & Tinker, P.B. (1971) Mechanism of absorption of phosphate from soil by Endogone mycorrhizas. Nature 233 278-279. Schler A. (2006) Glomeromycota species list. http://www.tudarmstadt.de/fb/bio/bo t/schuessler/amphylo/amphylo_species.html Accessed 8/28/06. Schler, A., Schwarzott, D. & Walker C. (2001) A new fungal phylum, the Glomeromycota : phylogeny and evolution. Mycological Research 105 1413-1421. Schubert, A. & Hayman, D.S. (1986) Plant gr owth responses to vesicular-arbuscular mycorrhizae. XVI. Effectiveness of different endophytes at different levels of soil phosphate. New Phytologist 103 79-80. Sieber, T.N. (2002). Fungal root endophytes. Plant Roots: The hidden half (eds Y. Waisel, A. Eshel & U. Kafkafi), pp. 887-917. Marcel Dekker, New York, NY. Sieber, T.N., Riesen, T.K., Mller, E. & Fried, P.M. (1988) Endophytic fungi in four winter wheat cultivars ( Triticum aestivum L.) differing in resistance against Stagonospora nodorum (Berk.) Cast. & Germ. = Septoria nodorum (Berk.) Journal of Phytopathology 122 289-306. Skipp, R.A. & Christensen, M.J. (1989) F ungi invading roots of perennial ryegrass ( Lolium perenne L.) in pasture. New Zealand Journal of Agricultural Research 32 423-431. Solaiman, M.Z. & Hirata, H. (1997) Respons es of directly seed ed wetland rice to arbuscular mycorrhizal fungi inoculation. Journal of Plant Nutrition 20 1479-1487. Solaiman, M.Z. & Hirata, H. (1998) Glomus -wetland rice mycorrhizas influenced by nursery inoculation techniques unde r high fertility soil conditions. Biology and Fertility of Soils 27 92-96.

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113 Sylvia, D.M. & Chellemi, D.O. (2001) Interactions among root inhabiting fungi and their implications for biological c ontrol of root pathogens. Advances in Agronomy 73 133. Tawaraya, K., Naito, M. & Wagatsuma, T. ( 2006) Solubilization of insoluble inorganic phosphate by hyphal exudates of arbuscular mycorrhizal fungi. Journal of Plant Nutrition 29 657-665. Thingstrup, I., Rubk, G., Sibbesen, E. & Jakobsen, I. (1998) Flax ( Linum usitatissimum L.) depends on arbuscular mycorrhizal fungi for growth and P uptake at intermediate but not high soil P levels in the field. Plant and Soil 203 37-46. Thygesen, K., Larsen, J. & Bdker, L. ( 2004) Arbuscular mycorrhizal fungi reduce development of pea root-rot caused by Aphanomyces euteiches using oospores as pathogen inoculum. European Journal of Plant Pathology 110 411-419. Tisdale, S.L., Nelson, W.L., Beaton, J.D. & Ha vlin, J.L. (1993a) Micronutrients and other beneficial elements in soils and fertilizers. Soil fertility and fertilizers (ed P.F. Corey), pp. 304-363. Prentice-Hall, In c., Upper Saddle River, NJ. Tisdale, S.L., Nelson, W.L., Beaton, J.D. & Ha vlin, J.L. (1993b) Soil and fertilizer sulfur, calcium and magnesium. Soil fertility and fertilizers (ed P.F. Corey), pp. 266-303. Prentice-Hall, Inc., Upper Saddle River, NJ. Trappe, J.M., Molina, R. & Castellano, M. (1984) Reactions of mycorrhizal fungi and mycorrhiza formation to pesticides. Annual Review of Phytopathology 22 331359. Treseder, K.K. (2004) A meta-analysis of mycorrhizal responses to nitrogen, phosphorus, and atmospheric CO2 in field studies. New Phytologist 164 347-355. Treu, R., Laursen, G.A., Stephenson, S.L ., Landolt, J.C. & Densmore, R. (1996) Mycorrhizae from Denali National Park and Preserve, Alaska. Mycorrhiza 6 2129. van Bruggen, A.H.C. & Semenov, A.M. (2000) In search of biological indicators for soil health and disease suppression. Applied Soil Ecology 15 13-24. van der Heijden, M.G.A., Klironomos, J.N., Ursic, M., Moutoglis, P., Streitwolf-Engel, R., Boller, T., Wiemken, A. & Sanders, I. R. (1998) Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity. Nature 396 69-72. Vierheilig, H., Schweiger, P. & Brundrett, M. (2005) An overview of methods for the detection and observation of arbuscu lar mycorrhizal fungi in roots. Physiologia Plantarum 125 393-404.

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114 Wang, C.J.K. & Wilcox, H.E. (1985) New species of ectendomycorrhizal and pseudomycorrhizal fungi: Philophora finlandia, Chloridium paucisporum and Phialocephala fortinii. Mycologia 77 951-958. Watts, F.C. & Stankey, D.L. (1980) Soil survey of St. Luci e County area, Florida USDA, SCS in cooperation with University of Florida, IFAS and AES, Soil Science Department and Florida Department of Agriculture and Consumer Services. Wennstrm, A. (1994) Endophyte – the misuse of an old term. Oikos 71 535-536. Yu, T., Nassuth, A. & Peterson, R.L. (2001) Ch aracterization of the interaction between the dark septate fungus Phialocephala fortini and Asparagus officinalis roots. Canadian Journal of Microbiology 47 741-753. Zelles, L. (1999) Fatty acid patterns of phospholipids and lipopolysaccharides in the characterization of microbial co mmunities in soil: a review. Biology and Fertility of Soils 29 111-129.

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115 BIOGRAPHICAL SKETCH Chris Rasmann was born in Olympia, Wa shington, but grew up in central and southern Maryland. While attending the Univers ity of Maryland at College Park pursuing an undergraduate degree in agronomy, he worked with Dr. Judith Abbott at the USDA Horticultural Crops Quality Lab at the Beltsv ille Agricultural Res earch Center (BARC), and later assisted with the research of Dr. Les Vough, the University of Maryland’s forage crop specialist. Prior to graduating, Chris took a sabba tical from school to seek a practical application of the theory he ha d been studying. After visiting farms in the eastern provinces of Canada and the northeastern United States as a WWOOFer (Willing Worker On Organic Farms), Chris manage d a small organic farm in Greeneville, Tennessee. Returning to the Univ ersity of Maryland to finish his undergraduate degree in 2001, he began to work with Dr. Michel Ca vigelli on the USDA Farming Systems Project at BARC. Upon graduation in 2003, Chris began pursuing a Master of Science degree in the Plant Pathology Department at the University of Florida.


Permanent Link: http://ufdc.ufl.edu/UFE0017800/00001

Material Information

Title: Alternative tomato farming system practices and their influence on the persistence and infectivity of Arbuscular Mycorrhizal fungi and other fungal root endophytes
Physical Description: Mixed Material
Language: English
Creator: Rasmann, Christopher ( Dissertant )
Graham, James H. ( Thesis advisor )
Chellemi, Daniel O. ( Thesis advisor )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2006
Copyright Date: 2006

Subjects

Subjects / Keywords: Plant Pathology thesis, M.S
Dissertations, Academic -- UF -- Plant Pathology

Notes

Abstract: Farming practices are known to significantly impact arbuscular mycorrhizal (AM) fungi, as well as other soil microbial communities in agroecosystems. The effects of highly divergent land management strategies on the incidence and infectivity of AM fungi and other fungal endophytes were evaluated in a 5-year cropping systems study at the USDA ARS Header Canal farm site in Ft. Pierce, FL. Two treatments, the conventional and disk fallow systems, represented farming practices considered to be detrimental to AM fungal populations. The remaining three treatments, the organic, bahia pasture and weed fallow systems, used practices thought to be more conducive to the presence of AM fungi. In December of year four and five of the study, tomato rhizosphere soil and fibrous roots of tomato were collected. Roots were cleared, stained and examined for colonization by AM fungi and other fungal endophytes. Rhizosphere soil was assayed for infection potential of AM and other root endophytic fungi based on maize seedling colonization at the five-leaf stage. Tomato roots and rhizosphere soil from year five were also analyzed for AM fungal biomass using a fatty acid biomarker 16:1ω5c. Sudangrass trap cultures were set up to determine abundance and diversity of AM fungal spore morphotypes. Of all investigated agricultural practices, phosphorus (P) fertilization and soil disturbance had the greatest influences on AM fungal infectivity and abundance. All plots had very high levels of available soil P, which resulted in low levels of colonization across treatments. The bahia pasture system was the only treatment without regular extensive soil mixing, and these plots had the highest level of field root colonization by AM fungi. Field roots were more heavily colonized by fungal endophytes other than AM fungi in all treatments; tomato plants from organic plots, though, were unique in encouraging colonization by Microdochium bolleyi. Populations of root-associated AM fungi and other fungal root endophytes were positively correlated in all studies. Hurricane-associated flooding and a shortened growing season played a role in reducing the infection potential in all treatments except for the organic plots in year five compared to year four. The levels of primary inoculum were maintained in organic plots, apparently through improved soil quality which resulted in decreased resistance to root development. Areas under active disturbance in disk fallow plots had the lowest levels of primary inoculum, but recovery to levels comparable to less disturbed treatments occurred after a single season of host root growth. Diversity of AM fungal morphotypes was typical of agricultural fields, with at least 11 species present across treatments. Glomus spp. were the dominant spore type recovered in all treatments.
Subject: Arbuscular, bahia, biomarker, colonization, endophyte, fallow, fame, flooding, fumigation, fungal, fungi, glomus, microdochium, morphotype, mycorrhizal, organic, phosphorus, rhizosphere, root, tillage
General Note: Title from title page of source document.
General Note: Document formatted into pages; contains 126 pages.
General Note: Includes vita.
Thesis: Thesis (M.S.)--University of Florida, 2006.
Bibliography: Includes bibliographical references.
General Note: Text (Electronic thesis) in PDF format.

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Holding Location: University of Florida
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Permanent Link: http://ufdc.ufl.edu/UFE0017800/00001

Material Information

Title: Alternative tomato farming system practices and their influence on the persistence and infectivity of Arbuscular Mycorrhizal fungi and other fungal root endophytes
Physical Description: Mixed Material
Language: English
Creator: Rasmann, Christopher ( Dissertant )
Graham, James H. ( Thesis advisor )
Chellemi, Daniel O. ( Thesis advisor )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2006
Copyright Date: 2006

Subjects

Subjects / Keywords: Plant Pathology thesis, M.S
Dissertations, Academic -- UF -- Plant Pathology

Notes

Abstract: Farming practices are known to significantly impact arbuscular mycorrhizal (AM) fungi, as well as other soil microbial communities in agroecosystems. The effects of highly divergent land management strategies on the incidence and infectivity of AM fungi and other fungal endophytes were evaluated in a 5-year cropping systems study at the USDA ARS Header Canal farm site in Ft. Pierce, FL. Two treatments, the conventional and disk fallow systems, represented farming practices considered to be detrimental to AM fungal populations. The remaining three treatments, the organic, bahia pasture and weed fallow systems, used practices thought to be more conducive to the presence of AM fungi. In December of year four and five of the study, tomato rhizosphere soil and fibrous roots of tomato were collected. Roots were cleared, stained and examined for colonization by AM fungi and other fungal endophytes. Rhizosphere soil was assayed for infection potential of AM and other root endophytic fungi based on maize seedling colonization at the five-leaf stage. Tomato roots and rhizosphere soil from year five were also analyzed for AM fungal biomass using a fatty acid biomarker 16:1ω5c. Sudangrass trap cultures were set up to determine abundance and diversity of AM fungal spore morphotypes. Of all investigated agricultural practices, phosphorus (P) fertilization and soil disturbance had the greatest influences on AM fungal infectivity and abundance. All plots had very high levels of available soil P, which resulted in low levels of colonization across treatments. The bahia pasture system was the only treatment without regular extensive soil mixing, and these plots had the highest level of field root colonization by AM fungi. Field roots were more heavily colonized by fungal endophytes other than AM fungi in all treatments; tomato plants from organic plots, though, were unique in encouraging colonization by Microdochium bolleyi. Populations of root-associated AM fungi and other fungal root endophytes were positively correlated in all studies. Hurricane-associated flooding and a shortened growing season played a role in reducing the infection potential in all treatments except for the organic plots in year five compared to year four. The levels of primary inoculum were maintained in organic plots, apparently through improved soil quality which resulted in decreased resistance to root development. Areas under active disturbance in disk fallow plots had the lowest levels of primary inoculum, but recovery to levels comparable to less disturbed treatments occurred after a single season of host root growth. Diversity of AM fungal morphotypes was typical of agricultural fields, with at least 11 species present across treatments. Glomus spp. were the dominant spore type recovered in all treatments.
Subject: Arbuscular, bahia, biomarker, colonization, endophyte, fallow, fame, flooding, fumigation, fungal, fungi, glomus, microdochium, morphotype, mycorrhizal, organic, phosphorus, rhizosphere, root, tillage
General Note: Title from title page of source document.
General Note: Document formatted into pages; contains 126 pages.
General Note: Includes vita.
Thesis: Thesis (M.S.)--University of Florida, 2006.
Bibliography: Includes bibliographical references.
General Note: Text (Electronic thesis) in PDF format.

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0017800:00001


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ALTERNATIVE TOMATO FARMING SYSTEM PRACTICES AND THEIR
INFLUENCE ON THE PERSISTENCE AND INFECTIVITY OF ARBUSCULAR
MYCORRHIZAL FUNGI AND OTHER FUNGAL ROOT ENDOPHYTES














By

CHRISTOPHER RASMANN


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2006
































Copyright 2006

by

Christopher Rasmann







































To Nita.
















ACKNOWLEDGMENTS

I appreciate the opportunity my committee co-chairs, Drs. Jim Graham and Dan

Chellemi, have provided in allowing me to pursue this research proj ect. Their support

and patience were important to me and instrumental in my ability to complete this work.

Additionally, I am grateful to have been the recipient of a Hunt Brothers endowment in

support of my research. I want to thank Dr. Lawrence Datnoff for opening his lab for me

to use as a home base and providing me with guidance and with many of the tools needed

to accomplish my research. I would also like to express my gratitude to Dr. David

Mitchell for his guidance and insight throughout my time at the University of Florida and

for the editorial input he provided with my research proposal and thesis.

I am grateful for the support and assistance I received from many individuals, all of

whom contributed to this proj ect. I would like to thank Diane Bright, Dr. Jeff Buyer, Dr.

Michel Cavigelli, Gloria Darlington, Dr. James Kimbrough, Dr. John Larsen, Dr. Ramon

Littell, Gary Marlow, Dr. Joe Morton, Patty Rayside, Dr. Fabricio Rodrigues, Dr. Jeff

Rollins, Mauricio Rubio, Brenda Rutherford and Dr. Carol Stiles.




















TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. .................... iv


LI ST OF T ABLE S ................. ................. vii...._._....


LIST OF FIGURES ...._.._ ................ .......__. .........vi


AB S TRAC T ......_ ................. ............_........x


CHAPTER


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


Arbuscular Mycorrhizal and Other Fungal Root Endophytes in Agroecosystems:
Context and Importance ................ ...............1............ ....
Study Site ........._._..... ........._ _. ...............3.......
Conventional Production ......__................. .........__ .......... 5

Organic Production............... ...............
Disk Fallow .............. ...............6.....
Bahia Pasture ................. ...............6........ ......
Weed Fallow............... ...............6.
Reasoning and Obj ectives. ........._.___..... .__. ...............7....

2 LITERATURE REVIEW ........._.___..... .__. ...............10....


Research on Arbuscular Mycorrhizal Fungi ................. ...............................10
Studying Arbuscular Mycorrhizae ................. ...............11........... ....
Non-M olecular Tools .............. ......... .. .............1

Quantification of fungal colonization in roots using microscopy ................13
Spore extraction for identification and enumeration ................. .. ...............18
Molecular Tools and Studying the Species Concept and Genetic Condition of
Arbuscular Mycorrhizal Fungi ................. ...............22........... ....
Fatty Acid Methyl Ester Analysis .............. ...............29....
Other Tool s ............... .......... ...............3 1.....
Arbuscular Mycorrhizal Fungi in Agroecosystems ......____ ..... ...__ ...............32
Phosphorus Fertilization. .............. ...............32....
Soil Disturbance .............. ...............34....
Pesticides ................. ...............35.......... .....
Flooding............... ...............36











Organic, Conventional, and Grassland Farming Management Strategies...........37
Other Fungal Root Endophytes Known to Occur in Herbaceous Plant Roots ...........40

3 MATERIALS AND METHODS .............. ...............43....

4 RESULTS AND DISCUSSION............... ...............5


Agricultural and Environmental Influences Impacting Edaphic Conditions at the
Header Canal Study Site ................ ... .... .... ... ... ...........5
Colonization of Roots of Tomato and Cover Crops by Arbuscular Mycorrhizal
Fungi and Other Fungal Endophytes............... .....................5
Arbuscular Mycorrhizal Fungi in Tomato and Cover Crop Roots ................... ...56
Tomato Root Colonization by Non-Arbuscular Mycorrhizal Fungal
Endophyte s........._..._... .. .... ..._. ... .. .. ..._.._ ..... ... .. .......6
M~icrodochium bolleyi in Tomato Roots from the Organic Treatment ................67
Infection Potential of Field Soil on Greenhouse-Grown Maize ................ ...............73
Arbuscular Mycorrhizal Fungal Infection Potential ................. .................. ......73
Non-Arbuscular Mycorrhizal Fungal Root Endophyte Infection Potential ........80
Spores: Enumeration and Identification of Arbuscular Mycorrhizal Fungal
M orphotypes ............. ... .. ........ ....... ...............8
Spore Enumeration of Year Five Trap Culture Soil ................ .....................83
Spore Morphotype Identification .............. ...............85....

