Effect of composted municipal waste on infection of citrus by Phytophthora nicotianae and the infection of citrus roots ...

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Effect of composted municipal waste on infection of citrus by Phytophthora nicotianae and the infection of citrus roots by Phytophthora spp
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vi, 212 leaves : ill. ; 29 cm.
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Widmer, Timothy Lee, 1964-
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Phytophthora diseases   ( lcsh )
Phytophthora nicotianae   ( lcsh )
Citrus -- Diseases and pests -- Control   ( lcsh )
Plant Pathology thesis, Ph. D   ( lcsh )
Dissertations, Academic -- Plant Pathology -- UF   ( lcsh )
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Thesis:
Thesis (Ph. D.)--University of Florida, 1996.
Bibliography:
Includes bibliographical references (leaves 189-211).
Statement of Responsibility:
by Timothy Lee Widmer.
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Typescript.
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Vita.

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University of Florida
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EFFECT OF COMPOSTED MUNICIPAL WASTE ON INFECTION OF CITRUS
BY PHYTOPHTHORA NICOTIANAE AND THE INFECTION OF CITRUS ROOTS
BY PHYTOPHTHORA SPP.
















By

TIMOTHY LEE WIDMER


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

UNIVERSITY OF FLORIDA


1996












ACKNOWLEDGEMENTS


I would first like to acknowledge the power of God, Who created this wonderful

universe and gave me the inspiration and curiosity to study it. Without God's direction

and power I would not have proceeded.

I would especially like to thank Dr. Dave Mitchell for all of his friendship and

guidance. His high ethical standards and philosophical views will always be

remembered. Without his friendship and consultation, I would not have continued at the

University of Florida. I would also like to thank Dr. Beth Kannwischer-Mitchell for her

warmth and hospitality. I also thank my cochairman, Dr. James Graham, for all of his

support, academically and personally. His initial and continued support enabled me to

gain the knowledge necessary to obtain this degree. I also thank the other members of

my supervisory committee, Dr. Don Graetz, Dr. Pete Timmer, and Dr. Jim Kimbrough,

for their assistance. Appreciation is extended to the Hunt Brother's Fellowship for their

financial support. I appreciate the assistance and patience of Patti Rayside, Diana

Drouillard, Diann Achor, and Craig Davis in the laboratory. I also thank the many other

people who helped me in my work and who let me use equipment in their laboratories.

The personal friendships of Greg and Diana Drouillard, Leandro Freitas, Erin Rosskopf,

Mario Serracin, Georgina Sydenham, and countless others will never be forgotten. It was

these friendships that made the time enjoyable. Finally I would like to thank my parents,

Dan and Edna Widmer, for all of their love and support. Without their continuous support

throughout my life, none of this would have been possible.

ii














TABLE OF CONTENTS


ACKNOWLEDGEMENTS ................... .................... ii

A B STR A C T ..................................... ............. v

CHAPTERS

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

2 LITERATURE REVIEW ................ .................... 3
History and Introduction ................... ............ 3
Importance of Phytophthora spp. on Citrus .............. .. 5
Biology of Phytophthora spp ........................ ... 7
Compost Preparation and Utilization .................... .. 9
Compost Effects on Pathosystems ..................... 14
Mechanisms of Suppression ............................ 16
Host Response to Pathogens ................ ............. 21

3 THE EFFECT OF COMPOSTED MUNICIPAL WASTE
ON INFECTION OF CITRUS SEEDLINGS AND GROWTH OF
PHYTOPHTHORA NICOTIANAE.............................. 29
Introduction ............................ ........... 29
M materials and M ethods .............................. 32
R results .............................. ............ 43
D discussion ..................... ........ ........... 62

4 THE EFFECT OF COMPOSTED MUNICIPAL WASTE AS A
SOIL AMENDMENT ON THE GROWTH OF YOUNG CITRUS
TREES AND PHYTOPHTHORA NICOTIANAE ................. 67
Introduction ............................. .......... 67
M materials and M ethods ............................. 70
Results ...................... ......... ........... 79
D discussion ............................. ........... 99

5 THE EFFECT OF COMPOSTED MUNICIPAL WASTE ON
MANAGEMENT OF PHYTOPHTHORA ROOT ROT IN
MATURE CITRUS TREES .............................. 103









Introduction ........................... ............ 103
M materials and M ethods ........................... 106
R results ............................... ............. 110
D discussion ......................................... 123

6 THE INFECTION OF CITRUS ROOTS BY PHYTOPHTHORA
NICOTIANAE AND P. PALMIVORA AT THE
ULTRASTRUCTURAL LEVEL.............................. 126
Introduction ............. .... ...................... 126
M materials and M ethods ................... ............ 130
R results .......... ........... ..... ............. 135
D discussion .......... .............. .............. 166

7 CON CLU SION .............................. ............ 171

APPENDICES

A ANALYSES OF COMPOSTED MUNICIPAL WASTE ............ 173

B EFFECT OF COMPOSTED MUNICIPAL WASTE ON
SOIL TEMPERATURE AND MOISTURE ...................... 178

C EFFECT OF COMPOSTED MUNICIPAL WASTE ON
CITRUS ROOT INFECTION BY PHYTOPHTHORA
PALMIVORA .......................................... 183

D EFFECT OF ACETIC ACID ON CITRUS ROOT INFECTION ...... 185

E EFFECT OF ACREMONIUM SP. ON CITRUS ROOT INFECTION .. 187

REFERENCE LIST .................. ........................ 189

BIOGRAPHICAL SKETCH ....................................... 212














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

EFFECT OF COMPOSTED MUNICIPAL WASTE ON INFECTION OF CITRUS
BY PHYTOPHTHORA NICOTIANAE AND THE INFECTION OF CITRUS ROOTS
BY PHYTOPHTHORA SPP.

By
Timothy Lee Widmer
December 1996

Chairperson: Dr. D.J. Mitchell
Cochairperson: Dr. J.H. Graham
Major Department: Plant Pathology

Phytophthora root rot, caused by Phytophthora nicotianae, results in a decline in

tree health and a reduction in yields in Florida. Chemical applications to control

Phytophthora root rot may not always be economically feasible and may be detrimental

to the environment. Use of composted municipal waste (CMW) to manage this disease

was examined. Citrus seedlings were grown for 3 weeks in noninfested soil or soil

infested with P. nicotianae, and nonamended or amended with 20% (v/v) CMW.

Incidence of infection was significantly reduced from 80 to 100% in infested,

nonamended controls to 0 to 35% in infested soil amended with CMW. Suppression of

infection was variable among batches and ages of CMW. An Acremonium species

isolated from suppressive CMW was antagonist towards P. nicotianae hyphae. Acetic

acid was produced in suppressive CMW and inhibited growth of P. nicotianae.









Field trials using 1-year-old Cleopatra mandarin and Sun Chu Sha rootstocks

were conducted at two different locations at two different times. Half of the trees were

infested with P. nicotianae, and CMW from two different sources was either

incorporated into the backfill or layered on top of the soil at the time of planting. The

growth rate of trees amended with CMW was significantly higher than that of

nonamended trees, even in the presence of P. nicotianae. Additional field plots of 15-

year-old Valencia orange on Carrizo citrange rootstock and 25-year-old white grapefruit

on sour orange rootstock were selected based upon high soil populations ofP. nicotianae

and poor tree health. Applications of CMW were applied under the canopies at rates of

180 or 360 tons/ha. Population densities of P. nicotianae were not reduced by treatment

with CMW, and differences in root densities among treatments were variable. Fruit

yields were not different among the treatments; however, fruit size was significantly

larger in two of the three plots amended with CMW than in nonamended plots.

Infection of susceptible and tolerant citrus varieties by P. nicotianae and P.

palmivora was compared by light and electron microscopy. No differences were

observed in the pre- and post-penetration phases between Phytophthora spp. After 24

hours the susceptible variety was colonized more extensively than the tolerant variety.

Differences in the hosts' cellular responses to the Phytophthora spp. were observed at 24

hours.














CHAPTER 1
INTRODUCTION


Crop production began around 8,000 to 10,000 years ago with maize, beans, and

cucurbits in the Americas and peas in the Near East (Harlan 1975). The most important

advantage in the agrarian societies, compared to the hunting and gathering societies, was

a more stable food base. With the invention of the plow in the Near East (3000 2500

B.C.), more land could be cultivated. By 2000 B.C. agriculture in this region was

relatively advanced; the knowledge of animal manures and nitrogen-fixing legumes, for

example, helped maintain soil fertility (Parr and Hornick, 1992). As humans moved

around the globe, they took their crops with them. Many of the crops that have been

successful were planted in regions far away from their areas of origin (Kloppenburg and

Kleimann, 1987). This movement has exposed crops to organisms that did not coevolve

with the crop and, thus, may be potentially more destructive.

Today, agriculture primarily involves the management of the ecosystem in order

to maximize the production of a certain crop. The individual crop is influenced by the

system in which the crop is a component. Thus, cropping conditions include not only the

atmospheric environment, soil, and the modifications made to this environment by the

farmer; but the preceding crop as well (Norman et al., 1995). It is difficult to effectively

manage and produce optimum yields of crops for which there is little understanding of











the ecosystem in which they are grown. Therefore, research is essential to the

understanding of a system and better crop management.

The use of organic amendments provides one method for manipulating the

environment of the crop. Not only does the amendment alter the physical properties of

the soil, but it influences the surrounding microflora. In some cases, this microflora may

protect the plant from harmful diseases (Hoitink and Fahy, 1986). The present work was

conducted to evaluate the effects of composted municipal waste applied as a soil

amendment on the growth and yields of citrus trees and on the root pathogen,

Phytophthora nicotianae. The specific objectives of the study included the development

of a greenhouse bioassay to determine the effectiveness of composted municipal waste

(CMW), as a soil amendment, to suppress Phytophthora root rot of citrus; the

examination of potential mechanisms involved in disease suppression; the evaluation of

the effect of CMW on P. nicotianae and young citrus trees transplanted under field

conditions; determination of the effect of CMW on P. nicotianae and mature citrus trees

in an established grove; and the evaluation of the host-parasite interaction of P.

nicotianae or P. palmivora and citrus root cells at the cellular level using light and

electron microscopy techniques. With this knowledge, new management strategies for

potential disease control and growth enhancement that are less harmful to the

environment may be employed.














CHAPTER 2
LITERATURE REVIEW


History and Taxonomy

Ever since Anton de Bary first recognized and described the genus Phytophthora,

considerable research has been devoted to determining the biology of this plant pathogen

(Brasier and Hansen, 1992; Erwin et al., 1983; Mitchell and Kannwischer-Mitchell,

1992; Ribeiro, 1978; and Stamps et al., 1990). In Latin the word Phytophthora means

"plant destroyer." Anton de Bary first identified this organism in 1876 as the causal

agent of potato blight (de Bary, 1876). He characterized the type specimen, P. infestans

(Mont.) de Bary, as having branched sporangiophores, sporangia which were shed,

zoospores that formed within the sporangium, and sporangia germinating by zoospores or

by a tube (de Bary, 1887). Rosenbaum (1917) constructed the first key to the species of

the genus. Between this time and 1960, several monographs and keys were produced

(Frezzi, 1950; Leonian, 1934; Schwinn, 1959; Tucker, 1931), but they varied in

characteristics deemed important for identification. In 1963, Waterhouse (1963) devised

a key that was accepted worldwide. From this key, 43 taxa were recognized. The genus

was divided into six groups, based upon different characteristics, to aid in the

identification (Waterhouse et al., 1983). This key is still used today, and a revised

tabular key recognizes 51 species (Stamps et al., 1990).











The genus Phytophthora was placed most commonly in the family Pythiaceae,

order Peronosporales, and class Oomycetes in the kingdom Fungi or Myceteae

(Alexopoulos and Mims, 1979). However, there are many characteristics of the genus

Phytophthora and other "water molds" which do not fit with the "true" fungi. These

characteristics include cellulose in the hyphal wall; lysine synthesis by the

diaminopimelic acid pathway instead of the a--amino adipic acid pathway; tubular cristae

instead of flattened cristae in the mitochondria; and motile zoospores with an anterior,

tinsel-type cilium and, if present, a single, posterior, whiplash cilium (Hawksworth et al.,

1995; D.J. Mitchell, unpublished).

In 1981, Cavalier-Smith (1981) proposed that the Eukaryotes be divided into six

kingdoms. This was later updated to include eight kingdoms (Cavalier-Smith, 1989a). In

this classification system, Phytophthora spp. are placed in the kingdom Chromista

(Cavalier-Smith, 1986, 1989b) along with other Oomycetes, Hyphochytriomycetes,

Labyrinthulea, and other organisms formerly in the kingdoms Plantae and Protista, such

as brown algae and some diatoms (Barr, 1992; Leadbetter, 1989; Patterson, 1989).

Organisms in this kingdom have at least one of two, unique, highly conserved characters:

i) rigid, tripartite, tubular mastigonemes (retronemes) on the cilia of zoospores; or ii)

chloroplasts, when present, inside the rough endoplasmic reticulum (Cavalier-Smith,

1986, 1989b; D.J. Mitchell, unpublished).

Phytophthora spp. have been the causal agent of many devastating epidemics.

The great Irish potato famine in 1845, caused by P. infestans, resulted in the loss of two

million people from Ireland, either by death or emigration (Klinkowski, 1970). The











destruction of the jarrah forest in Western Australia, caused by P. cinnamomi Rands,

devastated complex forest woodland communities on more than 100,000 hectares in

Western Australia (Newhook and Podger, 1972). Species of Phytophthora attack over

2000 plant species worldwide, including Citrus spp. and other members of the Rutaceae

family, (Timmer and Menge, 1988).

Importance ofPhytophthora spp. on Citrus

Citrus is one of the most economically important crops in Florida, with revenues

exceeding one billion dollars each year. Most of the 273,000 hectares of citrus in Florida

are planted on sandy soils low in organic matter and natural fertility. These soils have a

low exchange capacity and retain only small amounts of applied plant nutrients against

the leaching action of rainfall and irrigation (Tucker et al., 1995). Even under these poor

soil conditions, citrus is still able to produce acceptable yields. However, the climate in

Florida encourages pest problems greater than those in many other citrus producing

areas, and production may be limited (Jackson, 1991).

Diseases of citrus are probably the most important limiting factor in production.

A disease is defined, according to Bateman (1978), as an:

... injurious alteration of one or more ordered processes of energy
utilization in a living system, caused by the continued irritation of a
primary causal factor or factors (p. 59).