5 CONCLUSIONS .............. ...............99....

LI ST OF REFERENCE S ................. ...............105................

BIOGRAPHICAL SKETCH ................. ...............115......... ......
















LIST OF TABLES


Table pg

4-1 Results of soil analyses from year four and five of a 5-year cropping systems
study at the Header Canal experimental farm site in Fort Pierce, FL. .....................53

4-2 Field tomato root colonization by arbuscular mycorrhizal fungi and other fungal
root endophytes from year four and five of a 5-year cropping systems study at
the Header Canal experimental farm site in Fort Pierce, FL. ................. ..............57

4-3 Field soil infection potential of arbuscular mycorrhizal fungi and other fungal
root endophytes on maize from year four and five of a 5-year cropping systems
study at the Header Canal experimental farm site in Fort Pierce, FL. .....................62

4-4 Diversity of arbuscular mycorrhizal fungal morphotypes in year four and five of
a 5-year cropping systems study at the Header Canal experimental farm site in
Fort Pierce, FL................ ...............89..

















LIST OF FIGURES


Figure pg

4-1 Header Canal experimental farm site plots after the hurricanes of 2004. Photo
taken prior to Einal tomato transplantation in year Hyve of a 5-year cropping
systems study in Fort Pierce, FL. ............ ...............55.....

4-2 Field tomato root colonization by arbuscular mycorrhizal fungi in years four and
fiye of a 5-year cropping systems study at the Header Canal experimental farm
site in Fort Pierce, FL. .............. ...............58....

4-3 Correlation with least squares regression of the fatty acid 16: 1co5 content in
tomato roots to the observed proportion of colonization of tomato roots in Hield
plots by arbuscular mycorrhizal (AM) fungi ................. ............... ......... ...59

4-4 Bahia pasture plots with rows of tomato plants. ................ .......... ...............61

4-5 Field tomato root colonization by all non-arbuscular mycorrhizal fungal root
endophytes in year Hyve of a 5-year cropping systems study at the Header Canal
experimental farm site in Fort Pierce, FL............... ...............64...

4-6 Mass of enlarged fungal cells produced by Phialophora spp. within the cortex of
tomato roots; blue color is a result of staining with trypan blue .............. ...............65

4-7 Mass of enlarged fungal cells and hyphae produced by Phialophora spp. within
the cortex of maize roots; blue color is a result of staining with trypan blue. .........66

4-8 Clusters of resting spores (cystosori) formed by Polynzyxa spp. within the cortex
of a tomato root; blue color is a result of staining with trypan blue.........................68

4-9 Pigmented cells formed by M~icrodochium bolleyi within tomato roots. .................70

4-10 Close-up of pigmented cells formed by M~icrodochium bolleyi within tomato
roots. ............. ...............71.....

4-11 Close-up of pigmented and trypan blue-stained cells formed by M~icrodochium
bolleyi within tomato roots ................. ...............72................

4-12 Infection potential of treatment soil sampled in year four of a 5-year cropping
systems study at the Header Canal experimental farm site in Fort Pierce, FL. .......74










4-13 Infection potential of treatment soil sampled in year five of a 5-year cropping
systems study at the Header Canal experimental farm site in Fort Pierce, FL ........75

4-14 Infection potential of disk fallow treatment soil sampled in year four and five of
a 5-year cropping systems study at the Header Canal experimental farm site in
Fort Pierce, FL............... ...............76...

4-15 Results from Fatty Acid Methyl Ester (FAME) analysis of soil from year five of
a 5-year cropping systems study at the Header Canal experimental farm site in
Fort Pierce, FL............... ...............79...

4-16 Maize roots with colonization of a chytrid Olpidium spp. stained with trypan
blue. Fungal structures are resting spores and zoospore cysts. ............... ...............82

4-17 Mean arbuscular mycorrhizal spore numbers extracted / cm3 Of undiluted
treatment soil from year five of a 5-year cropping systems study at the Header
Canal experimental farm site in Fort Pierce, FL. ............. ........ .............8

4-18 Arbuscular mycorrhizal fungal spore with substantial parasitism evident through
dark internal swirling; no morphotypic designation could be assigned. ..................86

4-19 Damaged arbuscular mycorrhizal fungal spore with evidence of a defense
response initiated by parasitism. ............. ...............87.....

4-20 Surface ornamentations on the spore wall of an Acaulospora sp. ...........................91

4-21 Multiple spore walls continuous with the layers of the subtending hypha can be
seen in this example of the arbuscular mycorrhizal fungal morphotype Glonaus
intraradices. ............. ...............93.....

4-22 Sporocarp of Glonaus sinuosunt covered by peridial hyphae. ................ ...............94

4-23 Portion of a Glonaus a~nbisporunt sporocarp with an associated dense mass of
mycelium; individual subtending hyphae lead to each spore ................. ...............95

4-24 Gigaspora sp. with its large sporogenous cell still attached ................. ...............96

4-25 Echinulate auxiliary cells diagnostic for a Gigaspora spp. These structures were
only found produced on external hyphae associated with maize roots in the
infection potential study. ............. ...............97.....

4-26 Smooth surfaced auxiliary cell diagnostic for a Scutellospora spp.; these
structures were only found produced on external hyphae associated with maize
roots in the infection potential study. ............. ...............98.....
















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

ALTERNATIVE TOMATO FARMING SYSTEM PRACTICES AND THEIR
INFLUENCE ON THE PERSISTENCE AND INFECTIVITY OF ARBUSCULAR
MYCORRHIZAL FUNGI AND OTHER FUNGAL ROOT ENDOPHYTES

By

Christopher Rasmann

December, 2006

Chair: James H. Graham
Cochair: Daniel O. Chellemi
Major Department: Plant Pathology

Farming practices are known to significantly impact arbuscular mycorrhizal (AM)

fungi, as well as other soil microbial communities in agroecosystems. The effects of

highly divergent land management strategies on the incidence and infectivity of AM

fungi and other fungal endophytes were evaluated in a 5-year cropping systems study at

the USDA ARS Header Canal farm site in Ft. Pierce, FL. Two treatments, the

conventional and disk fallow systems, represented farming practices considered to be

detrimental to AM fungal populations. The remaining three treatments, the organic,

bahia pasture and weed fallow systems, used practices thought to be more conducive to

the presence of AM fungi.

In December of year four and Hyve of the study, tomato rhizosphere soil and fibrous

roots of tomato were collected. Roots were cleared, stained and examined for

colonization by AM fungi and other fungal endophytes. Rhizosphere soil was assayed









for infection potential of AM and other root endophytic fungi based on maize seedling

colonization at the five-leaf stage. Tomato roots and rhizosphere soil from year five were

also analyzed for the AM fungal fatty acid biomarker 16:1co5c. Sudangrass trap cultures

were set up to determine abundance and diversity of AM fungal spore morphotypes.

Of all investigated agricultural practices, phosphorus (P) fertilization and soil

disturbance had the greatest influences on AM fungal infectivity and abundance. All

plots had very high levels of available soil P, which resulted in low levels of colonization

across treatments. The bahia pasture system was the only treatment without regular

extensive soil mixing, and these plots had the highest level of field root colonization by

AM fungi. Field roots were more heavily colonized by fungal endophytes other than AM

fungi in all treatments; tomato plants from organic plots, though, were unique in

encouraging colonization by M~icrodochium bolleyi. Populations of root-associated AM

fungi and other fungal root endophytes were positively correlated in all studies.

Hurricane-associated flooding and a shortened growing season played a role in

reducing the infection potential in all treatments except for the organic plots in year five

compared to year four. The levels of primary inoculum were maintained in organic plots,

apparently through improved soil quality which resulted in decreased resistance to root

development. Areas under active disturbance in disk fallow plots had the lowest levels of

primary inoculum, but recovery to levels comparable to less disturbed treatments

occurred after a single season of host root growth.

Diversity of AM fungal morphotypes was typical of agricultural fields, with at least

11 species present across treatments. Glomus spp. were the dominant spore type

recovered in all treatments.















CHAPTER 1
INTTRODUCTION

Arbuscular Mycorrhizal and Other Fungal Root Endophytes in Agroecosystems:
Context and Importance

Arbuscular mycorrhizal (AM) fungi form symbioses with most plants, are present

widely in terrestrial environments, and have been identified as performing a variety of

functions in their associations within natural ecosystems and agroecosystems (Douds &

Millner 1999; Jeffries et al. 2003). These mycorrhizal associations are believed to be

capable of making a significant positive contribution to plant health as well as positively

affecting soil quality. Among the benefits arbuscular mycorrhizae may provide to plants

include increasing nutrient uptake, particularly of P and Zn; improving plant access to

water; positively modifying rhizosphere populations; and increasing both pest and soil

borne disease resistance in plants (Jeffries et al. 2003; Johansson et al. 2004). Additional

potential positive effects that have been described as a result of the symbiosis between

mycorrhizal fungi and plants are the improvement and maintenance of soil structure and

alleviation of plant stress in otherwise inhospitable environments, such as recently

disturbed or polluted soils (Douds & Millner 1999; Jeffries et al. 2003; Johansson et al.

2004).

The AM fungi have a central role in ecosystems and experimental evidence shows

that considerable functional diversity occurs among taxa within the phylum

Glomeromycota (Munkvold et al. 2004; Gosling et al. 2006). This diversity, however,

does not necessarily constitute benefits provided to all plants in all environments (Bever









et al. 2001; Jeffries et al. 2003; Munkvold et al. 2004). Based on experimental evidence,

the ability of AM fungi to function and maintain the mycorrhizal condition under some

field conditions common in industrialized agriculture appears limited (Kabir et al. 1999;

Oehl et al. 2004; Gosling et al. 2006), and while the symbiosis is generally perceived as

mutualistic, there are "occasional commensal and parasitic excursions from this norm"

(Johnson et al. 1997, p. 583). Management practices such as high P fertilizer

applications; tillage-mediated soil disturbance; and the application of pesticides,

including the use of soil fumigants are considered potentially detrimental to populations

of AM fungi. In fact, when considering the widespread use of and production benefits

gained from practices that may be deleterious to AM fungal populations, as well as the

carbon cost inherent to plants in interactions with these obligately biotrophic fungi, the

question can be raised as to whether or not AM fungi should be considered when making

management decisions in many agricultural systems (Ryan & Graham 2002).

The AM fungi are not the only organisms widely capable of occupying the root

cortex; there are many other fungal endophytes, some of which may also exist within the

stele (Yu et al. 2001; Barrow 2003). Several definitions for the term endophyte exist and

different uses of the word can sometimes be points of controversy (Wennstroim 1994;

Sieber 2002). When used in this work, a relatively narrow definition will be adopted.

The endophytes referred to herein are those fungi observed existing within the roots of

plants without causing apparent symptoms of disease. Recognizing that AM fungi fit this

definition of an endophytic organism, when the term endophyte is used in this work it

will be qualified to distinguish fungi not recognized as residing within the phylum

Glomeromycota. The individuals that make up this group of root-occupying fungi may









function as obligate or facultative biotrophs and exist within a range of plant-microbe

interactions from mutualism to, in some instances, parasitism or pathogenicity, as in the

case of some formae specials of Fusarium oxysporum (Morton 1998; Sylvia & Chellemi

2001; Sieber 2002). These forms are pathogenic on select taxa of plants, and are

seemingly innocuous or beneficial when associated with most other plants.

The data for this study were collected from the USDA, ARS cropping systems

proj ect at Header Canal in Fort Pierce, Florida. The proj ect provides, among other

obj ectives, an opportunity to examine the effects varied farming management practices

have upon the components that define soil quality. Part of determining the status of soil

quality and health involves monitoring biological indicators; this process begins by

determining the condition of resident plant health, and evaluating components such as

soil microbial biomass, activity, and diversity (van Bruggen & Semenov 2000; Hill et al.

2000; Chellemi & Porter 2001). As discussed above, both the AM fungi and other root

endophytes occur globally, and it is probable that all plants form intimate associations

with at least one taxa of fungal endophyte, while in many cases plants may be more or

less indiscriminate in their associations (Sieber 2002). The value of these organisms as

biological indicators of soil quality and health may be highly significant, and their impact

on ecosystem functioning needs to be better understood. An integral aspect of that

understanding in the context of agroecosystems involves the elucidation of the capability

of fungal root endophytes to persist and remain infective under intensive management

practices.

Study Site

The 5-year cropping systems study at the Header Canal Farm in Fort Pierce, FL,

has five land-management systems that are highly divergent, yet all use management









methods not uncommon in the region. The land which is being farmed in this proj ect had

been in conventional tomato production using raised plastic-mulched beds fumigated

with methyl bromide plus chloropicrin (MeBr + CP) annually for 10 years prior to being

leased for research. The entire research site has soil that has been classified as a Pineda

fine sand (loamy, siliceous, hyperthermic, Arenic Glossaqualfs), with a sandy texture

from the surface to a depth of 96 cm (Lewis et al. 2003; Watts & Stankey 1980). The

average pH among system plots over years four and five ranged from 6.8 to 7.7. Soil

phosphorus levels were extremely high due to the previous fertilization history and

nutrient applications made subsequent to the initiation of the land management

treatments. Mean Mehlich-1 extracted phosphorus ranged from 794 to 1831 kg hal

among systems. During the initiation of tomato crop production in year five of the study,

two hurricanes (Frances and Jeanne) struck the area of Florida where the experimental

farm site was located, causing significant damage to the farm and surrounding

communities. Rainfall totals exceeded 50 cm during the month of September, resulting

in widespread flooding. Tomato transplantation was delayed approximately a month due

to the hurricanes and the resultant field conditions.

Systems were initiated in the summer of 2000 and, with the exception of one

treatment, as will be explained below, were allowed 3 years to establish prior to

resumption of tomato production. The five treatments were established in a randomized

complete block design, with each treatment replicated six times for a total of 30 plots,

occupying 0.16 ha individually (plots are 68.5 x 23 m). Tomatoes in all treatments,

except the bahia pasture system, were grown in raised beds with plastic mulch. The five

systems are described below.









Conventional Production

This system continued the management practices of the preceding farmer, with the

exception being replacement of the original soil fumigant with Telone C-3 5, a 62:3 5

formulation of 1, 3-dichloropropene (1, 3-D) and chloropicrin (CP). Trifluralin (Treflan)

and napropamide (Devrinol) were spread and incorporated for weed control. Tomatoes in

this treatment received an initial fertilizer application during bed formation (20% of total

N and K), with subsequent fertilization provided during the growing season via inj section

through drip irrigation. In this system the 3-year transition was not necessary, so fall

tomato production continued throughout system plots. During the spring and summer of

2001 this treatment had a cover crop of Japanese Millet (Echinochloa crusgalli var

fr~umentacea); it was weed fallowed in 2002, and disk fallowed in 2003.

Organic Production

During the first 3 years no tomato crop was raised on the plots in this treatment.

Broiler litter and urban plant debris were broadcast and incorporated into the soil in July.

In August, a cover crop of sunn hemp (Crotalariajuncea) was planted, while in the

following spring, Japanese millet was grown. After the transition period, starting in the

fourth year, the south half of each plot was used to produce a tomato crop following

approximately 6 weeks of soil solarization of the beds, while the other half remained

under a sunn hemp cover crop. During the fifth year, the entire plot was put into tomato

production using the techniques just described.

The production plan for these plots was not certified organic due to their close

proximity to treatments that use technology prohibited by certifying bodies. However,

the practices used in this treatment are aligned with methods that are in favor with

advocates for organic agriculture.









Disk Fallow

The entirety of the plots in this treatment were disked regularly year-round to keep

them free of vegetation. Disking depth was about 25 cm. Tomatoes were not grown in

years one through three. In August of year four, the south half of these plots received

fertilizer and herbicides identical to those applied to the conventional system. After the

final harvest, both halves were again disked regularly. In year five, the entire plot was

put into production using the same methods to integrate the northern half as were used in

the southern half the year before.