More than 100 biotic and abiotic factors cause diseases of citrus trees (Whiteside et al.,

1988). The most important biotic factors include nematodes, bacteria, viruses, and fungi.

Fungal diseases can cause severe damage to young and mature citrus trees. Diseases

caused by soilborne fungi, such as Phytophthora spp. result in root rot, foot rot, brown











rot of fruit, reduced fruit quality and yield, and under optimum conditions trees may be

killed (Timmer et al., 1989).

The most common and important Phytophthora spp. that attack citrus are P.

nicotianae Breda de Haan (synonym = P. parasitica Dastur (Hall, 1993)) and P.

citrophthora (R.E. Sm. & E.H. Sm.) Leonian. Other species that have been reported

from citrus in limited geographical areas include P. hibernalis Came and P. syringae

Kleb. in areas with cool, moist winters; and P. palmivora (Butler) Butler and P. citricola

Saw. in tropical areas (Timmer and Menge, 1988). Phytophthora palmivora has been

isolated from citrus in Puerto Rico and more recently in Florida, where it was found to be

more pathogenic than P. nicotianae (Zitko and Timmer, 1994). In California, P.

megasperma Drechs. and P. cinnamomi reportedly have been isolated from citrus (Farr et

al., 1989), but these species are not commonly recognized as significant citrus pathogens.

Phytophthora citrophthora causes gummosis, root rot and brown rot of fruit. This

normally occurs where seasonal rainfall occurs during the cooler winter months

(Whiteside, 1970). This pathogen is generally controlled by fungicides. In addition,

cultural practices, such as pruning low-hanging branches and mowing or disking the

cover crop, will be helpful in disease control by permitting better circulation and

lowering humidity (Jackson, 1991).

Optimum temperature for P. nicotianae development is 30-32 C. In subtropical

climates, such as in south Florida, seasonal fluctuations in the population density are not

consistent, although an overwinter decline does occur (Duncan et al., 1993).

Phytophthora nicotianae is widespread in most citrus areas and causes foot rot,











gummosis, and root rot (Graham, 1990). Phytophthorapalmivora causes similar disease

symptoms but is more restricted in distribution (Zitko and Timmer, 1994). Fibrous root

rot is a common problem in citrus nurseries (Sandier et al., 1989), and about 90% of the

field nurseries assayed in Florida are infested with P. nicotianae (Fisher, 1993).

Foot rot is usually controlled by budding the susceptible scion cultivars on

resistant rootstocks and keeping the bud union dry above the soil line (Grimm and

Timmer, 1981). Commonly used rootstocks, such as sour orange (Citrus aurantium L.),

trifoliate orange (Poncirus trifoliata [L.] Raf.), Troyer and Carrizo citranges (C sinensis

[L.] Osbeck X P. trifoliata [L.] Raf), and Swingle citrumelo (P. trifoliata [L.] Raf. X C.

paradise Macf), range from tolerant to nearly immune. When over watered in infested

nurseries, even these rootstocks will suffer serious root rot damage (Timmer et al., 1989).

Applications of metalaxyl and fosetyl-Al fungicides have proven highly effective for

control of fibrous root rot problems in nurseries (Davis, 1982; Farih et al., 1981).

However, it may not be economically beneficial to apply fungicides if populations of

Phytophthora spp. are less than 10-15 propagules per cubic centimeter of soil (Sandier et

al., 1989). Also, isolates ofP. nicotianae which are resistant to metalaxyl have been

found in some citrus groves (Fisher, 1993). Other alternatives to control Phytophthora

root rot, such as the addition of composted municipal waste as a soil amendment, need to

be examined.

Biology of Phytophthora spp.

Species of Phytophthora that attack citrus are highly evolved root parasites, but

are not effective saprophytes. Phytophthora nicotianae does not live freely in the soil











(Tsao, 1969). The fungus must obtain its nutrients from living plant tissue (Lutz and

Menge, 1986). Phytophthora nicotianae survives in soil or root debris as

chlamydospores and oospores (Tsao, 1969).

Chlamydospores are produced when temperatures are cool and soils are poorly

aerated (Tsao, 1971). Production is stimulated when carbon dioxide levels increase

(Ioannou and Grogan, 1985). Chlamydospores can survive in moist, cool soil for several

months (Lutz and Menge, 1986; Malajczuk, 1983). Under favorable environmental

conditions of good aeration and low carbon dioxide levels, and in the presence of

nutrients from root exudates, chlamydospores will germinate and produce mycelium

(Mircetich and Zentmeyer, 1970).

Oospores are usually produced in lower numbers than chlamydospores, have thick

walls, and are resistant to drying and cold temperatures (Lutz and Menge, 1986). They

require a longer time to mature (Ribeiro, 1983) and can remain dormant for extended

periods (Malajczuk, 1983). Oospores form when two P. nicotianae isolates of opposite

mating types are paired. Both mating types are present in Florida nurseries, but the role

of oospores in the disease cycle is unclear (Zitko et al., 1987). In California citrus soils,

oospores have been observed throughout the year (Lutz and Menge, 1991). Oospore

production also can be stimulated in the absence of both mating types by other soil

microorganisms and factors (Brasier, 1971; Mukerjee and Roy, 1962; Shen et al., 1983).

Sporangia are the primary reproductive structures and form best under normal

atmospheric concentrations of oxygen and carbon dioxide (Mitchell and Zentmeyer,

1971). Well-aerated, moist conditions are optimal for both production and germination











(Sommers et al., 1970). Germinated propagules quickly form sporangia, which may

either germinate and form mycelium or release motile zoospores in saturated soils

(MacDonald and Duniway, 1978). Each sporangium releases from 5-40 zoospores,

which can swim or be carried by moving water to roots. Zoospores are attracted to root

exudates, particularly amino acids, sugars, and other organic acids, which are excreted

from wounds or the zone of root elongation (Morris and Ward, 1992; Schwab et al.,

1984). Amino acids, at high concentrations which occur near the root, induce the

zoospores to encyst (Khew and Zentmeyer, 1973). The movement of zoospores over

long distances between trees is due to free water movement from rainfall or irrigation.

Chemotaxis results in efficient dissemination over short distances between roots.

During the growing season many generations of sporangia are produced.

Formation is correlated with temporary soil saturation due to irrigation or rain. Under

flooded conditions sporangia formed on germ tubes produce zoospores that cause new

infections (Lutz and Menge, 1986). Epidemics caused by Phytophthora spp. have been

shown to be polycyclic, and disease can increase at explosive rates (MacKenzie et al.,

1983).

Compost Preparation and Utilization

In the United States, society is annually generating, on a dry weight basis, 7.7

million metric tons of sewage sludge and 165 million metric tons of garbage (Parr and

Hornick, 1992). In Florida, the amount of solid waste produced was about 18.5 million

metric tons in 1992 (DEP, 1993), which is over 4 kilograms per resident per day. In the

past, disposal of this waste has been through incineration, ocean dumping, and land











filling, with only 10% being recycled (USEPA, 1989a). Biodegradable organic that

could be composted comprise almost 60% of the total municipal solid waste (MSW) or

about 10.2 million metric tons annually (Smith, 1994). Since the amount of waste is

predicted to rise, economical and environmentally safe waste disposal alternatives need

to be examined. The U.S. Environmental Protection Agency has listed composting as an

acceptable practice to ensure the safe and beneficial use of sludge on land (USEPA,

1989b). The U.S. House of Representatives investigated whether cocomposting, which is

based on combining certain waste materials such as sewage sludge and waste paper or

yard wastes, is a viable option for alleviating the waste problem (U.S. House of

Representatives, 1990).

Composting is defined as "the biological decomposition of organic constituents in

wastes under controlled conditions" (Hoitink and Fahy, 1986). The control of

environmental conditions distinguishes the process from natural rotting or putrefaction,

which occurs in open dumps, manure heaps, or field soil. The main products of aerobic

composting are carbon dioxide, water, heat, mineral ions, and stabilized organic matter,

often called humus (Inbar et al., 1993). The process can be divided into three phases

(Hoitink and Fahy, 1986). The initial phase, during which temperatures rise to 40-50 C.,

lasts approximately 1-2 days. Sugars and other readily degradable compounds are

decomposed during this phase. The thermophilic phase can last for months. Microbial

decay of organic matter results in considerable heat production, with temperatures

reaching 40-60* C. In this phase, cellulose and most other complex substrates are

degraded, while lignins break down more slowly. During this phase the high









11
temperatures kill plant pathogens, weed seeds, and most biocontrol agents. Bacillus spp.,

which have been examined as potential biocontrol agents (Baker et al., 1985; Broadbent

et al., 1971; Handelsman et al., 1990; Kommedahl et al., 1975), survive due to the

formation of highly resistant spores that are killed only at higher temperatures.

Temperature is an important factor in composting, and the process is generally controlled

by manipulation of air flow and addition of water (Hoitink and Kuter, 1986). The

composting system can be in windows which require turning to enhance natural airflow

and ensure that all sections of a window reach high temperatures, or it can be a system

with forced aeration. Without complete aeration, the compost pile can "sour" from

anaerobic metabolism, which results in the production of methane, carbon dioxide, and

low molecular weight organic acids and alcohols. Temperatures decline as the compost

reaches the curing phase, decomposition rates decrease and mesophilic microorganisms

recolonize the compost. The mature compost is composed of humic materials, lignins

and other biomass materials.

For agricultural uses, compost has to be transformed to a humus-like product that

is sufficiently stable when the composting is complete (Inbar et al., 1993); otherwise,

negative plant responses, caused by root injury, can occur (Cook and Baker, 1989;

Hoitink and Fahy, 1986). When immature compost was incorporated into a tomato field,

plant growth was inhibited (Obreza, 1995). When stable, mature compost was added,

there was an increase in extra-large tomato fruit sizes and watermelon yields. Other

studies also showed an increase in tomato and squash yields when mature compost was

incorporated into planting soil (Bryan et al., 1995).









12
Although it is impractical to incorporate compost into the soil of established citrus

trees without substantially damaging the root system, a mulch layer may be beneficial.

Mulching is defined as any covering placed over the soil surface to modify soil physical

properties, create favorable environments for root development and nutrient uptake, and

reduce soil erosion and degradation (Thurston, 1992). Mulches can include materials

such as manure, sludge, sawdust, woodchips, bark, straw, shredded prunings, plant

foliage, paper, plastic, sand, and gravel.

Mulches are beneficial in many ways. Mulching conserves water use by reducing

evaporation from the soil, increasing the permeability of the soil surface, and increasing

the water holding capacity of the soil (Bengtson and Cornette, 1973; Gregoriou and

Rajkumar, 1984; Stephenson and Schuster, 1945). For quality citrus production it is

necessary to have a high water infiltration rate which supplies water to the plant and

removes salts from the soil (Jones et al., 1961). Organic matter increases the number of

macropores (Pagliai et al., 1981), and, thus, allows better water movement and aeration.

The soil structure is improved by the addition of mulches and organic matter

(Gallardo-Laro and Nogales, 1987). Clay particles aggregate into larger granules when

organic mulches are added (Stephenson and Schuster, 1945). As organic matter

decomposes, compounds are formed that cement soil particles together into stable

aggregates (Buckman and Brady, 1960). This permits better movement of carbon

dioxide and oxygen into and out of the soil.

Mulching may reduce or eliminate ground water nitrate contamination. Nitrogen

from fertilizer may have a large impact on the deterioration of groundwater (Embleton et









13

al., 1978), especially in Florida where high annual rainfall, sandy soils, and shallow water

tables result in a high risk for groundwater contamination (Calvert and Phung, 1972).

There have been many studies on ground water contamination due to fertilization in

citrus groves (Calvert and Phung, 1972; Dasberg, 1978; Embleton et al., 1978; Hubbard

and Sheridan, 1989; Lea-Cox and Syvertsten, 1992; Willis et al., 1990). Mulch provides

a continuous slow release of nitrogen, and therefore, reduces the amount of chemical

fertilizer that needs to be applied (Maynard, 1989; Stephenson and Schuster, 1945). In a

study involving mulched apple plots (Weeks et al., 1950), as an example, the treated plots

maintained a reserve of nitrogen 9 years after the last application of mulch.

Mulching can reduce wide fluctuations in soil temperature (Gregoriou and

Rajkumar, 1984). This can improve root growth, especially in young trees, where

summer temperatures can be very high. As an example, the mean dry weights of citrus

roots maintained at 350 C were reduced in comparison to those at 280 C (Reuther, 1973).

In the early days of citrus production, mulching was a common practice. There

were many reports that the use of mulches on citrus improved yields and soil conditions

(Craig, 1916; Hodgson, 1925; Lefferts, 1919; McNees, 1916). Before the late 1940s, it

was recommended that the source for half of the nitrogen applied to citrus groves come

from bulky organic (Hinkley, 1941). However, in the 1940s, chemical fertilizers that

were cheap and easy to apply became available and replaced organic materials. Also,

organic materials became scarce and more expensive due to diversion to other uses

(Camp, 1951). From 1936-1941 the average application of fertilizer was 260 kilograms

per hectare (230 pounds per acre). During 1946-1951 the average application was 450










kilograms per hectare (400 pounds per acre) (Florida Citrus Mutual, 1957). In 1993,

fertilizer rates were still approximately 450 kilograms per hectare (400 pounds per acre)

(Florida Agricultural Statistics Service, 1994).

Along with chemical fertilizer applications, chemical pesticides were also applied

at high rates to minimize disease losses. In 1993, 685 kilograms per hectare (610 pounds

per acre) of pesticides were applied on Florida citrus groves (Florida Agricultural

Statistics Service, 1994). As a more comprehensive understanding of pest biology is

acquired, alternative methods and strategies for disease management can be applied.

Compost Effects on Pathosystems

Whether compost is incorporated as a soil amendment or applied as a mulch, it

sometimes has been shown to suppress plant diseases. Chinese agriculture has

implemented the use of composts in farming for thousands of years (Cook and Baker,

1983; Kelman and Cook, 1977). It is estimated that half of the nutrients applied to crops

in China are from organic sources (Thurston, 1992). Other ancient societies also used

organic amendments for optimum crop yields. In Mexico, large quantities of organic

material were used in the chinampas (Thurston, 1992). These soils showed suppressive

behavior to damping-off, caused by Pythium spp. (Lumsden et al., 1987). However,

with the introduction of cheap chemicals, and, during the 1960's and 1970's when

agriculture became intensified, interest in compost declined. Recently, the discovery of

disease suppression by certain bark composts has increased interest in using compost for

disease management (Hoitink and Fahy, 1986).