Bahia Pasture

A bahia grass (Paspalum notatum var. Argentine bahiagrass) pasture was

established in these plots and managed through mowing and spring fertilization for the

first 3 years. In year four, the southern halves of the plots were put into tomato

production using a strip tillage production system (Chellemi et al. 1999). A 46-cm strip

was tilled into the bahia grass for planting, while the grass on either side of the row was

managed through mowing and use of the herbicide sethoxydim (Poast). The fifth year

involved cropping both halves of these plots using the strip-till technique.

Weed Fallow

These plots were left undisturbed for the first 3 years of transition, and weed

communities were allowed to establish. The southern halves of the treatment plots were

put into tomato production beginning in year four after disking and using the same

practices used in the conventional system. The entire plots were cultivated to tomato

during the fifth year.









Reasoning and Objectives

The main priority of this study is to characterize the impacts that varied

management practices have upon an important and highly influential group in the soil

community, the AM fungi. Across one soil type, a Hield with a uniform history of

intensive agricultural production using MeBr + CP was transitioned into five divergent

management strategies. Two treatments represented farming practices considered to be

highly detrimental to AM fungal populations, the conventional plots and the disk fallow

plots. The remaining three treatments used practices that are thought to be more

conducive to encouraging the presence of AM fungi. Organic plot management included

an intensive cover-crop regimen and nutrient sources that are more slowly available than

in the other systems, while weed fallow plots were left undisturbed during the transitional

period, allowing for the unrestricted development of an endemic plant population, and

were not exposed to the use of agrichemicals until the re-initiation of tomato production.

In the bahia pasture treatment, a sod known to be highly mycotrophic was established

during the transitional period, and was maintained after strip-tilled rows were generated

for tomato transplants.

Specifically of interest in this proj ect was whether systems encouraged or

discouraged the relative abundance and infectivity of fungal root endophytes. In

addition, while considerable literature exists detailing and speculating on the impacts of

certain agricultural practices on AM fungi, there is little information regarding the ability

and extent to which populations recover from adverse conditions once land management

practices are changed. Little is known about the long-term repercussions, if any, that

farming strategies such as repeated soil fumigation have on AM fungal presence and

infectivity. The quantifieation of the AM fungal presence among the systems during year









four and five is important in that it can provide information about the effects management

has upon these organisms with transition to production and on the differences in

populations occurring among the systems during production.

While considerable interest is developing in enhancing understanding of the

possible functional role of non-AM fungal root endophytes in agroecosystems,

particularly those fungi that have been designated as dark septate endophytes (DSE),

there is little information regarding their ability to persist under intensive agricultural

management. Gathering information regarding structures associated with the occurrence

of non-AM fungal root endophytes as they exist in roots can provide a provisional

identification or classification. Investigating the degree to which field tomatoes and other

host crops are colonized may help us determine the AM fungal and other root endophyte

abundance and aggressiveness within the various treatments and help us understand how

these have been affected by management strategies.

The infectivity of AM and other fungal root endophytes was quantified in end-of-

season field tomato roots, and their infection potential in field soil was assayed in a

greenhouse study. The non-AM fungal endophytes found existing in field roots were

identified when possible based on their morphological features within the root

association. Fatty acid methyl ester (FAME) analysis was performed on year five tomato

roots and field soil, providing an independent measure of biomass by quantification of the

AM fungal fatty acid biomarker, 16:1co5c. Spore extractions from a greenhouse study in

which trap crops were grown in field soil were completed, achieving a provisional

accounting of AM fungal morphotypes occurring in the field. In year five, spore numbers

from the harvested trap cultures were enumerated.









The above work was accomplished to elucidate the impact of industrialized

agricultural practices, as represented by the history of the farm site at Header Canal and

perpetuated in two of the five treatments, on the survival, infectivity and occurrence of

AM and other fungal root endophytes. In addition, it was important to determine the

degree to which farming strategies that have been considered more amenable to

encouraging the existence and functioning of AM fungi were able to affect these

populations after long-term exposure to intensive tomato production.














CHAPTER 2
LITERATURE REVIEW

Research on Arbuscular Mycorrhizal Fungi

Our knowledge of the existence of AM fungi is generally accepted to have begun

with descriptions Nageli made in 1842 (Koide & Mosse 2004). In the late 19th and early

20th century, detailed descriptions of the morphological results of the symbiosis were in

place, including both of the most important diagnostic structures: vesicles (Janse 1897)

and arbuscules (Gallaud 1905). While the relationship between plants and AM fungi has

been known to exist for well over 100 years, its widespread occurance in ecosystems and

intensity therein, although speculated upon in some earlier literature, has been established

much more recently (Read et al. 1976). A partial list of ensuing work, occurring on a

broad range of scales while spanning multiple disciplines, includes: attempting to develop

a taxonomic system and understand the phylogeny of the AM fungi; ascertaining and

characterizing the effects of the mycorrhizal relationship on the symbionts and on the

broader environment, as well as investigating the natural and anthropogenically derived

edaphic and environmental conditions that impact the partnership; understanding the

regulating factors in and the physiology of mycorrhizae; and the attempt at development

of AM fungi as an agricultural technology (Koide & Mosse 2004). Research that is of

particular interest in the context of this proj ect includes studies that relate to the ecology

of mycorrhizae, especially in managed systems.









Studying Arbuscular Mycorrhizae

Accurate methods to detect, quantify and identify AM fungi are paramount to

developing an understanding of mycorrhizae in any environment and context. A variety

of obstacles exist in studying arbuscular mycorrhizae, however, stemming mostly from

their obligately biotrophic nature and complex, not yet fully understood, genetic

condition. Classical, biochemical and molecular tools have been developed and used

with varying degrees of success by researchers.

While tools and techniques exist both for quantifieation and identification of AM

fungi as well as for measuring their metabolic activity, linking identified active taxa to

possible ecological roles is more complicated (Millner & Wright 2002). There appears,

based on experimental evidence, to be considerable inter- and even intra-phylo and

morphotypic functional diversity in the AM fungi. Munkvold et al. (2004) compared the

hyphal growth of 24 isolates from four described morphotypes and their ability to

influence the growth and P-nutrition of Cucumis sativus, and found considerable

variability existing within morphotypes in their mycelial extention and in the ability to

promote growth. Another study comparing the capabilities of 16 isolates of Glomus

intraradices from a single Hield produced similar results, with five-fold differences in

final hyphal lengths (Koch et al. 2004).

Further confounding understanding of the functional diversity of AM fungi is that

when taken out of the microcosm of a controlled experiment, the fungal role in

mycorrhizal functioning seems plastic, depending largely on its environment (Johnson et

al. 1997). If functional richness abounds in the AM fungal genotype, and is shown to not

be dependent upon species diversity, but rather on isolates as influenced by their

environment, it would represent a shift in a standard view of microbial ecology as it









relates to this type of mycorrhiza. The same uniquely diverse genetic makeup of AM

fungi that makes them difficult to characterize, and in some cases investigate, may serve

to place the organism in position to ensure the perpetuity of the relationship between

symbionts in the presence of adversity through functional redundancy in the larger

population. This would be especially important to understand in the context of

agricultural or other disturbed environments where alteration of habitat occurs.

There remains substantial debate as to whether maintaining taxonomic diversity is

important if functional diversity is preserved and is active. Our understanding of these

concepts with regards to AM fungi and the scale on which we judge them has changed as

our tools for research have developed and improved. In the early stages of mycorrhizal

research the AM fungi were considered functionally redundant; we now are beginning to

better understand the influence that the edaphic and climatic environment as well as the

host plant imparts on the functional phenotype of the fungi. Likewise, in natural systems,

the phenotype of the fungal component in mycorrhizal associations can directly affect the

structure of plant communities (van der Heij den et al. 1998; Klironomos et al. 2000;

Graham & Miller 2005). Future research determining the extent to which functional

diversity and resiliency exists among AM fungal species and isolates as well as the

degree to which it is present in given populations will help clarify the association

between these factors (Kirk et al. 2004). Furthermore, when considering ecological

studies of the mycorrhizal relationship it is vital that, where possible, experimental results

from controlled environments be investigated on the broadest scale possible to verify

their real world significance (Graham & Miller 2005).










Prior to discussing research on AM fungi in ecosystems in greater depth, it is

important to describe some of the more important and commonly used tools and methods

available for the research of these organisms, including problems associated with their

use.

Non-Molecular Tools

Quantification of fungal colonization in roots using microscopy

Assessing the levels of fungal infection in host plants is performed in most studies

involving AM fungi. Barring the use of biochemical or molecular techniques, both of

which are still often correlated with classical methods, examination of roots takes place

under a microscope. In order to best prepare samples for observation, harvested roots

should be processed rapidly after taking any necessary measurements from the fresh root

system followed by drying the roots to be examined as soon as possible to stop metabolic

activity. To visualize intra-cortical fungi when using non-vital stains, the plant' s

pigmentation and cellular constituents need to be clarified prior to staining.

Phillips and Hayman (1970) were among the first to use a procedure that is still

commonly used today, due to its low cost, low technical requirements and consistent

results; host cytoplasm is cleared through heating roots in 10% (w/v) potassium

hydroxide (KOH) followed by rinsing, with roots undergoing subsequent staining using

trypan blue. An exceptional alternative clearing agent is concentrated chloral hydrate,

which renders the roots transparent, although its legal status makes it difficult to acquire.

Several variations on the basic procedure have been developed. The length of time

required to clear roots varies with the type and age of the root, the size of the root sample

and the temperature at which clearing takes place. Calibration is essential when

establishing clearing procedures, as too little clearing will reveal little in terms of fungal









structures, while too much will destroy roots, stripping the cortex from the vascular

tissue. The root samples to be processed are typically best kept to < 2 g, with segment

lengths cut to 1- 3 cm. Heating of samples in KOH generally is accomplished in an oven

or hot water bath (typically 60-950C), but is fastest when autoclaved. It can also be done

at room temperature, if extending the process to hours or days is not problematic. Further

clearing with acidified bleach (1.7% NaOCl with 1% diluted HCL v/v in water) or

alkaline hydrogen peroxide (0.5% NH40H with 0.5% H202 V/V in water) at room

temperature provides additional removal of pigments in root tissue that contain

particularly recalcitrant obscuring compounds. This step is especially valuable when

further clearing in KOH would lead to destruction of the sample, but, once again, in this

procedure the sample should be observed frequently to achieve the best results for a given

type of root. If bleaching is performed using alkaline H202, the sample should

subsequently be acidified with diluted HCL to enhance the staining that follows. Roots

should be thoroughly rinsed with water after each clearing and bleaching step.

Non-vital stains are still the most commonly used technology for enhancing

visualization of AM fungi in roots. These stains are valuable in that they provide a

means to view the entirety of colonization at the time of plant harvest, and are therefore

useful for observation of both viable and non-viable fungal tissue. Trypan blue (0.05%

w/v) was used in the study that popularized the hot KOH clearing technique and is still

widely used today (Phillips & Hayman 1970). This, and similar stains such as 0.05%

(w/v) cotton blue, 0.01% (w/v) acid fuschin and 0.03% (w/v) Chlorazol black E (CBE),

are typically accomplished by placing the roots in dye that has been suspended in lacto-

glycerol (lactic acid glycerol water, 1:1:1 or 1:2:2) solution and heating. Stained









roots can then be stored in lacto-glycerol or diluted glycerol without dye, a step that can

also enhance visualization of fungal tissue by removing excess stain, especially important

when using trypan blue or CBE. Stain concentrations and staining time should be

assessed and adjusted according to the root type and the needs of a particular study.

Higher concentrations and longer staining times are more likely to be required with

cotton blue and acid fuschin, while over-staining can often occur when CBE is used.

Trypan blue is particularly useful when examining roots under a dissecting microscope

for determination of colonization levels, while a higher contrast stain such as CBE is

probably more appropriate for morphological studies.

In addition to the destructive, non-vital staining techniques described above, non-

destructive and vital staining methods have been developed. Technologies such as

epifluorescence and confocal laser-scanning microscopy (CLSM) allow for the

examination of living tissue, albeit with inherent limitations in the use of both.

Epifluroescence microscopy appears to only reveal collapsed and therefore inactive

arbuscules, providing information of select value, while CLSM requires specialized and

expensive equipment and supplies. Vital staining using nitro blue tetrazolium chloride

(NBT), diaminobenzidine (DAB) and phosphatase assay kits can also be useful to

visualize tissue that was metabolically active at the time of plant harvest. These methods

are considerably more expensive than non-vital stains, their use can be an involved

process, and investigators may still require or benefit from the use of specialized

equipment.









An excellent general review of clearing and staining techniques was recently

written by Vierheilig et al. (2005). Brundrett et al. (1996) provides detailed guidance

with regards to non-vital staining materials and procedures.

There are two general approaches to studying AM fungi colonization levels that

have evolved and generally supplanted a multitude of other techniques, favored because

they minimize compromises researchers need to make with regards to accuracy, ease of

execution, and reproducibility. One approach was adapted from a procedure developed

by Newman (1966) to improve the ability of researchers to estimate total sample root

lengths, which he called the "line intersection method," now often called the grid-line

intersect or just the line-intersect method. This procedure was devised to provide another

quantitative evaluation besides root weights for assessing the amount of root tissue in a

sample, and yields accurate, reproducible estimations. As commonly adapted for the

study of AM fungi, this method was described, as well as tested for accuracy and

compared to several other techniques by Giovannetti and Mosse (1980). In the line-

intersect method, previously cleared and stained roots are spread on a Petri dish onto

which a grid of straight lines are affixed, drawn or scored. The grid of lines is then used

as a reference, where intersections between them and the root sample are tallied and

observed for evidence of infection using a dissecting microscope. This will yield data

representing both the total root intersections and the total infected intersections, from

which the proportion of infected tissue can be derived. An appropriate volume of roots is

chosen, not so many that individual roots can not be observed, but not so few that at least

100 intersections can not be tallied. This means that when fine roots are being observed,

the dry weight of roots to be examined will rarely exceed 0.2 g.










The grid of straight lines is usually laid out so that 1.27- cm (0.5- in.) squares are

formed. The line-intersect method can be used to not only estimate the proportion of

infected root, but also the total root length, using the formula

xnNA

2H

where R = total root length, A = the area of the surface over which the roots are observed,

N = the number of intersections between roots and lines, and H = the length of all straight

lines. When 1.27- cm squares are used, the total root length in cm = the number of root-

line intersections, or R = N. Giovannetti and Mosse (1980) determined that increasing

the number of observations from 100 to 200 improved the standard error of percent

infection measurements from & 4 to a 3 when one set of observations was performed,

while increasing the number to 300 yields a smaller improvement. This number can

therefore be adjusted according to a researcher' s need for accuracy.

McGonigle et al. (1990) developed a method that not only allows the

determination of colonization levels and separation according to structures encountered,

it additionally assists with problems encountered in distinguishing AM fungal structures

from other cortical obj ects by virtue of its reliance on the use of a compound microscope

to examine intersections. The use of dissecting microscopes to study root endophyte

interactions is inherently limited due to the relatively lower magnifications available

when compared to compound microscope. A root system, even when adequately cleared

and stained, and with its cortex intact after processing, can be a confusing mess of colors

and shapes due to root cell content staining as well as the coexistence of a diversity of

microorganisms within the root. This problem can be multiplied when an investigator is










studying field roots or has limited experience differentiating among the varied

components of the root and its inhabitants.

The technique described as the magnified intersections method (McGonigle et al.

1990) examines intersections between the eyepiece crosshairs and roots at a

recommended magnification of x 200, higher than that capable of achievement using

dissecting scopes. In using this procedure, between two to four slides of roots from a

cleared and stained subsample are observed. The field of view is moved so four, six or

eight passes are made across a slide along its short axis. Where the crosshair intersects

with a root section, the field of view is adjusted so the vertical line is parallel with the

long axis of the root. Intersections are counted and classified according to their AM

fungal structural content.

Although the accuracy of individual intersection assessment increases with this

method, there are drawbacks. The authors themselves noted that the preparation of

sampling and subsequent observation is a much lengthier process using their technique

when compared to the line-intersect method. Further, it remains unclear as to whether

this represents an improvement in the assessment of the true colonization level of a root

system. The additional information yielded in determination of the level of types of AM

fungal structures present also seems to have limited value. Where these determinations

are perceived to be important or useful, the magnified intersections method is clearly a

superior investigative process to the line-intersect method; however, it requires more skill

on the part of the microscopist.

Spore extraction for identification and enumeration

The separation of spores from soil or other plant growth media is required for

classification, as AM fungal structures formed during the association with roots are not









suitable for differentiation among morphotypes except at the higher taxonomic levels.

Spore extraction is important not only for the determination of the presence of

morphotypes using classical techniques, but is also currently necessary for molecular

studies in identifying phylotypes. This section will detail the technique of spore removal

and subsequent procedures that relate to morphological classification and quantitative

evaluations, while the subj ect of molecular studies will be dealt with subsequently. Spore

extractions in ecological studies are typically accomplished for two reasons: 1) to

quantify the level at which sporulation is occurring in the Hield at different plant

phenological stages, or to check for fluctuations in production related to seasonal or plant

successional factors; and 2) for identification purposes, either from Hield soil, or from

greenhouse pot cultures.

The processes of spore extraction that are most commonly used are adaptations of a

method derived to extract nematodes, first modified by Gerdemann (1955). The method

varies among researchers, but the general process involves homogenization of a soil or

root-soil complex in water followed by sieving, centrifugation and decanting. Some

investigators additionally include steps involving separation in sucrose density gradients

with additional centrifugation (Brundrett et al. 1996). The specific method used in this

study will be detailed in the Materials and Methods section.

Enumeration of spores is generally associated with directly or recently sampled

Hield soil, although investigators in some studies count spores concurrently or exclusively

from trap cultures (Liu & Wang 2003). Spore enumeration data is best observed with

reservation and understood with qualification in ecological research. It does not

necessarily represent an accurate indication of AM fungal presence, since the degree to









which spores are present in field soil or in pot cultures depends on many variables.

Individual isolates are known to sporulate more heavily than others, with some isolates

rarely forming spores under any conditions. Sporulation generally appears to occur in

predisposed isolates after reaching a threshold of colonization within a plant and can be

affected by a multitude of environmental conditions. Additionally, spores are not the

most important infective propagule in many systems (Clapp et al. 1995; Klironomos &

Hart 2002), and are only a small fraction of the total AM fungal biomass in any system.

Identification based on spores directly harvested from the field can be a difficult

ordeal, owing to the large proportion of parasitized spores and associated organic

material that is generally present, complicating both morphological and molecular

identification. This problem is commonly resolved by segregating healthy-looking

morphotypes from field soil extractions, then inoculating greenhouse or potted field

grown host crops with the individual morphotypes. Growth media in pot cultures is

usually sterilized sandy or sandy loam soil fertilized during crop growth using a weak

Hoagland's solution without phosphorus. The host crop is then harvested at a particular

developmental stage or at senescence, and a second extraction is performed, yielding

spores more suited for identification.

There are currently two systems of taxonomic classification broadly recognized for

AM fungi, based primarily on morphological and genetic variation among groups,

respectively. The revisions to taxonomy have been continuous during the long history of

research of these organisms, so it is with great relief that most researchers note that

considerable homology has occurred, and that effort has been made to maintain

congruence where possible thus far between the systems (Koide & Mosse 2004).









The taxonomic system built on the foundation of the morphological variation of

spores exists in descriptions associated with the International Culture Collection of VA

Mycorrhizal Fungi (INVAM), currently held at West Virginia University and maintained

by Joe Morton. The system is not isolated from advances in molecular research, and has

accounted for the elevation of the AM fungi to the level of division Glomeromycota that

occurred based based on SSU rDNA sequence analysis by Schtiiler et al. (2001).

Additionally, results of SSU rDNA analysis helped elucidate the existence of the two

most recently recognized families within this taxonomic system: the Paraglomaceae and

Archaeosporaceae (Morton & Redecker 2001).

The INVAM collection currently recognizes one order, Glomales, within the

phylum Glomeromycota. This order is divided into two suborders: the Glomineae, which

contains four families and five genera; and the Gigasporineae, with one family and two

genera (INVAM 2006a). There are, indeed, many divergent anatomical and

morphological characteristics independent of spores that have been identified among

these groups, some of which have aided in the separation of taxa. Examples include the

formation of auxiliary cells associated with the Gigasporineae, the inter- and intra-

cellular vesicles formed by the Glomineae, as well as differences in the shape, size and

staining intensity of hyphae and arbuscules in the cortex. The ability to use these

characteristics for diagnostic purposes, though, is extremely limited and therefore they

are generally only noted in morphotype descriptions from pure culture. They can, in

certain circumstances, provide evidence of the existence of taxa that are otherwise not

evident through the primary means of morphotype identification: spore wall

characterization. The use of alternative characteristics as a means of identifying AM










fungal taxa would only be important when primary evidence was lacking, for example,

when spores are not extracted but colonized roots are observed. Identification with the

use of spores is preferred as morphotype descriptions are formed based on a variety of

spore attributes including the number of walls, external and internal anatomical feature

structure, size and staining characteristics, and spore ontogeny.

The system of classification based on SSU ribosomal gene analysis does not refer

in its descriptions to the other morphological, biochemical, phenological or other sources

of genetic variability that occur among and within identified taxa. This classification

groups the AM fungi to the species level (Schuinler et al. 2001; Schuinler 2006), and has

rearranged the taxonomic organization creating multiple orders, as well as adding and

shifting family-level classifications. Phylotypes currently recognized by this

classification include four orders: Glomerales, now divided into two groups;

Diversisporales, a grouping that includes the Acaulosporaceae, the Gigasporaceae, as

well as two new families, Diversisporaceae and Pacisporaceae; the Archaeosporales with

Archaeosporaceae and the newly formed Geosiphonaceae; and the Paraglomerales

containing the Paraglomeraceae. Some of the problems with speciating the AM fungi

and associated difficulties with molecular analyses are discussed in the next section. The

separation of AM fungi from Zycomycota into their own phylum, the Glomeromycota, is

widely accepted; finer taxonomic levels, however, will ideally ultimately depend on a

more comprehensive knowledge of morphological and molecular variability.

Molecular Tools and Studying the Species Concept and Genetic Condition of
Arbuscular Mycorrhizal Fungi

While the knowledge of the existence of mycorrhizae is over a century old, and

interest in studying these organisms has long been high due to their ubiquitous nature and









perceived potential as an agricultural technology, the concept of species has yet to be

firmly established with consensus. As noted previously, spore morphology has

historically been the most important factor in AM fungal species determination, but there

are a number of problems associated with relying solely on spores for identification in

ecological studies. Some practical difficulties include rapid sample degradation, presence

of associated, but non-sporulating morphotypes, the lack of significant distinguishing

characteristics among some taxa, and the extreme laboriousness involved in processing

and examining numerous samples (Redecker et al. 2003). The use of molecular tools has

emerged and developed as an option for identification, but there are a number of

associated problems using molecular methods in trying to identify AM fungi.

Heterogeneous nuclear material exists within individual AM fungi, and the reasons for

this state remain yet to be definitively confirmed. Additionally, polymorphism occurs in

typical marker genes adding to the complications of studying an organism that is already

difficult to classify (Redecker et al. 2003).

Two important questions were raised by Landis et al. (2004) and also discussed by

Sanders (2004): what is the best way to quantify AM fungal richness in a given

ecosystem, the classical method that categorizes by spore morphotypes, a molecular

method, or some combination of both? Also, is there a most relevant level on which to

measure diversity when studying an organism which up to this point has had a

continuously transitioning species concept? Satisfactory answers to these questions still

elude us and will require a great deal of additional work.

Two published studies cited by Sanders (2004) highlight the problems associated

with using spore morphotype as the sole method for speciation. The first involved









research investigating AM fungal diversity in understory vegetation under forest cover in

England, and found that PCR amplification detected a Glomus spp. phylotype strongly,

yet it was not found sporulating during long-term soil and plant sampling (Clapp et al.

1995). Spore morphology as a measure of diversity was also found lacking in a study by

Rosendahl and Stukenbrock (2004), in which rDNA analysis of AM fungi on sand-dune-

occupying grasses in Denmark was performed. Phylogenetic analysis revealed 11 groups

of distinguishable AM fungi, but only four correlated with species identified by spore

morphotype. In addition, those identified clades were only "patchily distributed in the

grassland," suggesting that a significant presence and greater diversity could exist

undetected in the form of the non-sporulating population (Rosendahl & Stukenbrock

2004, p. 3184). Sanders, in his review, speculates that using spore morphotype counts

only give us the barest beginnings of the ecological story (2004).

While the complications related to morphotype characterization might seem

daunting, they are at least a concrete standard providing guidance, irrespective of

ecological significance. Molecular methods, despite years of attempts at development,

are still in the germinal stage in terms of adequately answering key ecological questions

conclusively. The main problem lies in the previously mentioned genetic variation which

occurs in the phylum and even within individual spores (Pawlowska & Taylor 2004;

Sanders 2004). Before delving into the problems associated with molecular

characterization, I will discuss some of the published evidence regarding the nuclear

condition and life history of AM fungi.

The vast maj ority of fungi exist vegetatively as haploids, and it has recently been

advanced that at least one of the AM fungi, Glomus intraradices, is also haploid; the









ploidy of the larger group of AM fungi remains to be positively determined however, and

may even be variable (Hijri & Sanders 2003). The DNA content of nuclei has been

estimated for 11 AM fungi other than G. intraradices, and ranged from approximately

127 Mb to 1058 Mb (Hosny et al. 1998). Interestingly, both extremes came from

morphotypes of the same genus, Scutellospora. In contrast, G. intratradicesrtrt~t~r~rtrt the species

used most extensively in molecular studies involving AM fungi, has a genome size

estimated to be about 16.5 Mb (Hijri & Sanders 2003). Also, spores and hyphae have

been determined to be multinucleate, with the number of nuclei in each spore ranging

from 800 to 50,000 (Sanders 2002).

When Pawlowska and Taylor (2004) looked at the ploidy status of isolates of

Glomus etunicatum, starkly different conclusions were derived compared to the study

describing G. intratradices~rtrt~t~r~rtr as an organism with a haploid genome. Their methods

involved investigation of the transmission of polymorphic markers as well as examining

the rDNA variant distribution among spore nuclei using PCR to clone the ITS1 region.

That study looked at the variation within four field isolates of G. etunicatum, the results

of which supported the case for the homokaryotic condition, and therefore the conclusion

that high variability is most likely a product of polyploidy. One of the most intriguing

aspects of this study is their additional investigation of a G. intratradices~rtrt~t~r~rtr strain, the same

morphotype described in the 2003 work of Hijri and Sanders as haploid, and using the

same PCR amplification technique predicted it was polyploidic.

The AM fungi have defied almost every attempt at their culturing due to their

obligately symbiotic nature, although developments in monoxenic culture using

transformed root tissue have been successful (de la Providencia et al. 2004). Studies









involving fungal persistence in soil subsequent to spore germination generated results

showing only an ephemeral existence in the soil in the absence of a host (Sanders 2002).

There has been evidence, however, for longer-term survival in soil after initial

germination, though with arrested development (Logi et al. 1998). Experimental

evidence revealed that isolates of the morphotype Glomus caledonium germinated

without a host, stopped growth after 15 days, and subsequently retracted its protoplasm

from hyphal tips, and then isolated empty hyphae by forming septations. These AM

fungal isolates retained infectivity for extended periods, with some remaining viable 6

months after initial germination (Logi et al. 1998).

The AM fungi have been assumed to exist as asexual organisms, although this has

not yet been definitively proven, but is instead a result of the lack of any evidence for the

occurrence of sexual processes. The origin of the high levels of genetic variation within

individuals remains an obj ect of speculation in the absence of sexual recombination, with

anastomosis and subsequent nuclear exchange among different isolates remaining under

investigation as a possibility. Anastomosis has been known to occur in AM fungi for

almost 50 years (Godfrey 1957), and has been periodically observed and quantified since

that time as occurring extensively in various AM fungi. The process of anastomosis has

not been seen to occur between different morphotypes, nor to this date in isolates of the

same morphotype from different geographical locations. This has led to speculation upon

the existence, and nature of unidentified recognition factors that seem to exist, during in

vitro studies, on a finer level than any current taxonomic system describes (de la

Providencia et al. 2004; Sanders 2002). Both de la Providencia et al. (2004) and

Giovannetti et al. (2001) have looked at the nature and levels of anastomosis in AM fungi









in isolates from families of both described suborders. They produced contradictory data

with regards to the number of anastomoses formed per unit length of hyphae; however,

they agreed that Glomeraceae strains formed anastomoses between individual hyphae. In

their work, de la Providencia et al. (2004) found Gigasporaceae strains formed bridges

only upon the same hyphal strand. These studies, and another by Giovannetti et al.

(2004), provided important information through the visualization of cytoplasmic

movement and nuclear exchange between anastomosing hyphae in both symbiotic and

presymbiotic growth (de la Providencia et al. 2004; Sanders 2002). This suggests a

mechanism whereby resource and genetic sharing might exist, although not yet detected

on a level higher than intra-isolate.

There are two widely accepted yet conceptually divergent schools of thought

regarding the nuclear condition of AM fungi that can explain detected polymorphism in

single spore rDNA and the genetic diversity in these apparently asexual multinucleate

fungi. The first model involves a collection of coexisting genetically diverse nuclei

existing within each fungal individual that is passed on to successive generations. Not

every spore in the next generation would receive the same complement of nuclei with the

same frequency as the parent generation. Anastomosis, in this heterokaryotic model,

would result in the transfer of intact nuclei from any donating fungi. Karyogamy would

not have to occur to change a phenotype, as the hundreds to thousands of nuclei present

in AM fungal cells would gradually accumulate mutations during replication (Sanders

2002; Pawlowska & Taylor 2004). The other model argues for a genome that contains

more variability within each nuclei, the homokaryotic model. This would involve an

assemblage of similar nuclei with variance manifested through the presence of multiple










gene copies either on individual or, in the case of polyploidy, multiple chromosomes.

Genetic diversity could arise from either errors in mitotic recombination and

accumulation of other mutations, or through as yet undescribed genetic exchanges

involving sexual recombination (Sanders 2002; Pawlowska & Taylor 2004).

Taking into account the complexity and our lack of understanding regarding the

AM fungal genome, the use of molecular techniques for ecological studies of AM fungi

can be perceived as tools under development. Identification in such research almost

exclusively targets the SSU rDNA, therefore revealing an inherently limited picture of

diversity. Considering the high degree of polymorphism of rDNA that exists in these

fungi, and the possibility that considerable variation may even occur within individuals,

interpretation of results can prove problematic. Reliance on a single genetic locus for

identification may prove less popular as our understanding of the source of the incredible

diversity within this organism increases.

Quantifieation using molecular techniques in ecological studies of arbuscular

mycorrrhizae is no less difficult than identification. While quantitative techniques like

Real-Time PCR (RT-PCR) have proved invaluable in the study of other organisms, it

probably has the most potential for the study of AM fungi when the presence of only one

isolate is being measured. So far, quantitative measures with this technology have been

limited to root-associated AM fungi, where it shows some promise for specialized

studies.

Alkan et al. (2004) describe the use of RT-PCR to achieve quantitative results in

detecting Glomus intratradices~rtrt~t~r~rtr in tomato and alfalfa. Their RT-PCR data was calibrated

with microscopic observations, thus enabling them to subsequently screen large numbers









of samples with reduced labor. They suggested that a similar application of using

calibration curves to allow for high-throughput colonization assays using RT-PCR. This

method appears likely to be useful for rapid identification and quantifieation of individual

fungi for which reliable, established primers exist, though less useful for quantifieation in

Hield plants, where a complex of diverse AM fungi are present.

Ishii and Loynachan (2004) described two methods using nested-PCR, in which

root segments infected with AM fungi previously stained by trypan blue were used in the

extraction of DNA. The four greenhouse-grown plants inoculated with an isolate of

Glonaus nzosseae acquired from INVAM were maize (Zea nzays), alfalfa (M~edicago

sativa), soybean (Glycine nzax) and sudangrass (Sorghum sudanense).~~~dd~~~dd~~~dd Both methods

used bead beating to expedite sample processing. The use of this technique would allow

correlation with microscopic observations of the same samples, and could confirm

colonization levels if phylum-specific primers were available and quantitative PCR was

used.

Further results from the use of molecular and other investigative tools will be

presented in a subsequent section where ecological research is discussed.

Fatty Acid Methyl Ester Analysis

Fatty acids occur at a reasonably constant proportion of cell biomass as they exist

largely as components of membrane lipids. Signature fatty acids are biomarkers that can

differentiate groups of organisms within studied communities and can provide

quantitative estimates of their microbial biomass. These compounds are represented in

literature by the number of carbon atoms with the degree of their unsaturation separated

from the carbon chain length by a colon. The position of a double bond is indicated next,

following the co symbol indicating the number of carbon atoms from the terminal methyl









end of the molecule to the first occurrence of unsaturation (Zelles 1999). The suffixes c

and t stand for the cis and trans isomers of the unsaturation, respectively.