Composts and other organic soil amendments have been shown to suppress

certain soilborne diseases caused by fungi, including those caused by Rhizoctonia solani

Kuhn, Pythium ultimum Trow, Fusarium oxysporum Schlechtend. :Fr f. sp. conglutinans

(Wollenweb.) Snyder & Hans. and Phytophthora spp. (Borst, 1983; Broadbent and

Baker, 1974b; Chen et al., 1987; Nelson and Hoitink, 1982; Trillas-Gay et al., 1986);

bacteria (Chellemi et al., 1992; Hartman and Yang, 1990; Sun and Huang, 1985); and

nematodes (Gallardo-Lara and Nogales, 1987; Hunt et al., 1973; Malek and Gartner,

1975). Compost extracts also have been shown to suppress some foliar diseases (Hoitink

and Grebus, 1994; Weltzien, 1989; Weltzien, 1991). Water extracts of composts

suppressed downy mildew, caused by Plasmopara viticola (Berk. & M.A. Curtis ex de

Bary) Berl. & de Toni, and powdery mildew, caused by Uncinula necator (Schwein.)

Burr, on grape (Vitis vinifera L.); late blight of potato (Solanum tuberosum L. cv.

'Grata') and tomatoes (Lycopersicon esculentum Miller cv. 'Rheinglut'), caused by

Phytophthora infestans; powdery mildew of barley (Hordeum vulgare L. emend. Bowden

cv. 'Gerbel'), caused by Blumeria graminis (DC) Speer f. sp. hordei Em. Marchal; and

white mold, caused by Botrytis cinerea Pers., on beans (Phaseolus spp.) and strawberries

(Fragaria X ananassa Duchesne cv. 'Corona').

Floricultural crops grown in nurseries are prime candidates for the application of

composts to suppress soilborne diseases. Certain composts have proven effective in

suppressing soilborne diseases caused by Fusarium spp., Phytophthora spp., Pythium

spp., and Rhizoctonia solani on cyclamen, azaleas, poinsettias, and other ornamentals











(Boehm and Hoitink, 1992; Daft et al., 1979; Hardy and Sivasithamparam, 1991b;

Hoitink et al., 1991; Ownley and Benson, 1992).

Utilization of composts and amendments to suppress diseases in field crops has

been investigated with mixed results. As mentioned before, P. cinnamomi was

suppressed in field soils in Australian avocado groves by the application of mulches in

combination with gypsum (Broadbent and Baker, 1974b). Compost added to nematode-

infested citrus groves improved yield and fruit size (Tarjan, 1977). In Taiwan, Fusarium

wilt of watermelon, caused by Fusarium oxysporum f sp. niveum (E.F. Sm.) Snyder &

Hans., was reduced by 61% with the addition of organic amendments (Sun and Huang,

1985). Amendments in other field trials by the same authors in Taiwan reduced the

incidence of disease in radish, mustard cabbage, Chinese cabbage, cucumber, pepper,

bean, rice, and tomato caused by F. oxysporum f. sp. raphani Kendrick & Snyder, F.

oxysporum f. sp. conglutinans, Plasmodiophora brassicae Woron., Phytophthora melonis

Katsura, Sclerotium rolfsii Sacc., Rhizoctonia solani, and Pseudomonas solanacearum,

respectively. Site selection may play an important part in the effectiveness of mulches

for control. In Costa Rica, web blight of bean, caused by Thanatephorus cucumeris

(Frank) Donk (anamorph = Rhizoctonia solani), was effectively managed with mulches

(Galindo et al., 1983); however, in Colombia with cooler temperatures at higher

elevations, mulching was of no value (Thurston, 1992).

Mechanisms of Suppression

The exact mechanisms involved in suppression of plant diseases with composts

are unclear. However, the chemical and physical properties of compost and the biology











of microorganisms colonizing it may affect suppression of fungal plant pathogens or the

diseases caused by them. Physical properties of the soil, such as texture, structure,

porosity, and consistency, may be altered by the addition of compost. These properties

influence rooting depth, aeration, water movement, and chemical and biological activities

(Lyda, 1982). As an example, Phytophthora root rots are more prevalent in media with a

lower air capacity, typically caused by smaller pore space, than in media with greater air

capacity, such as those amended with tree barks (Hoitink and Kuter, 1986).

There is evidence that suppression can be attributed to microorganisms.

Broadbent and Baker (1975) found that incorporation of green plant material into the soil

reduced disease caused by P. cinnamomi on avocado in Australia. This suppression was

destroyed by aerated steam for 30 minutes at 1000 C, but not at 600 C. They attributed

the suppression to spore-forming microorganisms, such as Bacillus spp., that tolerated

600 C rather than to nonsporulating bacteria or actinomycetes. Other pathosystem models

indicate that different microorganisms are involved in suppression (Rovira, 1982).

Potential biocontrol agents that recolonize composts after peak heating include Bacillus

spp., Enterobacter spp., Flavobacterium balustinum Harrison, Pseudomonas spp.,

Streptomyces spp., Trichoderma spp., and Gliocladium virens Miller, Giddens, & Foster

(Chung and Hoitink, 1990; Hardy and Sivasithamparam, 1991 la; Hoitink and Fahy, 1986).

The suppressive mechanisms involved may affect growth of the pathogen or the

production of reproductive structures involved in survival. (Hoitink et al., 1977; Spencer

and Benson, 1982). Composts also have been shown to stimulate growth of certain











microorganisms which colonize roots and induce protection in the leaves of plants to

foliar pathogens (Hoitink and Grebus, 1994; Maurhofer et al., 1994; Wei et al., 1991).

The earliest report on the suppression of a disease caused by a Phytophthora sp.,

with composted amendments, was with tree bark applied to strawberry plants (Vaughn et

al., 1954). Incorporation of composted Douglas fir bark into soil controlled strawberry

red stele disease caused by P. fragariae Hickman for the first 2 years after application.

In Australia, soils suppressive to P. cinnamomi have been maintained for decades by

developing a soil mulching system for avocado (Broadbent and Baker, 1974b). Plant

production systems in nurseries may utilize composts to control Phytophthora root rot in

azaleas caused by P. cinnamomi (Ownley and Benson, 1992). Diseases of a wide range

of other container-grown plants caused by five different species of Phytophthora also

may be suppressed by compost applications (Hardy and Sivasithamparam, 1991b).

Phytophthora nicotianae, like many other Phytophthora species, is a soilborne

organism that completes most of its life cycle in the soil or the roots of its host. This

environment, under natural conditions, is full of a wide range of other microorganisms.

Some of these microorganisms are potential antagonists to Phytophthora spp. The

addition of composts, as soil amendments, favors an increase in the antagonistic soil

microflora (Cook and Baker, 1989; Hunt et al., 1973; Nesbitt et al., 1979; Rothwell and

Hortenstine, 1969).

Every part in the life cycle of Phytophthora spp. is vulnerable to antagonists

(Malajczuk, 1983). Mycelium of P. nicotianae, in untreated soil, has a relatively short

survival period of less than 7 days (Tsao, 1969). Lysis of mycelium occurs rapidly in











natural soils (Hine and Trujillo, 1966). A considerable amount of work has been

conducted on hyphal lysis and parasitism (Brasier, 1975; Dennis and Webster, 1971;

Durrell, 1968; El-Goorani et al., 1976; Kelley and Rodriguez-Kabana, 1976; Lacey,

1965; Reeves, 1975; Sneh et al., 1977; Vaartaja et al., 1979). Usually antagonistic fungi

contact and coil around the host hyphae before penetration and the host mycelium is

cleared of cytoplasm (Malajczuk, 1983).