Fatty acid analysis using gas chromatography (GC) has proven to be a useful tool

for structural analysis of soil and plant-associated microbial communities. Fatty acid

methyl ester (FAME) analysis has been developed into an automated process by MIDI

Inc. (Newark, Del.), whereby whole cell fatty acids are extracted, then analyzed using

appropriate software. Analysis of whole cell fatty acids to study broad microbial

communities that occur within varying agricultural management strategies has been well

established (Cavigelli et al. 1995; Buyer & Drinkwater 1997). The use of FAME

extraction analyzed using the MIDI system is widespread due to the ease of sample

processing; however, careful interpretation of the results is necessary, since both cellular

and extracellular lipids are detected with this technique. Extracellular lipids can be

derived predominantly from organic matter as opposed to the active fraction of the

targeted organism, and therefore, data should be considered accordingly (Zelles 1999).

The fatty acid 16:1mo5c has been confirmed as a biomarker for AM fungi in studies

investigating colonization of roots (Graham et al. 1995), in estimations of fungal biomass

in the soil (Olsson et al. 1995), and in work that requires quantification of the AM fungal

presence associated with both substrates (Thygesen et al. 2004). The AM morphotypes

within the Glomineae are reported to have higher concentrations of 16: 1m5c compared

with some members of the Gigasporineae, within which it appears to negligible.

Therefore, the use of this particular fatty acid will not detect comprehensively at the

phylum level; however, it is capable of being used for detection and quantification of the

AM fungal morphotypes that are considered the most important in agricultural










ecosystems. As a result, the use of 16:mIc~ has good potential for use in studies to

reasonably quantify AM fungal abundance of members of the Glominaeae in roots and

soil when finer levels of identification are not deemed necessary; either in lieu of using

labor-intensive root, spore and hyphal quantification, or as a means of complementing

classical techniques.

The fatty acids 18:1mo9c and 20: 1m9c have been identified as possible biomarkers

for members of the suborder Gigasporineae, yielding useful results in studies on plant

roots (Graham et al. 1995) and from soil (Madan et al. 2002), although their extensive

use has yet to be undertaken. Perhaps complicating the matter of the use of fatty acids

that appear to be produced in abundance other than 16:1mo5c, is the evidence that fatty

acids seem to be differentially produced in the various AM fungal structures (Graham et

al. 1995; Sakamoto et al. 2004). For example, some evidence shows that the external

hyphae of Gigaspora rosea seems to lack 20: 1m9c, while it has been shown to be

abundant within spores (Sakamoto et al. 2004).

Other Tools

There are additional techniques for the characterization of AM fungi in soil and

roots, including variations of the methods listed previously. Those described above,

however, are the most commonly found in research currently. Undoubtedly, as our

understanding of these organisms and the technology used to study them advances, a

more distinct picture of the ecological role of mycorrhizae will similarly develop. A

good introductory overview of some of the other techniques used in AM fungal research,

such as the quantification of extraradical hyphae, assays for functional activity, and

identification using immunological methods is given in Millner and Wright (2002), and

references contained therein.









Arbuscular Mycorrhizal Fungi in Agroecosystems

There has been an abundance of work published focusing on the existence,

aggressiveness and state of functioning of AM fungi within ecosystems, including a

substantial number of reviews and even meta-analyses of other researchers' results. In a

review of the diversity of AM fungi in a large variety of managed and natural

ecosystems, the morphotype numbers found among the various studies ranged from 3 to

46 per site (Douds & Millner, 1999). Other studies that were cited in that work have

shown, as was discussed in the first chapter, that the choice of crop rotation (or lack

thereof), tillage and fertilization strategies all can profoundly affect populations of AM

fungi. A meta-analysis of data from independently published Hield studies was

accomplished by Treseder (2004), looking at the impacts that fertilization and CO2

enrichment have on the mycorrhizal state. The author found that across studies,

mycorrhizal abundance was reduced by 15% due to N fertilization, 32% by P fertilization

and increased 47% as a result of elevated CO2 leVOIS. While ectomycorrhizal fungi were

included in the meta-analysis, 18 of 20 studies compared for impact of P fertilization

focused on the AM fungi.

In the following sections, I will undertake a brief review of literature, and will

concentrate on research that focuses on anthropogenically derived alterations to the

environment under agricultural production and their impact on AM fungal biomass and

infectivity. In addition, an emphasis will be placed on Hield conditions that were

important in this study.

Phosphorus Fertilization.

If such a thing as common knowledge exists for AM fungi, it is related to their role

in plant P nutrition and, to a lesser degree, the impact high P levels can have on the









organism. This does not mean accurate characterizations of these fungi occur in

discourse as most, if not all, public awareness of mycorrhizae is due to the marketing of

these fungi as an agricultural cure-all. Within the agricultural sciences it is widely

accepted that they are capable of providing P to plants, especially, and perhaps

exclusively, when soil availability of the nutrient is moderate to low (Schubert &

Hayman 1986; Thingstrup et al. 1998), and that high levels of available soil P contribute

to depressed colonization of roots by AM fungi (Daft & Nicholson 1969; Thingstrup et

al. 1998).

The mechanism for increased absorption of soil P by mycorrhizal roots is likely

twofold. The primary mechanism seems to be due to the increased soil exploration that

roots with the association are capable of undertaking (Sanders & Tinker 1971; Koide

1991). There is also evidence that AM fungal hyphae may be able to excrete compounds

increasing the solubility of the inorganic P (Joner et al. 2000; Tawaraya et al. 2006). The

decrease in levels of the association when high levels of available soil P are present is

apparently more a result of the P concentration in the plant than it is related to actual soil

levels (Koide & Li 1990; Koide 1991), although the two are obviously connected.

Additionally, sporulation has been shown to be related to a threshold of AM fungal root

biomass and possibly plant P concentration (Douds & Schenk 1990), providing an

explanation for why spore numbers are dramatically reduced when available soil P is

high. This suppression of the mycorrhizal condition has been perceived as a self-

regulatory process, whereby the plant reduces C allocation to the fungi when it is more P

self-sufficient (Graham et al. 1981). The mechanisms through which plants inhibit

infection by AM fungi are probably quite varied, considering the length of time the










organisms have had to coevolve and the diversity of the many plant and fungal genotypes

involved. There is evidence, though, that both biochemical means and anatomical

structures can be involved (Brundrett & Kendrick 1990; Fortin et al. 2002). The

continuance of the mycorrhizal relationship when soil P is readily available can lead to a

reduction in crop yield, through the misapplied C investment without a commensurate

benefit in terms of plant P acquisition (Graham 2000; Kahiluoto et al. 2001; Ryan &

Graham, 2002).

Soil Disturbance

The external hyphal network of AM fungi in soil is an important inoculum source

for plant roots, so it comes as no surprise that disruption of soil through agricultural

practices such as primary tillage and cultivation can reduce the overall biomass and root

colonization levels. This has been shown in many studies with similar results; only a few

will be mentioned.

A study by Drijber et al. (2000) considered changes in the broad soil microbial

community in moldboard plowed, reduced tillage, no-tilled and sod plots. Using FAME

analysis, AM fungal biomass was determined to have decreased significantly in the more

heavily disturbed treatments compared to the no-till and sod plots. Jasper et al. (1991)

investigated the infection potential of annual pasture, heathland and forest soil sown with

clover plants after disturbance. There was a significant decrease in the AM fungal

infection potential in heathland and forest soils after soil mixing, which led them to

speculate that disturbance is probably most important where the initial propagule number

is already low, a condition that was overcome in the pasture soil. More recently, an on-

going tillage-rotation experiment in its sixth year in Chile compared different levels of

soil disturbance on a variety of AM fungal-related parameters (Borie et al. 2006). The









less disturbed treatments had higher spore numbers and greater levels of root

colonization, as well as higher total content of the AM fungal-derived glycoprotein,

glomalin, when compared the more highly disturbed treatments.

Pesticides

One might anticipate that the use of pesticides, particularly broad range biocides

and fungicides, would have a negative effect on mycorrhizal survival and functioning.

While this is generally the case, the impact of many of these agrichemicals on AM fungal

communities is often slight and usually appears to be only ephemeral in nature, and it can

be difficult to predict the response to an individual pesticide. The range of responses has

varied from deleterious to negligible to stimulatory (Koide & Mosse 2004). It has, in

fact, proven difficult to control AM fungi with chemical methods for research purposes, a

situation that has made the development of non-mycorrhizal mutants all the more

welcome.

One of the fungicides considered most effective, benomyl, gives only partial

control and requires frequent reapplication. In a study looking at the P nutrition of the

perennial common bluebell (Hyac~I imbarides~ non-scripta), individual colonies were grown

in 28-L cubic blocks of soil (Merryweather & Fitter, 1996). The colonies receiving

fungicide were immersed monthly in a solution of benomyl (50% w.p.) for control of AM

fungi. While a significant reduction occurred, colonization was still measurable, reaching

approximately 40% at the final assessment.

In a recent work, six soil fumigants were investigated for their effects on the

structure and functionality of the microbial community, including AM fungi, using

FAME profie analysis (Klose et al. 2006). The sandy loam soil used in the microcosm

study was fumigated for 24 hours, and then subj ected to FAME extraction periodically









over the next 90 days. Four of the six fumigants had a significant depressive effect on

AM fungi biomass for the duration of the experiment, while the other two did not differ

significantly post-fumigation from control soil; neither methyl bromide with chloropicrin,

nor methyl isothiocyanate had a significant impact. Although the authors imply a lack of

community recovery in the case of the four effective fumigants, the absence of change in

the AM fungal biomarker during the course of this study is not surprising, considering

that no host was available for colonization in the microcosm.

A succinct review of the impact that some pesticides have on AM fungal

populations is contained in a section from a publication by Gosling et al. (2006), while a

more comprehensive, albeit dated, treatment is undertaken in Trappe et al. (1984).

Flooding

There has been less information published regarding the mycorrhizal status of

agricultural crops during flooded conditions than there has of wetland plants. It has been

demonstrated repeatedly that AM fungal associations can persist and benefit rice in

wetland production (Solaiman & Hirata 1997; Solaiman & Hirata 1998), and it is well

established that AM fungi colonize many wetland plants. While they are known to be

aerobic organisms, there is an apparent means through which at least some taxa of these

fungi are capable of surviving and functioning in flooded conditions. However, in native

wetland plants, colonization levels have been reported to slow during periods of flooding,

with substantial increases in mycorrhizal development during any extended periods

between flooding (Miller 2000; Ray & Inouye 2006). Even when soils have been

submerged for long stretches, resulting in little development of AM fungal colonization

within roots, levels rebound in field plants and infection potential studies after drawdown

of water levels (Miller 2000; Ray & Inouye 2006).









In a study of post flood syndrome in the Midwest and Great Plains, Ellis (1998)

examined maize and soybean plants that were grown in Hields that had been flooded for

long periods the previous year. Plants were sampled three times during the growing

season. Additionally, a greenhouse experiment was undertaken in which maize plants

were grown in sterilized soil that was subsequently inoculated with Glomus intraradices

spores and then flooded for either 0, 1 or 3 weeks. While flooded Hields initially had low

infection potentials the following year, colonization levels steadily increased as the

season progressed. There was no significant difference among the levels of colonization

in the greenhouse study, and overall levels were actually highest in the two flooded

treatments. The author attributed the low levels of infection in fields during seasons

subsequent to flooding episodes not to the extended periods of saturated soil, but rather to

the absence of host tissue after the near complete crop death.

These and other (Sah et al. 2006) studies indicate that flooding conditions do not

preclude the presence of a functional population of AM fungi. Mechanisms for coping

with both long-term saturated conditions in wetlands and short-term flooding in

agricultural Hields and other land exist, and infective propagules persist after wet

conditions subside. The reduced colonization during times of flooding is likely due to

severely stressed plant root systems, which is relieved when drier conditions prevail and

normal root growth can proceed.

Organic, Conventional, and Grassland Farming Management Strategies

In distinguishing among studies that focus on individual agricultural practices, it is

easy to neglect research of a broader scope that compares pertinent management

strategies. Of particular interest here is research that focuses on organic and grassland (or

pasture) agriculture. Both of these represent types of farming that have been considered









to be relatively amenable to the encouragement of AM fungal populations in terms of

biomass, functioning and diversity.

Organic agriculture is not a static management style and farmers incorporate a

tremendous diversity of practices with yield goals, type of crop, certifying body, soil type

and geographic location among some of the more important variables involved in

deciding practices. Farming strategies in organic agriculture that are thought to have the

potential to minimize disruption of AM fungal populations include crop rotations and

cover cropping, the absence of highly soluble chemical fertilizers, and reduced reliance

on pesticides with use restricted to botanically derived or mined sources. While not

exclusive of either organic or conventional management, but rather an integral part of

both in many instances, grassland or forage agriculture differs from agronomic or

horticultural row crop production. Agroecosystems dedicated to or integrating long-term

stands of native grasses or forage crops are generally distinguished by a lower relative

agrichemical input, reduced traffic and soil disturbance and increased levels of plant-

derived organic compounds in the soil when compared to row crops. It is important to

bear in mind that there can be considerable overlap among farming management

strategies and that what differentiates one from the other, in terms of what specifically

influences AM fungal populations in a given work, may not be readily deduced.

Oehl et al. (2004) found 35 morphotypes of AM fungi in one Hield site in

Switzerland where the systems trial had been in place for longer than 22 years. This

large number of identified taxa was a product of their trapping techniques, in which

cultures were kept for extended periods of time under different trap plant species.

Morphotype richness, spore abundance and infection potential were all higher in two










organically managed systems when compared to two conventional systems. They

attributed these differences, as well as the significantly higher colonization levels in bio-

dynamic plots and the development of different morphotype populations in the organic

treatments, to qualitative and quantitative variation in fertilizers.

Thirty-seven morphotypes of AM fungi were collected from a single one-hectare

field under mixed grasses and forbs in North Carolina (Bever et al. 2001). The method

used to extract so many species from a single field was a rigorous variation of trapping

techniques; long, successive trapping periods, multiple trap plant species and other

changes in environmental conditions were used in order to simulate the year-round field

conditions from which the samples were taken. The authors speculated that when a

mycorrhizal complex exists, as opposed to individual strains associating with one plant

species, the host specificity of AM fungi is high with respect to fungal growth. That is,

on certain plants, host specificity is manifested through more or less dominant AM fungal

types developing. Similarly, the study lent credence to the idea that AM fungi exert an

influence on plant community structure, but only in particular plant-fungal combinations.

A central philosophy of organic farming is that management practices should strive

to maximize biological functioning and diversity in order to maintain the health of the

broader ecosystem, and AM fungi are perceived as key players in these systems (Oehl et

al. 2004; Harrier & Watson 2004). Most studies comparing organic and conventional

management systems have shown higher relative colonization levels (Douds et al. 1993;

Ryan et al. 2000), as well as spore number and morphotype diversity (Douds et al. 1993;

Oehl et al. 2004) in organic farming. This is typically interpreted as a product of the

lower levels of slowly available soil P that comes from the use of fertilizers acceptable in










organic agriculture (Ryan & Graham 2002). Where nutrient applications are in excess of

crop needs and build to high levels in the soil, and when tillage is a frequent occurrence

for seedbed preparation and cultivation purposes, this ideal of enhancing mycorrhizal

relationships may be no more likely than in other high-input systems. Communities of

AM fungi in the extensive root network in grassland and forage agriculture, on the other

hand, can be expected to develop a considerable external hyphal network, undisturbed by

tillage (except during periods of establishment) and with reduced occurrence of

inhibition due to excessive nutrient levels.

Other Fungal Root Endophytes Known to Occur in Herbaceous Plant Roots

As non-AM fungal root endophytes represent a diverse range of fungal taxa, and

probably exist within most plant species around the world, it is speculated that they

contribute substantially to plant health and soil ecosystem functioning (Sylvia &

Chellemi 2001; Sieber 2002). However, as mentioned in the introduction, the ecological

roles of these endophytes can not be ascribed as being clear cut or uniform. In order to

begin to understand them, a great deal of further work needs to be undertaken to ascertain

both the phylogenetic status of many of these organisms and their host ranges

(Jumpponen & Trappe 1998; Sieber 2002). Some of the non-AM fungal endophytes

identified as commonly occurring in herbaceous plant roots include Philophora spp. and

their teleomorphs, as well as species of Fusarium, M~icrodochium, Trichoderma, and the

loosely described group of anamorphic fungi currently known as DSE' s (Sieber et al.

1988; Skipp & Christensen 1989; Jumpponen 2001; Sylvia & Chellemi 2001; Sieber

2002). Phialophora spp. are some of the few endophytes commonly inhabiting

herbaceous plants that have been identified and classified as belonging in that group.