The large populations of bacteria that inhabit soil under natural conditions (Paul

and Clark, 1989) can have a significant impact on Phytophthora spp. Nesbitt et al.

(1979) demonstrated that bacterial populations increased as organic matter was increased.

This has been correlated positively with hyphal lysis. A light and electron microscope

study of P. cinnamomi hyphae in untreated soil showed an accumulation of a wide range

of morphologically distinct types of bacteria near fungal hyphae (Malajczuk et al., 1977).

These bacteria appear to be attracted to hyphae by a chemotactic response (Nesbitt et al.,

1981 a), perhaps to phenylalanine and glucose (Nesbitt et al., 198 1b) or other compounds

associated with root extracts (Morris and Ward, 1992). Isolates of Pseudomonas

fluorescens Migula and P. putida (Trev.) Migula were shown to colonize fungal hyphae

and inhibit P. cinnamomi on agar media (Yang et al., 1994). However, no antibiotics

were associated with this inhibition.

Some bacteria may produce antifungal compounds that can inhibit the growth of

Phytophthora spp. Growth of P. cactorum (Leb. & Cohn) Schr6t., the causal agent of

crown rot of apple trees, was inhibited by a bacterial isolate (Utkhede and Gaunce, 1983).

Autoclaved bacterial extract also completely inhibited the growth. Sterile filtrates from a









20

culture of Bacillus cereus Frank and Frank reduced mortality of alfalfa (Medicago sativa

L. cv. 'Iroquois') seedlings caused by P. megasperma Drechsl. f sp. medicaginis Kuan &

Erwin (Handlesman et al., 1990). Planting seeds coated with the antagonist, B. cereus,

significantly increased the emergence of alfalfa in soil infested with P. megasperma f. sp.

medicaginis in a small-scale field trial. However, the bacterial isolate did not inhibit

growth of P. megasperma f. sp. medicaginis on agar plates. Bacterial isolates collected

from citrus rhizosphere soil inhibited growth of P. nicotianae on agar plates (Tumey et

al., 1992).

Sporangia are also subject to lysis and parasitism. Bacteria, identified as Bacillus

subtilis Cohn emend. Praz., have been associated with the breakdown of sporangia

(Broadbent and Baker, 1974a). The bacteria are chemotactically attracted to the

sporangium and attach themselves to the sporangial wall. Electron micrographs show

that the outer, thin, electron-dense layer of the sporangial wall disappears in the vicinity

of each bacterium. This is followed by withdrawal of the sporangial cytoplasm from the

sporangial wall (Broadbent and Baker, 1974a). Chytrids also have been observed

parasitizing P. cinnamomi sporangia (Malajczuk, 1983), but this is rarely observed in

natural soil.

Possibly due to their thick walls, chlamydospores and oospores are more resistant

to bacterial antagonists (Malajczuk, 1983). Although colonization of some

chlamydospores of P. cinnamomi by bacteria was observed, their viability was

unaffected. No bacterium has been isolated that is capable of secreting extracellular

enzymes which break down fungal cell walls of Phytophthora spp. Sneh et al. (1977)









21

observed oospores of P. megasperma Drechsl. var. sojae Hild. and P. cactorum that had

been parasitized by oomycetes, chytridiomycetes, hyphomycetes, actinomycetes, and

bacteria. Holes observed in the cell walls of oospores (Old and Darbyshire, 1978) and

chlamydospores (Old and Oros, 1980) are characteristic of spore destruction by

mycophagous amoebae. Amoebae appear to have a nonspecific effect on a wide range of

fungal spores, but, although they are numerous in soils, their specific role in disease

suppression is unknown (Malajczuk, 1983).

Phytophthora root rot can be a very difficult disease to manage in the field.

Additions of organic amendments or mulches increase the microbial activity of the soil,

which then may have antagonistic activity against Phytophthora spp. and may reduce the

effect that the pathogen has on the citrus trees. Amendments or mulches also could have

a positive effect on the growth of the tree physiologically. In addition, the garbage that

society is generating may be used as a resource rather than accumulating in landfills.

Host Response to Pathogens

An understanding of the pathogenicity of a parasite is important in finding ways

to manage diseases caused by it. Depending upon the pathosystem, the host responds by

a resistant, tolerant, or susceptible reaction. A plant can also be immune to a pathogen,

where even under the most favorable conditions it is not attacked. At the macro-level,

visible symptoms of the reaction may be obvious, but this does not really explain what is

happening. Observations at the cellular level of the host-parasite interface allow insight

into the host's response to invasion.











Disease resistance may be defined as the ability of a plant to inhibit the pathogen,

to any degree, at one or more stages during the disease cycle (Hooker, 1967). Resistance

is generally believed to be controlled by genetics and has been classified into four broad

mechanistic classes (Michelmore, 1995). The first category involves resistance genes

encoding components of receptor systems that detect the presence of the pathogen, which

initiates a signal transduction pathway. The gene-for-gene interactions are believed to

belong in this class. The mechanisms responsible for induction of resistance are not

known. Lysis and growth inhibition of several plant pathogenetic fungi in vitro by

chitinase and P-1,3 glucanase suggest that induced proteins are also capable of acting

directly on the invading pathogen in vivo (Mauch et al., 1988; Schlumbaum et al., 1986).

However, there is no direct evidence in vivo of the involvement of these enzymes in

resistance of plants to fungi. Phytophthora cactorum was shown to be insensitive in vitro

to a mixture of chitinase and P-1,3 glucanase (Mauch et al., 1988). Woloshuk et al.

(1991) purified similar proteins from tobacco and tomato plants induced by tobacco

mosaic virus and P. infestans, respectively. These proteins caused lysing of P. infestans

sporangia, at a concentration of 40 nM, and hyphal growth inhibition at a concentration

of 400 nM.

The other classes of resistance genes are not involved in signal transduction. A

second type of resistance genes encodes products that detoxify and deactivate compounds

that the pathogen requires to cause disease (Michelmore, 1995). An example of this is

the gene Hml from corn, which encodes a reductase that deactivates HC-toxin of

Cochliobolus carbonum Nelson. The third type of resistance genes encodes altered











targets for pathogen-derived molecules required for pathogenicity. The fourth type of

resistance genes encodes structural or constitutive biochemical barriers to the pathogen.

Physical barriers and chemical defenses that are natural components of the host

may also be involved in resistance. The defensive barriers help prevent potential

pathogens from initiating infection or prevent further spread. Components of the

protective coverings of plant parts may prevent penetration because of thickness,

hardness, hydrophobicity, or resistance to enzymatic attack (Campbell et al., 1980). If

the pathogen is able to penetrate the surface of the host, there are preformed, internal

physical defenses, such as suberized endodermis, lignified tissues, cellulosic walls, and

other components (Akai and Fukutomi, 1980). There are also internal chemical defenses

in the host which are expressed in host tissue before infection and do not rise to higher

levels in response to invading microorganisms (Schlosser, 1980). These chemicals may

be enzyme inhibitors, hydrolytic enzymes, or antifungal compounds.