The DSE are an informal grouping that comprises Melin' s (Melin 1922; Wang &

Wilcox 1985; Jumpponen & Trappe 1998) form taxon M~ycelium radicis atrovirens

(MRA) and other root-occupying fungi that meet a morphological standard and are

apparently non-deleterious to the host plant. Melin described a fungus isolated from

Pinus sylvestris and Picea abies roots that did not sporulate, which he believed was non-

mycorrhizal and possibly harmful to the plant. Further, it grew on media, forming "grey

air mycelium of tenuous hyphae" and produced a "dark olive-green pigment" when

cultured (Melin 1922, p. 256; Wang & Wilcox 1985). The term MRA has continued to

be used by some researchers for sterile fungi with darkly pigmented, septate hyphae

found both growing in root cells and isolated from the soil (Wang & Wilcox 1985; Sieber

et al. 1988; Jumpponen & Trappe 1998).

In a study examining mycorrhizal colonization of plant communities at different

altitudes in Austria, Read and Haselwandter (1981) noted the existence of these

endophytic fungi throughout the range of their survey and referred to the group as DS

fungi. Thereafter, the term DSE has come into favor for dark septate endophytes and is

used most frequently, although the exact meaning and appropriateness of this term is

sometimes questioned. A commonly used description for DSE includes those root-

occupying, biotrophic, mitosporic fungi with septate, melanized hyphae that typically

also produce melanized microsclerotia within the root (Treu et al. 1996; Jumpponen &

Trappe 1998; Sieber 2002). The search for a universally accepted name, or perhaps the

dissolution of the need for a group, should come as taxonomic affinities are determined.

It is interesting to take note of Barrow' s (2003) suggested refining of the designation

DSE to systemic endophytic fungi (SEF). His and others' work take note of the difficulty










in quantifying colonization by these endophytes due to their extensive network of non-

staining, hyaline hyphae (Yu et al. 2001; Barrow 2003). In the present study a great deal

of hyaline hyphae was encountered while observing samples; the abundance of hyaline

hyphae with lesser amounts of dark, septate hyphae occurring, as well as the existence of

other poorly defined structures made grouping into classes difficult. Therefore, these

non-AM fungal endophytes were not otherwise designated in this study, except where

species or other taxonomic units could be assigned. It is unclear whether changing the

designation to SEF would truly be a refinement or if there is instead an overlap of two or

more groups of endophytes. Additionally, it would seem such a change would likely

open the door to greatly expanding the number of fungi included, potentially leading to

more confusion.















CHAPTER 3
MATERIALS AND METHODS

Root and soil sampling. Soil samples were collected twice for use in infection

potential and trap culture studies. Soil and root sampling for this study took place in

years four and five of the Header Canal cropping systems proj ect, and occurred in

December of 2003 and 2004. While plant and soil sampling were completed on the same

day in both years, plots in year five were planted 36 days later than in year four (October

7, 2004 compared to September 2, 2003) due to the severe weather. Four beds of tomato

plants were in each half plot under production in 2003, but the hurricanes of 2004

destroyed a substantial portion of the beds that had been formed in September; only two

beds were established in each plot half when tomatoes were planted in October.

To approximate the rhizosphere of the plant, for each sample that was taken, the

probe entered at the base of a plant and was angled slightly to capture a substantial

portion of the root system along with the soil. Soil cores were taken from beneath plants

in the middle beds and from within the middle 100 plants in each row. Thirty cores from

each plot were taken to a depth of about 20 cm, with 15 samples collected walking East

to West in one center row, and the second 15 samples gathered walking West to East in

the other center row. The cores from each plot were combined and homogenized in

plastic bags, with the composite sample volume being approximately 2 L. Each of the

five tomato production treatments was replicated six times, producing a total of 30

samples from the portion of the plots then used for tomato production in year four, and 60

samples in year five.









Soil sampling was also conducted within the transition areas in the organic, bahia

pasture, weed fallow and disk fallow treatments in year four. Thirty cores were taken in

each plot from the area parallel to the portion of the plots from which the tomato

production soil samples were taken. Cores were taken from beneath the cover crop sunn

hemp plants in the organic system, and from the bahia sod in the bahia system. Ragweed

(Ambrosia artemisiifolia) was one of the most abundant and consistently present weeds

across the weed fallow system plots, therefore, soil samples were taken to approximate

this plant' s rhizosphere in that treatment. Cores were also taken from the disk fallow

plots in the areas being actively disked. There were 24 total composite samples taken

from these transitional portions of the systems (four treatments x six replications).

In year five, all previously transitioning portions of the plots were put into

production. However, in the bahia pasture and disk fallow treatments, a considerable

portion of the plots were under sod or being actively disked, respectively. The section

between each half plot remained free of tomato production throughout the experiment,

and in these two systems this area approximated the continuance of the rotation.

Therefore, soil samples were taken from this middle portion of the plots in these two

systems using the same method described for sampling the transitional areas from the

previous year, generating a total of 12 composite samples.

Root samples were taken from all plots with tomato plants and treatment plots with

cover crops and were used to assess colonization by AM fungi and other fungal

endophytes. Four tomato plants from each plot were dug from the center 100 plants of

the middle beds. Loose soil was removed by gently shaking roots prior to cutting the

entire root system from the stem. A total of 120 tomato plants were removed in year four










(four plants x 30 plots), and 240 plants were collected in year five (both halves of plots,

eight plants x 30 plots). In year four, root samples were also taken from cover crops from

the plots with sunn hemp (organic), ragweed (weed fallow) and bahia plants. The bahia

root samples were extracted from the paths between the tomato rows and not the tomato

sod. The root systems in both year four and five were placed in paper bags and dried at

70 oC. Fine roots were subsampled for clearing and staining, roots were dried and

subsample weights were between 0.05 and 0.07 g.

Infection potential study sample preparation. Infection potential studies provide

a method to evaluate the relative amounts of viable inoculum by growing a mycotrophic

crop that forms abundant roots quickly in either diluted (with sterilized sand), or

undiluted soil (as in this study). Quite different from full-season studies, infection

potential results are typically timed to provide data regarding levels of primary infection

on host crop roots. Harvested roots are examined for either the percentage colonization

by AM fungi or total mycorrhizal root length as described in the section on quantification

of fungal colonization in roots using microscopy. In preparation for this and other

evaluations, each of the composite soil samples from individual plots was sieved to

remove structural roots and to further homogenize soil. Fibrous roots were cut into small

pieces (< 2 cm) and mixed with the soil. A portion of soil from each plot was removed

for fertility and other analyses. The remaining soil and roots from individual plots were

divided in half, with one half designated for the spore trap culture described below, and

the rest used for the infection potential study. For the infection potential test, soil was

distributed at a volume of 125 mL among five to seven 150-mL containers based upon

the amount of soil in the initial composite sample. Two germinated maize seeds were










planted and watered into each tube, then later thinned, selecting the healthiest looking

plant. Infection potential plants were not fertilized, but were watered as necessary. Trays

holding the containers were randomly rearranged weekly. Maize plants were harvested

approximately 30 to 40 days after planting, at the five-leaf stage of development. Soil

was gently washed from the roots prior to their separation from the plant, and root

systems were then each placed in paper bags and dried at 70 oC. Dried roots were

sub sampled for clearing and staining, with subsample weight typically ranging from 0.03

to 0.05 g.

Trap crop sample preparation. Acquisition of fresh spores for morphotype

identification through culturing diluted field soil or previously extracted spores using live

plant roots is highly desirable, if not necessary, as the rapid degradation in quality of field

collected spores can render them useless (Millner & Wright 2002; Redecker et al. 2003).

The portion of soil not used in the infection potential study was used to grow trap plants

for subsequent isolation of AM fungal spores. The soil designated for the trap culturing

in each of the composite samples was divided between two 450-mL tubes. Five

sudangrass (Sorghum sudanense)ddd~~~ddd~~~ddd seeds were planted and then watered into the soil in

each tube, later thinned to three plants. Trays holding the containers were randomly

rearranged biweekly through the first 6 months of growth, and were fertilized weekly

using 35 mL of a Hoagland solution with a very low concentration of P (0.25 mM) after

nutrient deficiency symptoms first developed. The plants were allowed to naturally

senesce, and were harvested approximately seven months after planting.

Trap culture processing, spore extraction and observation. The method used to

extract spores from trap cultures was an adaptation of the process mentioned in the










previous chapter (Gerdemann 1955; Brundrett et al. 1996). After the sudangrass plants

had senesced, the aboveground biomass was removed and the soil within each container

was allowed to air dry. After removal from the tube, the soil and root system from each

pot was broken down, with roots cut into small (< 2 cm) fragments. A representative 100

cm3 Of soil and root mixture were suspended in 500 mL of DI water for 5 minutes with

agitation. The soil-root suspension was then poured over a set of three sieves. The sieve

openings, top to bottom were 2 mm, 1 mm, and 38 pm. The material on the top sieve

was discarded, while the contents in the Imm sieve were gently washed over the bottom

sieve; the material remaining after rinsing was thrown away. The material in the bottom

sieve was then distributed equally among six 50-mL centrifugation vials followed by the

addition of equal parts DI water and 50% sucrose (w/v). The sucrose was inj ected into

the soil and associated material at the bottom of each tube after adding water, using a

syringe with a piece of tubing attached, creating two layers with a clear interface. The six

tubes were put in an International model HN centrifuge and spun for 5 minutes at

approximately 771 x g. After centrifugation, the more dense soil was pelleted at the

bottom of each tube, while the remaining root tissue and other organic debris became

suspended at the top. From each of the six tubes, the 20-mL of solution surrounding the

interface between sucrose and DI water was removed individually using a syringe, taking

equal proportions from above and below the interface to maximize spore recovery. This

collected solution was drained from the spores in suspension using vacuum filtration.

The filtration system consisted of 55-mm circles of Whatman 50 hardened fi1ter paper

within a Biichner funnel and flask attached to a KNF Laboport vacuum pump. During

filtration, after spores from all six tubes were extracted, the syringe carrying the solution









with spores was rinsed several times by loading and ej ecting DI water from the syringe

onto the filter paper. This served several purposes; it was a method of clearing the

contents of the syringe for the next sample, it ensured that spores remaining in the syringe

after extraction were recovered, and it washed the sucrose from the spores and the filter

paper, which improved the subsequent handling of spores.

The filter paper was then moved to a Petri dish and observed under an Olympus

SZH10 stereo microscope for enumeration and identification. Enumeration was

undertaken using year five trap cultures and was based on the method used by INVAM

(2006b). The area of the ocular field at 4x magnification was determined to be 23.8 mm2,

and the area of the 55-mm diameter filter paper was 2375.8 mm2, prOViding 100 possible

fields of view for each spore extraction. The average number of spores counted in 40

randomly chosen fields was determined, yielding an approximation of the spores / cm3 Of

soil (average number of spores found x total possible fields / 100 cm3 Of Soil from which

spores were extracted).

Spore morphotypes were segregated after enumeration using the sharpened end of a

thin wooden dowel. Spores were transferred to a slide, and observed after covering them

with the several drops of a semi-permanent mounting agent consisting of polyvinyl-lacto-

glycerol (PVLG) with Melzers reagent (1:1 v/v). Spore morphotypes were observed and

identified to the extent possible using an Olympus BX51 compound microscope with

brightfield and differential interference contrast (DIC) optics. Joe Morton, the curator of

INVAM at West Virginia University, provided assistance with morphotype delineations,

and the INTVAM collection descriptions were used for classification.









Clearing and staining roots. Roots were cleared and stained for examination of

colonization levels both in the IP study and for field roots. The procedure used for

clearing and staining was a modified version of that described by Brundett et al. (1996).

Fibrous roots were subsampled and placed into scintillation vials. Roots were covered

with 10% KOH (w/v) and placed in an oven at 95 oC until cellular contents and pigments

were largely removed, and before significant damage occurred to the root system. This

time period varied among the different plant roots. Maize roots from the IP study were

heated for 15 minutes, while roots of tomato, sunn hemp, and rag weed were cleared for

25 minutes, and bahia roots were removed after 30 minutes of heating.

After rinsing several times with tap water, all roots, with the exception of those

from maize plants, received an additional bleaching step. An acidified bleach solution

was prepared using diluted Ultra bleach (1.7 % NaOC1) and 1 mL of diluted HCL per 100

mL of bleach. The bleach was added until the sample was covered, and then the sample

was swirled in the solution for up to 30 seconds. Total bleaching time varied among

samples with additional applications initiated until the roots became noticeably lighter in

color; however, bleaching never exceeded 90 seconds. Roots were rinsed repeatedly after

bleaching, then soaked in water for 5 minutes before staining.

Staining was with 0.05% (w/v) trypan blue in lacto-glycerol (1:2:2 lactic acid -

glycerol water). Maize and tomato samples were covered with stain and placed in a 95

oC oven for 10 minutes, while the rest of the root samples were heated with stain for 12

minutes. Stained roots were rinsed and then destined in lacto-glycerol for at least 24

hours before examination.









Determination of % colonized root-length. Colonization levels were determined

using the line-intersect method described in the section on quantifieation of fungal

colonization in roots using microscopy, and derived from Giovannetti and Mosse (1980).

Roots from both the infection potential study and Hield plants were observed using this

method. Examination was with an Olympus SZH10 stereo microscope using dark-Hield

illumination. Intersections were scored for presence of AM fungi and other endophytes.

When structures of questionable origin were encountered, root sections were removed

from the Petri dish, mounted on a slide, and observed under an Olympus BX51

compound microscope using brightfield and differential interference contrast (DIC)

optics.

Soil fertility and fatty acid analysis. Soil from each plot was sent to Waters

Agricultural Laboratories in Camille, GA for fertility analysis in both year four and five.

The Mehlich-1 extraction procedure was used to determine levels of available P, as well

as other nutrients.

Subsamples from composite soil and tomato roots were sent to John Larsen at the

Danish Institute of Agricultural Sciences, for FAME analysis in his lab. Approximately

0.2 g of tomato root tissue from each plot, a portion of fine roots from each of the four

tomato plants sampled, was stored in 2% (v/v) acetic acid, and a 10-g subsample of soil

from each plot composite was subj ected to lyophilization for preservation during

transport to Denmark. Whole cell fatty acids were extracted and methyl esters separated

using gas chromatography and subsequently identified using the automated procedure

developed by MIDI Inc. (Newark, DE).










Statistical analysis. One-way analysis of variance was used to test for differences among

treatments with regards to colonization levels. Means determined to be significantly

different (P < 0.05) were separated using Tukey's HSD test. Data were transformed

where necessary to achieve normal distribution. Pearson's correlations were used to

examine relationships between FAME results from soil and roots and colonization levels

in the IP and field plants, respectively, as well as interactions among endophytes and

between endophytes and edaphic factors. Statistical analyses were performed using SAS

release 8.02 for Windows.














CHAPTER 4
RESULTS AND DISCUSSION

Agricultural and Environmental Influences Impacting Edaphic Conditions at the
Header Canal Study Site

Over the production history of this field, including the period during this research

proj ect, multiple applications of fertilizer have resulted in extremely high P levels (Table

4-1). The relatively higher levels of this nutrient that occur in the organic treatment are

due to the annual addition of broiler litter in these plots, at the rate of approximately

22,400 kg ha l, which should result in about 800 kg of applied P20s ha- or 350 kg of

applied P ha- The increase of soil P levels by 600 kg ha-l between years four and five in

the organic plots as revealed by soil testing indicate higher P concentration in the manure,

greater relative mineralization leading up to the time of sampling, or a combination of

both of these factors. The over-application of P compared to plant need when using

broiler litter as a nutrient source is a common problem, resulting from the low N:P ratio

of the material, because fertilizer rates are often determined based on the N needs of a

crop. The plots in other treatments, during seasons of tomato production, received a

comparatively conservative 56 kg ha-l of applied P20s. As discussed in the literature

review, high levels of available P in the soil result in plant P sufficiency with

accompanying higher tissue concentrations and correlative lower levels of plant-

associated AM fungal biomass and spore production. All treatments had very high levels

of soil P, and this was exacerbated by applications that exceeded plant uptake, with the

most extreme example occurring in the organic plots.