Resistance may be expressed as a hypersensitive reaction; the death of only a few

host cells, usually near the point of pathogen invasion, limits the progression of the

infection (Goodman and Novacky, 1994). In the redox theory of hypersensitivity, the

necrotic response is the result of a disturbance of the balance between oxidative and

reductive processes that results in an excess of polyphenol compounds and a breakdown

of cellular and subcellular structures (Kiraly, 1980). The initial mechanism which

triggers the secondary biochemical event is not understood. This lack of understanding is

also true, despite many studies, for the hypersensitivity to infection by Phytophthora spp.

There have been extensive studies examining the reaction of potato tissue to P. infestans











(Doke et al., 1980; Keenan et al., 1985; Kiraly et al., 1972; Ricker and Bostock, 1994).

Kiraly et al. (1972) showed that P. infestans mycelium that had been killed still induced

necrosis in the potato tuber cell. Their work showed that in normal incompatible systems

the pathogen is first killed by an unknown mechanism, with a subsequent release of

endotoxins and induction of the hypersensitive response and necrosis. Although there is

uncertainty as to the role of particular enzymes present in the system, enzyme products

have been shown to inhibit P. infestans during the early stages of infection (Ricker and

Bostock, 1994). Studies of other hypersensitive reactions involving different

Phytophthora spp. are scarce. A hypersensitive-type reaction was evident in epidermal

and adjacent cortical root cells of a resistant tobacco line 3 hours after inoculation with P.

nicotianae (Hanchey and Wheeler, 1971).

A tolerant reaction may be defined as allowing the pathogen to develop within the

host while minimizing the unfavorable effects of the pathogen on host performance

(Mussel, 1980). In citrus, tolerance is defined as the condition in which plants are

infected but show little or no net root loss, either because infected roots do not rot or

because root mass density is maintained by root regeneration (Graham, 1990). Many

types of fungi that are not believed to be pathogenic have been associated with citrus

roots (Farr et al., 1989; Smith et al., 1989).

Susceptibility is defined as the inability of a host plant to resist disease or the

effect of a particular pathogen (Agrios, 1988). Susceptibility is generally considered to

be the exception rather than the rule in most systems. Although susceptibility is a general











term, different pathogens infect host tissue in various ways that can result in a different

response by the host.

There are three basic processes by which the pathogen can initially penetrate the

host's outer defenses. The first process is by a passive entrance through stomates and

other natural openings. In the case of root pathogens, this may be through natural

wounds or at the points where lateral rootlets emerge (Nemec et al., 1986; Sadasivan and

Subramanian, 1960). The second way is by mechanical force. The fungus produces an

infection peg which provides enough force to rupture the cell wall or enter between cells.

The third way is by production of tissue macerating enzymes that degrade and weaken

the cell wall and allow easy penetration. The three main enzyme systems found in fungi

are pectolytic, cellulytic, and lignolytic (Dickinson, 1960).

All three methods of host penetration have been demonstrated in systems

involving different Phytophthora spp. Penetration through leaf stomata by P. infestans is

reported as the usual mode of entry (Hohl and Suter, 1976). Most Phytophthora spp.

form germ tubes that usually penetrate through the periclinal wall of the epidermal cell or

through the middle lamella of the anticlinal walls of epidermal cells (Beagle-Ristaino and

Rissler, 1983; Coffey and Wilson, 1983; Hinch et al., 1985; Mourichon and Salle, 1981;

Phillips, 1993). Phytophthora spp. have been reported to produce cellulase (Benhamou

and Cote, 1992; McIntyre and Hankin, 1978), pectin esterase (Jarvis et al., 1981),

phospholipase (Moreau and Rawa, 1984) and other enzymes (Moreau and Seibles, 1985).

Phytophthora cinnamomi was also shown to dephenolize lignin enzymatically (Casares et

al., 1986). Colonization of tobacco roots by P. nicotianae involved pectin and cellulose











degrading enzymes; penetration into the roots was preceded either by dissolution of the

middle lamellae or by direct penetration of primary walls (Benhamou and Cote, 1992).

Although the pathosystems involving citrus and Phytophthora spp. have not been

examined in this detail, the epidermal and hypodermal cell walls of citrus roots contain

cellulose, pectic substances, suberin and lignin, and are similar in structure to those of

other plants (Hayward and Long, 1942; Wilson and Peterson, 1983). This opens the

possibility for enzymatic degradation in the infection of citrus roots. Phytophthora

citrophthora and P. nicotianae were able to form pectolytic enzymes in vitro, and their

esterase activity was high (Graniti, 1969). Also, production of cellulase was apparent

when these fungi were grown on carboxymethylcellulose.

After initial penetration has occurred, the hyphae can grow throughout the cortex

and stele. Advancement of the hyphae can be rapid. In soybean hypocotyls inoculated

with zoospores of P. megasperma Drechsl. f. sp. glycinea (Hildeb.) Kuan and Erwin, the

first layer of the cortex was invaded within 2 hours and the third layer within 3 hours

(Ward, 1989). It has been observed by inoculating avocado roots with P. cinnamomi that

infection was established in the cortex after 2-4 hours and in the endodermis after 16

hours (Philips, 1993). Again, there has been no previous work in examining the

ultrastructural changes of citrus cells in response to infection by Phytophthora spp.

However, a microscopic study of citrus roots infected with Fusarium solani (Mart.)

Appel & Wr. emend Snyd. & Hans showed that cortical infection was primarily

intracellular (Nemec et al., 1986).











There have been numerous studies on the ultrastructure of other hosts infected

with Phytophthora spp. Some of the earliest studies were on the response of potato after

infection by P. infestans. In potato cells, there is a formation of deposits of papilla-like

material on host walls next to hyphae (Aist, 1976). Callose-like material accumulated at

the penetration point and on the walls of adjacent host cells (Wilson and Coffey, 1980).

Callose deposits also were observed in hosts as a response to infection with P.

cinnamomi (Cahill and Weste, 1983). In resistant cultivars, the epidermal cell at the

penetration site turned brown and the underlying mesophyll cells became necrotic

(Pristou and Gallegly, 1954). In susceptible reactions, wall appositions were not present,

but encasements around small haustoria were well developed in resistant reactions (Hohl

and Stossel, 1976).

In various pathosystems involving Phytophthora spp., separation of the

plasmalemma from the cell wall or shrinkage of the protoplasts of host cells in advance

of the hyphae were commonly observed. In some cases, this occurred three to four cells

in advance of the pathogen (Hanchey and Wheeler, 1971; Slusher et al., 1974; Tippett et

al., 1977; Ward et al., 1989). In tobacco roots these observations were made just 6 hours

after infection (Hanchey and Wheeler, 1971). Tippet et al. (1977) suggested that this

might be the result of a diffusible toxin. Toxins have been isolated from P. citrophthora

(Breiman and Galun, 1981), P. dreschleri Tucker (Strange et al., 1982), P. infestans

(Keenan et al., 1985), P. megasperma Drechsl. var. sojae Hild. (Paxton, 1972) and P.

nicotianae (Ballio et al., 1972). These toxins can produce necrotic symptoms and cell

death when applied to host cells or protoplasts.









28

The host-parasite interaction is very complex and involves many factors. In a soil

environment, even the slightest changes in chemical and biological factors can alter their

interactions. Adding composted municipal waste can improve the soil environment and

favor plant growth. Studies of the host-parasite interaction at the cellular level may

provide a better understanding of infection and disease processes and may eventually

clarify the roles of compost in disease suppression.