Table 4-1. Results of soil analyses from year four and five of a 5-year cropping systems study at the Header Canal experimental
farm site in Fort Pierce, FL.
P Bulk density S Mn
Year Treatment %OM 1- H1.
(kg ha- ) (g cm') ) (kg ha- ) (kg ha )

Bahia pasture 0.75 (A 0.04) 795 (A 55) Not measured 7.14 (A 0.09) 77.90 (A 7.86) 23.15 (A 1.18)

Conventional 0.60 (A 0.04) 821 (A 51) Not measured 7.60 (A 0.16) 67.95 (A 18.35) 23.52 (A 2.09)

4 Disk fallow 0.53 (A 0.03) 819 (A 58) Not measured 7.70 (A 0.07) 44.20 (A 5.58) 25.59 (A 1.08)

Organic 1.70 (A 0.14) 1220 (A 90) Not measured 6.88 (A 0.07) 78.03 (A 11.40) 37.43 (A 2.61)

Weed fallow 0.82 (A 0.02) 884 (A 51) Not measured 7.40 (A 0.06) 58.24 (A 6.12) 26.76 (A 1.43)


39.48 (A 2.28)

11.29 (A 2.96)

40.25 (A 1.22)

76.26 (A 2.68)

47.88 (A 2.79)


0.73 (A 0.05)

0.55 (A 0.03)

0.44 (A 0.02)

2.36 (A 0.25)

0.69 (A 0.02)


794 (A 36)

849 (A 35)

899 (A 31)

1831 (A 80)

859 (A 52)


1.43 (A 0.02)

1.55 (A 0.02)

1.55 (A 0.02)

1.24 (A 0.02)

1.47 (A 0.02)


24.22 (A 7.19)

42.28 (A 9.02)

27.60 (A 4.83)

13.64 (A 10.28)

17.45 (A 8.65)


7.04 (A 0.10)

7.47 (A 0.10)

7.65 (A 0.04)

7.10 (A 0.03)

7.34 (A 0.07)


Bahia pasture

Conventional

5 Disk fallow

Organic

Weed fallow


Values are treatment means & SE.
a Bulk density was not determined in year four plots.









Organic matter levels were highest in the organic plots (Table 4-1), due to the

broadcast of urban plant debris at an approximate rate of 70,000 kg ha l, spread

concurrently with the broiler litter. The bahia pasture and weed fallow treatments both

had long-term stands of vegetation, resulting in substantial carbon input through root-

derived deposition, and therefore had higher organic matter levels than either the

conventional or disk fallow plots. As would be expected on a field with a homogeneous

soil type, the addition of ample organic matter had the corresponding effect of decreasing

soil bulk density in individual treatments, as was determined through soil testing in year

five. While bulk densities were high for a plow layer, they were not uncharacteri stically

so for sandy soils.

Additions of organic matter are known to have a number of positive effects on soil

structure. Water-stable aggregates are formed as organic matter is added from external

inputs or directly from plant and microbial deposition and decomposition. Many

physical, chemical and biological factors interact in forming soil aggregates, including a

substantial contribution derived from the activity of AM fungi (Rillig & Mummey 2006).

The benefits that organic matter lend to the development and maintenance of soil

structure were evident after two hurricanes swept over the experimental farm site in 2004.

While most treatment plots had their raised beds destroyed (Figure 4-1A), many of the

organic plots largely retained their bed structure (Figure 4-1B), despite substantial

flooding.

Soil fertility test results additionally indicated that flooding conditions persisted

long enough to have a substantial impact on nutrient availability, revealing the extent of























































Figure 4-1. Header Canal experimental farm site plots after the hurricanes of 2004. Photo
taken prior to final tomato transplantation in year five of a 5-year cropping
systems study in Fort Pierce, FL. A) Remnants of raised beds for tomato
production in conventional system. B) Raised beds still largely intact in
organic plots.









waterlogging. Manganese (Mn) is extremely soluble at the low redox potentials that

occur in soil with flooding (Tisdale et al. 1993a); this effect is evident at the farm site

when comparing extractable Mn2+ between year four, a season with normal rainfall, and

year five, a season with abnormally high rainfall. The status of sulfur (S) was also altered

in the Hield between the two seasons. The form of S absorbed by roots is SO42-, and is

easily leached from soils. Additionally, when soil moisture is high, mineralization from

organic matter and oxidation of inorganic S is decreased (Tisdale et al. 1993b). Reduced

amounts of S were extracted in treatments during year Hyve as a result of these processes.

Colonization of Roots of Tomato and Cover Crops by Arbuscular Mycorrhizal
Fungi and Other Fungal Endophytes

Arbuscular Mycorrhizal Fungi in Tomato and Cover Crop Roots

Tomato root colonization by AM fungi was very low to moderately low across

treatments, with significantly higher levels occurring in the bahia pasture plot in both

years (Table 4-2; Figure 4-2). The organic system had significantly lower levels of

colonization compared to all other treatments in year Hyve. The colonization levels

observed in year Hyve tomato roots were corroborated by FAME analysis (Figure 4-3).

There was a strong correlation between colonization levels and 16:1co5c content

measured from root samples on the plot level.

The overall pattern and levels of colonization in tomatoes among treatments did not

vary much between years (Table 4-2). There are two main distinguishing characteristics

that separate the bahia treatment, which had the highest levels of colonization, from the

others: the bahia pasture plots had neither extensive soil mixing nor raised bed formation

prior to tomato production. Instead, maintenance of a highly mycotrophic sod was

preserved throughout the 5-year experiment, except where narrow strips were tilled for










Table 4-2. Field tomato root colonization by arbuscular mycorrhizal (AM) fungi and
other fungal root endophytes from year four and five of a 5-year cropping
systems study at the Header Canal experimental farm site in Fort Pierce, FL,.


Other fungal
root
endophyte

colonization
ab


AM fungal

colonization


M. bolleyi %
colonization
acd


Year


Orientation


Treatment


Bahia pasture
Conventional
Disk fallow
Organic
Weed fallow


24.1 (A 3.5)
8.2 (A 3.4)
8.8 (A 1.8)
8.2 (A 2.3)
14.4 (A 3.5)


Was not
measured


Was not
measured


South


P values
Treatment


0.0039


Bahia pasture
Conventional
Disk fallow
Organic
Weed fallow


Bahia pasture
Conventional
Disk fallow
Organic
Weed fallow


32.6 (A 4.6)
9.2 (11.6)
9.9 (12.5)
6.7 (11.7)
12.7 (A 1.8)

30.3 (A 2.6)
15.2 (A 2.4)

4.3 (A 0.9)
13.0 (A 2.0)

<0.0001
0.4011
0.1541


52.1 (A 2.0)
39.6 (A 1.1)
47.9 (15.7)
44 (A 1.4)
37.9 (A 5.0)

58.4 (A 4.8)
47.8 (15.4)
48.8 (A 6.3)
37.5 (11.6)
36.2 (13.3)

0.0083
0.5263
0.2828


0.6 (A 0.4)

ND
12.8 (A 2.2)
ND


0.5 (A 0.4)
ND
ND
13.0 (13.1)
ND

<0.0001
0.7934
0.9856


North


South


P values
Treatment
Orientation
Interaction


aProportion data were transformed to the square root of acrsine prior to performing
ANOVA; values in table are actual mean proportions of root colonization of treatments
& SE.
b COlonization levels of non -arbuscular mycorrhizal fungal root endophytes were not
measured in year four tomato roots.
" Colonization levels of2~icrodochium bolleyi were not measured in year four roots.
dND indicates treatments in year five where M~icrodochium bolleyi was not detected.





Figure 4-2. Field tomato root colonization by arbuscular mycorrhiza (AM) fungi in years
four and five of a 5-year cropping systems study at the Header Canal
experimental farm site in Fort Pierce, FL. The treatments in the study were
bahia pasture (B), conventional (C), disk fallow (DF), organic (0), and weed
fallow (WF).


35 -

30 -
a

~ 25
CB
ff
~210 -
o
E,
1S -
o
a
Icl 10 -

5

0-


B3 C


AMfthagal caladantio d tomato roots


I yar 4

D ~aa S

















0.3 I 05




0.15




0 0 20 3 4 0 0 70 8
AMfrl es ooiaio ftmt


Fiue43 orltonwt es qae egeso fteftyacd1:c5cneti
toaorost h bevdpootono ooiaino oaorosi






Daa wrnoma lize by trnfomton to sq ar roots









tomato transplantation (Chellemi et al. 1999) (Figure 4-4). The minimal levels of

disturbance creating conditions to sustain preservation of the external hyphal network

associated with bahia roots provided transplants with a directly infective, thoroughly

distributed and effective source of inoculum.

The other treatments all had soil mixing to different degrees prior to tomato

transplantation, and, with the exception of organic plots in year five, this resulted in full

season colonization levels that were not statistically distinguishable from each other

(Table 4-2). The significantly lower levels of colonization in year five organic tomatoes

may be attributed to soil-related factors, including conditions that both enhance root

growth and reduce dependency on mycorrhizal fungi. It has already been established that

the P status in the field was well in excess of plant need; levels in the organic plots were

so high that it is surprising that AM fungi were able to consistently gain ingress.

Additionally, the lower bulk density and improved aggregate formation that develops

from organic matter additions in these plots would improve the physical environment for

root growth by reducing resistance for root extension and improving drainage after

waterlogging. Reduced relative colonization levels in these conditions may not be a

result of less inoculum or decreased infectivity of the inoculum present, but rather may be

derived from conditions that allowed roots to grow in the organic treatment at a rate that

outpaced the ability of AM fungi to develop; a faster rate than occurred in the other

treatments with regularly homogenized soil. This possibility is supported by the results

of the infection potential study that show that the levels of primary infection were not

comparatively depressed in the organic treatments (Table 4-3).






































Figure 4-4. Bahia pasture plots with rows of tomato plants. In this treatment, the only
soil disturbance after establishment of the sod occurred in years four and five
of the proj ect, when 46-cm rows were tilled for tomato transplants. Picture
taken during year five of a 5-year cropping study at the Header Canal farm
site in Fort Pierce, FL.









Table 4-3. Field soil infection potential of arbuscular mycorrhizal fungi and other fungal
root endophytes on maize from year four and five of a 5-year cropping
systems study at the Header Canal experimental farm site in Fort Pierce, FL,.


Non-AM fungal
root endophyte
% colonization"


AM fungal %
colonization


Year


Orientation


Treatment


Bahia pasture
Disk fallow
Organic
Weed fallow

Bahia pasture
Conventional
Disk fallow
Organic
Weed fallow


23.4 (11.7)
4.3 (A 0.8)
14.8 (12.3)
21.7 (A 2.5)

26.5 (A 2.3)
24.0 (A 1.5)
25.4 (A 5.7)
18.0 (A 2.0)
21.2 (A 3.5)


52.2 (A 1.2)
29.6 (A 3.8)
46.6 (A 3.3)
53.1 (A 3.8)

54.9 (A 3.3)
40.0 (A 3.1)
34.4 (A 4.5)
28.4 (A 3.0)
45.4 (A 6.5)


North


South


P values
Treatment
Orientation
Interaction


0.0040
0.0018
0.0010


0.0001
0.1668
0.0739


Bahia pasture
Conventional
Disk fallow
Organic
Weed fallow

Bahia pasture
Conventional
Disk fallow
Organic
Weed fallow

Bahia pasture
Disk fallow


16.9 (A 2.5)
8.3 (A 1.8)
8.8 (A 0.7)

8.8 (A 1.2)

18.1 (A 4.4)
9.0 (A 1.5)
9.7 (A 1.4)
22.7 (A 4.9)
7.6 (A 1.3)

13.4 (A 1.5)
3.7 (A 1.6)

< 0.0007
0.8685
0.1571


48.7 (A 2.9)
33.3 (A 6.9)
29.8 (A 3.1)
19.7 (A 2.5)
34.2 (A 3.0)

47.6 (A 4.7)
28.7 (A 6.1)
30.7 (A 3.1)
17.6 (A 1.8)
38.9 (A 3.0)

28.8 (A 2.9)
19.2 (A 1.4)

< 0.0001
0.8300
0.7773


North


South




Middle


P values
Treatment
Orientation
Interaction


aProportion data were transformed to the square root of acrsine prior to performing
ANOVA; values in table are actual mean proportions of root colonization of treatments
& SE.









Colonization levels of AM fungi were also determined for cover crop roots in the

northern half of year-four plots, the portion that remained out of production until the

following year. Mean root colonization levels were approximately 17.2 & 2.5 and 21.8 &

3.3 %, respectively, for bahia roots from the bahia pasture and ragweed roots from the

weed fallow plots. Sunn hemp was colonized to a much lesser extent, with average levels

among organic plots only reaching 3.4 & 0.6 %. Again, it is likely that a lack of soil

homogenization up to that point in the weed fallow plots probably allowed for the

development of an extensive mycelial network as in the bahia pasture treatment. This

consequently maximized the spatial distribution of AM fungal inoculum. Tillage was

performed in the organic plots to establish cover crops, however, and soil P had been

steadily building due to manure applications. Additionally, the mycorrhizal status of

sunn hemp under conditions of P sufficiency may simply be negligible.

Tomato Root Colonization by Non-Arbuscular Mycorrhizal Fungal Endophytes

The extensive occurrence of other endophytic fungi and fungal-like organisms was

noted in year-four tomato roots, and quantified in the year-five roots (Table 4-2; Figure

4-5). In addition, quantification of this group of organisms was determined through the

infection potential study using soil from treatments in both year four and five (Table 4-3).

When considered as a discrete group, the levels of infection of tomato roots in year five

by these other fungal endophytes was considerably greater than that of the AM fungi in

the same year.

The maj ority of non-AM fungal endophytes identifiable through the direct

observation of tomato and other field roots, appeared to be Phialophora spp., as

determined by the presence of distinctive, enlarged fungal cells within cortical tissue

(Figures 4-6 and 4-7). These structures are often called microsclerotia in studies where










TeaC3r 5 elhar endophlet coloi ~fat l e tomarte swai




60, B% Odrh






~ o108Id~







B C DFP 0 WFl


Figure 4-5. Field tomato root colonization by all non-arbuscular mycorrhizal fungal root
endophytes in year five of a 5-year cropping systems study at the Header
Canal experimental farm site in Fort Pierce, FL. The method used to
determine colonization levels was identical to and concurrent with
assessments for the presence of arbuscular mycorrhizal fungi. The treatments
in the study were bahia pasture (B), conventional (C), disk fallow (DF),
organic (0), and weed fallow (WF).





































Figure 4-6. Mass of enlarged fungal cells produced by Phialophora spp. within the cortex
of tomato roots; blue color is a result of staining with trypan blue.







































Figure 4-7. Mass of enlarged fungal cells and hyphae produced by Phialophora spp.
within the cortex of maize roots; blue color is a result of staining with trypan
blue.









the fungi are referred to as DSE. The hyphae associated with these structures were only

occasionally pigmented, although in individual roots the extent of naturally pigmented

hyphae could be considerable. These hyphae were often stained by procedures used to

detect AM fungi. Polymyxa spp. are endophytes classified in the order

Plasmodiophorales, and were found on several occasions in tomato roots; these

organisms were easily recognized by the clusters of resting spores formed within the

cortex (Figure 4-8). There was a diversity of other structures that were only occasionally

observed in Hield roots, although identification was not always achieved. Unidentified

structural features were often not positively related to taxa due to the lack of

distinguishing characteristics (an example being intracortical hyphae without distinctive

associated features), or the absence of confirming descriptions. Identifying taxa was not

a priority when designating colonization levels of non-AM fungal root endophytes,

although tissue had to clearly be fungal, or from fungal-like organisms, such as the

plasmodiophorids, in order to be positively designated during colonization assessments.

It is important to note that none of these observed organisms were determined to be

deleterious to the health of their host plants in any way, nor was there any obvious

antagonistic relationship among studied organisms. In the season that both groups of

fungi were quantified from field roots, their concurrence was positively correlated (r =

0.472, P < 0.0005). Similar observations were made in the infection potential study and

will be discussed below.

Microdochium bolleyi in Tomato Roots from the Organic Treatment

The distinctive, corn-cob-like structures produced by M. bolleyi within tomato

roots in this study were the stimulus for examining the occurrence of a broader range of







68


















Figre4-8 Cusersofresin sore (ystsoi)foredby olmya sp.witinth
cortex~~~~~~~~~~I ofatmt ot leclri esl fsann ihtya le










fungal endophytes than just the AM fungi (Figure 4-9 and 4-10). After clearing tomato

roots for observation, these masses were the most clearly distinguishable structures where

they occurred, irrespective of staining. These masses of fungal tissue did occasionally

absorb trypan blue and stain blue (Figure 4-11), but this did not enhance visualization

compared to naturally pigmented tissue. Tomato roots in the organic treatment were

unique in that they were the only roots in this study in which significant levels of

this organism were measured (Figure 4-5), a phenomena that occurred in both years,

although it was only measured in year five.

The diagnostic structures for this organism as it develops within the plant cortex

have been called alternatively, chlamydospores, dark cells, or microsclerotia, and are

known to preferentially occupy the inner and outer cortex (Murray & Gadd 1981; Kirk &

Deacon 1987; Sieber 2002). While these structures were obvious within roots when the

organism produced them, they appeared to be a small portion of the total biomass

produced by M. bolleyi, as extensive colonization occurred through hyaline hyphae that

did not stain with trypan blue (Figure 4-10). In fact, extension of the transparent

intracortical hyphae appeared to terminate with these pigmented structures, making their

origins difficult to discern.

The ability ofM~ bolleyi to flourish in the organic plots was not due to the tomato

plant host crop in which the organism was detected, as this was the production crop in all

systems and the same variety was used across the field. However, the use of cover crops

and organic matter applications in the organic treatment may provide the answer to its

selectively prolific nature. M~icrodochium bolleyi is a saprophytic fungus that is known to

occur widely within the roots of small grains, in which it has been reported to be a











*


.k

r


'`F

*9~2
~

60 ~m





r ~v
-



r 9



i


Figure 4-9. Pigmented cells formed by M~icrodochium bolleyi within tomato roots.






71


























Figure 4-10. Cloe-u ofpgetdclsfrmdb irdcimbol ihntmt
roos. raspaen celsandhyhaeca besee asocatd wththestuctre












12 plm





















Figure 4-11. Close-up of pigmented and trypan blue-stained cells formed by
M~icrodochium bolleyi within tomato roots.









weak parasite with a possible antagonistic interaction with pathogenic fungi and thus a

potential biocontrol agent (Murray & Gadd 1981; Kirk & Deacon 1987). The organic

treatment, with a spring crop of Japanese millet directly preceding tomato production,

was the only system with a cover crop similar to the small grains. Extensive sampling of

this cover crop did not occur, although an informal sampling found M. bolleyi to be very

common within its roots.

Infection Potential of Field Soil on Greenhouse-Grown Maize

Arbuscular Mycorrhizal Fungal Infection Potential

Whereas determining the mycorrhizal status of end-of-season roots provides a

measure of the ability of AM fungi to develop associations in the field over time, the

infection potential bioassay is used to provide an indication of the infective inoculum

available to create primary infections. In order to estimate this potential, it is crucial that

host plants are harvested in a timely manner. It has been determined previously that

secondary infection in maize roots can be minimized when plants are harvested at the

five-leaf stage of development (approximately 30 days after seed germination) (Moorman

& Reeves 1979).

The results from the study on the infection potential of AM fungi revealed

relationships between these organisms and management tactics that were not evident

from the full season tomato plants that were investigated (Table 4-3; Figures 4-12

through 4-14). Soil sampled from both halves of system plots produced generally

uniform levels of colonization on maize roots in year four, with the exception being the

northern halves of disk fallow plots which differed significantly from all other treatments

regardless of management strategy (Figure 4-12). The northern portion of the disk fallow





Figure 4-12. Infection potential of treatment soil sampled in year four of a 5-year
cropping systems study at the Header Canal experimental farm site in Fort
Pierce, FL; infection potential was determined by colonization levels of
arbuscular mycorrhizal (AM) fungi on greenhouse grown maize plant roots at
the five-leaf stage. Columns with the same letter above SE bars indicate no
significant difference (means separated at a = 0.05 using Tukey's HSD). The
treatments in the study were bahia pasture (B), conventional (C), disk fallow
(DF), organic (0), and weed fallow (WF).Treatment codes are followed by an
N or S, designating the plot half sampled (N = north, S = south). Conventional
plot soil was not sampled in the northern half in year four.


Yrear 4 AM Amapl ink~tim potenthnl

40]




ab6











BN BE CS DFN DF5 OR OS











ear 5 ~liAM Inga inisilbnp018Hld




















BN BS CN C:S DRIN F ON OS WF


Figure 4-13. Infection potential of treatment soil sampled in year five of a 5-year
cropping systems study at the Header Canal experimental farm site in Fort
Pierce, FL; infection potential was determined by colonization levels of
arbuscular mycorrhizal (AM) fungi on greenhouse grown maize plant roots at
the five-leaf stage. The treatments in the study were bahia pasture (B),
conventional (C), disk fallow (DF), organic (0), and weed fallow
(WF).Treatment codes are followed by an N or S, designating the plot half
sampled (N = north, S = south).




































Figure 4-14. Infection potential of disk fallow treatment soil sampled in year four and
fiye of a 5-year cropping systems study at the Header Canal experimental
farm site in Fort Pierce, FL; infection potential was determined by
colonization levels of arbuscular mycorrhizal (AM) fungi on greenhouse
grown maize plant roots at the Hyve-leaf stage. During this study, the southern
half of disk fallow plots produced their first tomato crop in year four and
Finished a second year of production at the end of year Hyve. Northern plot
halves only produced one tomato crop, harvested at the end of year five.
Actively disked areas were located in the northern half of plots in year four,
and in between plot halves in year five.









treatment was still actively disked at the time of sampling and had been managed

accordingly to eliminate plant growth for the four previous years. All other treatments

were statistically similar, with the exception of slightly lower levels of colonization in the

northern half of organic plots at the end of their first year of tomato production when

compared to bahia pasture and conventional system plots. This difference in organic

plots might be attributable to the impact of the high P levels in that treatment, although

the results do not carry over to the following year.

While the extremely low levels of AM colonization occurring on infection potential

hosts grown in actively disked soil is not surprising, especially considering the generally

adverse conditions due to other agricultural management practices in the field, a direct

comparison between the northern and southern halves of plots revealed an unexpected

response (Figures 4-12 and 4-13). After a single full season of host growth, such as

occurred in the southern half of disk fallow plots, the infection potential recovered to the

extent that it was indistinguishable from all other treatments. The implication is that AM

fungal inoculum density and infectivity following 14 years of intensive agricultural

management, including three consecutive years of continuous disturbance, was not

significantly impacted compared to the other farming practices investigated after host

roots were allowed to persist for several months. This indicates that inoculum recovers

from periods of intense disturbance in the absence of host tissue more rapidly than

previously suspected (Gosling et al. 2006).

The intense flooding and reduction in exposure to host roots in year five appears to

have altered treatment differences compared to the previous year (Table 4-3; Figure 4-

13). Both halves of the disk fallow plots had been put into production and at the end-of-









season sampling time they both had similar levels of colonization, albeit reduced from

colonization levels that occurred in disk fallow production plots from the year before.

However, the infection potential of that portion of plots where disking continued, in the

stretch of land between the northern and southern plot halves, colonization levels were

almost identical to actively disked soil from the year before (Figure 4-14). This suggests

a possible baseline level of viable inoculum in this Hield regardless of management, from

which recovery to more extensive levels occur after host roots prime the soil. Because

tomato roots were in the soil for 36 fewer days in year Hyve compared to year four, it is

possible to surmise the reduced recovery of infection potential in year Hyve was partially a

product of the shorter priming period.

The infection potential of all treatments was reduced in year Hyve, with the

exception of organic plots (Table 4-3). Determination of the 16: 1co5 content of field soil

using FAME analysis helped confirm the infection potential study results in year five

(Figure 4-15), and correlation at the treatment level was verified (r = 0.9001, P = 0.037).

When comparing the colonization levels of full season field tomatoes to infection

potential maize plants in the organic treatment, the discrepancy mentioned in the previous

chapter is clear; despite low levels of colonization occurring on tomato plants, the organic

plot had considerable active inoculum as evidenced by substantial primary infection on

maize in infection potential assays. The likely reasons for the low levels of field tomato

root colonization were discussed in the previous chapter, and are related to the clearest

explanations for higher infection potentials in the organic treatment. The improved soil

structure in these plots allowed for more rapid drainage from saturated conditions and

also reduced resistance to root growth when compared to the other treatments with higher




































Figure 4-15. Results from Fatty Acid Methyl Ester (FAME) analysis of soil from year
five of a 5-year cropping systems study at the Header Canal experimental
farm site in Fort Pierce, FL. Results show the levels of the arbuscular
mycorrhizal (AM) fungal biomarker 16:1co5 within treatments. Content of
treatment soil is relative to a fatty acid standard (19:0). The treatments in the
study were bahia pasture (B), conventional (C), disk fallow (DF), organic (0),
and weed fallow (WF).Treatment codes are followed by an N or S,
designating the plot half sampled (N = north, S = south).









bulk densities. This would lead to a quicker recovery of tomato plant root growth from

stressful conditions, and a generally more favorable habitat for roots in the organic plots.

Concurrent with increased relative root growth is a greater extension of an active AM

fungal mycelial soil network, and thus increased generation of inoculum. Where the pace

of root growth exceeds the ability of the AM fungi to develop and advance in the cortex,

lower overall colonization levels can be expected to occur. Additionally, extensive

development within plant tissue is more likely to be retarded in plants that have access to

abundant P, while the primary infection of newer fine roots may proceed unabated.

The bahia pasture plots had infection potential levels that were not clearly elevated

compared to other systems as occurred in field roots, although colonization levels were

significantly higher than conventional, disk fallow and weed fallow plots in year five

(Figure 4-13). The moderation of bahia pasture infection potentials compared to full

season field root colonization can be attributed to sample preparation for the greenhouse

study. Tomato transplant colonization levels in bahia pasture plots benefited from a

largely undisturbed, spatially dense mycelium in the soil; but after cores were sampled at

the end of each growing season, soil samples from each plot were bulked and thoroughly

homogenized. While colonization levels in infection potential maize plants were

decreased compared to field plants, the bahia pasture plots consistently generated levels

of inoculum that overcame the short-term disturbance with moderate levels of primary

infection.

Non-Arbuscular Mycorrhizal Fungal Root Endophyte Infection Potential

As in the field roots, no negative interaction among these groups of fungi was

detected. Instead, positive correlations occurred in year four (r = 0.441, P = 0.002) and

year five (r = 0.201, P = 0. 117) with respect to colonization levels of both groups of










endophytes within maize roots from the infection potential study. Levels of colonization

by non-AM fungal endophytes in the field were comparable to the results of the infection

potential study (Tables 4-2 and 4-3). Bahia pasture soils had the highest overall infection

potential for non-AM endophytes, while organic plots had significantly lower levels of

colonization in year five compared to other systems. In addition, there was a general

decline in the colonization levels in the infection potential study in year five compared to

the previous year, probably because of the widespread waterlogging of soils. There was

no evidence ofM~ bolleyi in maize roots, although this does not preclude the presence of

this endophyte. The maize roots were quite young at the time of harvest and M. bolleyi is

known to preferentially occupy naturally senescing root cells, generally forming the

darkly pigmented diagnostic structures where the plant forms papillae (Kirk & Deacon

1987), while hyphae are hyaline and do not stain. The morphological structures of fungal

root endophytes observed in maize were similar to those seen in tomato roots, although

extensive occurrence of the chytrid Olpidium spp. was observed. (Figure 4-16).

Making assessments on the status of such a broad group of organisms based on

their ability to initiate primary infection would be difficult and probably be of limited

value. However, it is clear from these data, in addition to that from full-season tomato

roots, that considerable root colonization by a diverse set of fungal taxa occurred in this

field and that colonization can develop quickly.





































Figure 4-16.Maize roots with colonization of a chytrid Olpidium spp. stained with trypan
blue. Fungal structures are resting spores and zoospore cysts.










Spores: Enumeration and Identification of Arbuscular Mycorrhizal Fungal
Morphotypes

Spore Enumeration of Year Five Trap Culture Soil

Sudangrass cultures were harvested and spores extracted from the root-soil

complex for each of the replicate pots that were established. Spores were then counted

and a mean derived for each replicate pot to develop an approximation of the number of

spores occurring in the rhizosphere of host plants in each treatment. Spore counts were

performed in both years of tomato production and in both halves of all plots.

Spores extracted and observed from year five soil were scarce (Figure 4-17). While

significant differences existed among treatments, with the bahia pasture and conventional

plots having more spores than the other treatments, and organic and disk fallow plots

having the fewest, spore numbers in all treatments were very low. Spore numbers may

have been slightly to moderately underestimated in both years, however, as a result of

adapted harvesting and enumeration procedures that were used to improve spore retrieval

for mounting. Also, the final step of spore extraction used vacuum filtration to drain and

wash away sucrose solution from spores. When on wet filter paper, spores were

remarkably easier to grasp and manipulate for mounting when compared to spores free

floating in water within a Petri dish. When observed associated with white filter paper,

though, juvenile and hyaline spores are much harder to see and therefore, were

undercounted

While year five sudangrass cultures were grown in undiluted field soil, year four

cultures were grown in soil diluted with very low P sand in order to stimulate spore

generation. Early in the study, it seemed questionable whether sporulation (or

colonization of plant roots in the infection potential test) would occur at all at the extreme






84





Hw sanlthomittse



14 h








0
B C DF 0 WF


Fiur 4-7 enabsua yoria uglsoenmesetatd/c3O
uniue tramn si l rmya fiveo -ercopigsse ssuya







FigursignificMantabsua difeec (mezans sepgarapr ubesetted at a=3 0.5uigTke' S)h



treatments in the study were bahia pasture (B), conventional (C), disk fallow
(DF), organic (0), and weed fallow (WF).









P contents that exist in the Header Canal field soil; therefore, the initial cultures were

diluted in half with low-P sand. Since results from trap culture spore enumerations

already have diminished value when compared to counts made directly from the field, the

spore numbers from the year four extraction using diluted soil, which further modified

the edaphic environment, are not included.

Sporulation was approximately three to nine times higher in the diluted year four soil

than in undiluted year five soil. It is difficult to ascribe the detected difference in

sporulation simply to the reduced P levels derived through soil dilution, however,

because the field conditions in year five were significantly altered by climatological

conditions. Considerable parasitism existed in spores extracted both years (Figures 4-18

and 4-19) and the extremely wet field conditions in the final year of tomato production

may have provided optimal conditions for the many organisms that prey on AM fungal

spores. Sporulation also may have been affected by phenological differences in tomato

plants that occurred at time of sampling between years, as a result of the later planting

date in year five due to the severe weather that was experienced in the end of 2004.

Spore Morphotype Identification

Identification of AM fungal spore morphotypes can be a challenging prospect in

the best of circumstances, such as when harvested in abundance from pure trap cultures

grown in sterilized soil. Differentiating among the more than 150 morphotypes described

in the INTVAM collection is a product of the ability of the investigator to capture

examples and visualize incremental variability within groups classified as genera. Even

inter-familial differences can be hard to ascertain, as crucial diagnostic structures or

developmental stages may be absent or otherwise missed from culture.




































Figure 4-18. Arbuscular mycorrhizal fungal spore with substantial parasitism evident
through dark internal swirling; no morphotypic designation could be assigned.




































Figure 4-19. Damaged arbuscular mycorrhizal fungal spore with evidence of a defense
response initiated by parasitism.









Cultures generated directly from Hield soil, as in this study, can be even more

problematic. In addition to the issues related to parasitism mentioned above,

considerable associated debris can obscure Hield soil cultures and competition among root

inhabiting fungi can cause alterations in AM fungal community spore production and

representation. Because of this, in addition to the associated sampling technique

limitations, the Hield diversity of AM fungal spore morphotypes detected in this study

must be considered as incomplete and may considerably under-represent the richness

present at the Header Canal site during the 2 years that sampling and identification took

place.

At least 11 individual morphotypes were identified as having sporulated between

the two sampling dates; it is likely that more existed, but were not captured or identified.

Joe Morton, the curator of the INVAM collection, identified two of the spore types

present, Archaeospora trappei and Glomus etunicatum, and additionally assisted with

multiple confirmations of other types. Morphotypes identified from the various

treatments are listed in Table 4-4, but the diversity present in the field or even that which

was encountered in this study is not definitively revealed. For example, the vast maj ority

of spores fell into one of the last four categories on the table, distinguished as a Glomus

spp. morphotype. In reality, since substantial variation was observed in each group, these

categories are constructs for spores that could represent considerable morphotypic

diversity. This variation, however, could not be confirmed to represent different

individual morphotypes. Additionally, it is important to understand that many of the

observed differences might be attributed to individual spore morphotypes encountered at










Table 4-4. Diversity of arbuscular mycorrhizal fungal morphotypes in year four and five
of a 5-year cropping systems study at the Header Canal experimental farm
site in Fort Pierce, FL. Morphotypic diversity was determined by extracting
spores from sudangrass trap cultures grown in field soil.


Treatments where morphotype recovered

Bahia, Conventional, Disk Fallow,
Organic
Bahia, Conventional, Disk Fallow

Bahia, Disk Fallow, Weed Fallow

Bahia


Conventional, Disk Fallow


Bahia, Weed Fallow


Bahia, Conventional, Disk Fallow,
Weed Fallow
all


Morphotypes extracted


Acaulospora spp.

Archaeospora turappei

Gigasporaceaeb

Glonaus a~nbisporunt

Glonaus clarunt

Glonaus etunicatunt

Glonaus geosporunt

Glonaus intratr adices~rtrt~t~r~rtr

Glonaus nzosseae


Glonaus sinuosunt


Glonaus spp. (orange)c

Glonaus spp. (small hyaline)c

Glonaus spp. (small pale yellow)c

Glonaus spp. (yellow)c


aHigh quality spores of these morphotypes were not acquired, and sporiferous saccules
were not retained after extraction; this grouping may therefore include multiple
morphotypes.
bFiner taxonomic designations were not possible with the spore types extracted.
cThese groupings are constructs that could be identified as Glonaus spp., but could not be
classified on a finer taxonomic level; multiple morphotypes may exist within each
category.