CHAPTER 3
THE EFFECT OF COMPOSTED MUNICIPAL WASTE ON INFECTION OF CITRUS
SEEDLINGS AND GROWTH OF PHYTOPHTHORA NICOTIANAE


Introduction


In the United States, society is generating more than 160 million metric tons of

garbage, on a dry weight basis, each year (Parr and Hornick, 1992). In Florida alone,

each resident produces 3.6 kilograms (8 pounds) per day (DEP, 1993). Reduction in

waste accumulation has become a primary concern of society. Recycling and composting

are some of the limited methods that may be employed to dispose of garbage in an

ecologically sound manner. However, in 1989, it was estimated that only 10% of

disposed waste was being recycled (USEPA, 1989a). The organic fraction of municipal

solid waste that could be composted is estimated to be 45% (Diener et al., 1993).

Composting is an accelerated biological process in which organic materials are

decomposed by microbial activity, resulting in a stable organic product. Composting and

the application of compost on agricultural lands completes a cycle whereby organic

matter removed by crops for human consumption is replaced.

Waste from the wood products industry and from human consumption

accumulates rapidly and landfills across the country are near capacity. Only a few

additional landfill facilities have been approved for construction (Rathje, 1991).

Economical profitability and marketability of the compost are two major concerns for

29










survival of a composting facility. Composting of municipal solid waste should be

considered as a cost avoidance activity by diverting waste from costly landfills (Diener et

al., 1993). A compost facility tipping fee likely will be required to make compost

facilities economically viable (Kashmanian and Spencer, 1993). Government regulations

have allowed composting to compete with landfilling as a means of disposal (Obreza and

Reeder, 1994); for example, in Florida, the Florida Solid Waste Act of 1988 (Chapter 88-

130, Laws of Florida) mandated a 30% reduction in landfilling by December, 1994, and

prohibited disposal of yard trimmings in landfills after January 1, 1992. As a result,

municipal solid waste composting facilities have increased. In 1992, there were 19

operating municipal solid waste composting facilities in the United States, seven were

under construction, and over 150 were in various stages of planning (Hyatt et al., 1992).

This is double the number of facilities operating in 1989 (Gillis, 1992). Increased

marketability for compost will probably be in agricultural applications, such as

landscaping, nurseries, gardens, sod and vegetable farms, and fruit groves (Donovon,

1990). There are over 32 million hectares (80 million acres) of cropland for potential

disposal of composted municipal waste (CMW) (Diener et al., 1993).

Currently, there are no regulations regarding the production of compost and the

quality of the final product. If the compost has not been decomposed completely to a

humus-like product when transported, the decomposition process continues; in addition

the product is difficult to handle, may have a repulsive odor, and may contain metabolites

that are toxic to plants (Zucconi et al., 1981). Certain tests have been developed to

determine the maturity of composts (Giusquiani et al., 1989; Zucconi et al., 1981). The











use of spectroscopy may be employed to evaluate the production of humus, which is

correlated to the completion of the composting process (Giusquiani et al., 1989). A

guideline for compost quality has been written, although compost manufacturers are not

legally bound to follow it (E & A Environmental Consultants, Inc., 1995). These factors

represent chemical, physical and biological aspects of composts. Seedling bioassays

have been developed to evaluate the effect of composts on plant growth and on

suppression of plant pathogens. Chemical assays also are used to determine the

suppressive behavior of composts (Chen et al., 1988).

Soil amendments of organic materials have been shown to be beneficial to plant

health by increasing and retaining plant nutrients in soils and by slowly releasing them

(Dick and McCoy, 1993). Organic materials are rich in nutrients in forms that are easily

utilized by beneficial microorganisms and plants. Increasing populations of beneficial

microorganisms can suppress other microorganisms that cause plant diseases. In Mexico,

as an example, the rich, organic muck from the chinampas was suppressive to Pythium

damping-off when incorporated into the planting soil (Lumsden et al., 1987; Thurston,

1992). This sustainable practice has been proven to be highly effective in producing

quality crops in this region. Recently, increasing attention has been given to the disease

suppressive behavior of these organic amendments (Hoitink and Fahy, 1986).

Composted organic material has been demonstrated to be especially effective in

suppressing diseases in containerized systems (Boehm and Hoitink, 1992; Daft et al.,

1979; Hardy and Sivasithamparam, 1991b; Hoitink et al., 1991; Ownley and Benson,

1992). Composts have been shown to be effective in the management of the following











pathogens in containerized systems: parasitic nematodes (Malek and Gartner, 1975),

Phytophthora spp. (Hoitink et al., 1977; Spencer and Benson, 1981, 1982), Rhizoctonia

solani (Stephens et al., 1981), Fusarium oxysporum (Chef et al., 1982) and Pythium spp.

(Chen et al., 1987; Lumsden et al., 1983; Mandelbaum et al., 1988).

The objective of this study was to develop a bioassay to evaluate the effectiveness

of CMW to suppress Phytophthora root rot of citrus. This disease is a problem

throughout the world, and especially in Florida, where serious damage can occur under

certain environmental conditions. If CMW proves to be suppressive under greenhouse

conditions, it may be effective in the field. Compost lots with tested suppressiveness

could then be applied to the over 273,000 hectares of citrus in Florida as an

environmentally sound way to manage Phytophthora root rot as well as provide for other

improvements in plant health. Suppressive CMWs will be studied further to examine the

mechanisms involved in disease suppression and their effect on Phytophthora nicotianae

Breda de Haan.


Materials and Methods

Media and Inoculum Production

Clarified V-8 medium was prepared by mixing 2.29 grams of CaCO3 with 163 mL

of V-8 juice (Campbell Soup Co., Camden, NJ) for 20 minutes with a magnetic stirrer.

The suspension was centrifuged for 15 minutes at 2500 x g. The supernatant was

carefully decanted and saved. Solid V-8 agar medium was prepared by mixing 200 mL

of the clarified V-8 juice with 800 mL of water and adding 17.0 grams of Bacto agar.











Half-strength V-8 broth was prepared by mixing 110 mL of the clarified V-8 juice with

890 mL of water. All media were autoclaved for 20 minutes at 0.1 MPa.

A selective medium (PARP-H) was prepared as a modification of the following

procedure by Mitchell and Kannwischer-Mitchell (1992). Seventeen grams of Difco

cornmeal agar (Difco Laboratories, Detroit, MI) were added to 1 L of deionized water

and autoclaved for 15 minutes at 0.1 MPa. After cooling to 500 C, 5.0 mg of pimaricin

(Delvocid, 50% active ingredient (a.i.), Gist-Brocades N.V., Delft, Holland), 250 mg of

ampicillin (98% a.i., Sigma Chemical Co., St. Louis, MO), 10.0 mg of rifampicin (100%

a.i., Sigma Chemical Co., St. Louis, MO), 100 mg of pentachloronitrobenzene (Terraclor,

75% a.i., Olin Mathieson Chemical Corp., Little Rock, AR), and 50 mg of hymexazol

(Tachigaren, 99.5% a.i., Sankyo Co., Ltd., Tokyo, Japan) were mixed into the medium.

The medium was poured into sterile petri plates (100 X 15 mm) and stored in the dark

until used.

Strains of P. nicotianae (Duda) and P. palmivora (Shaw), isolated from citrus

roots by L.W. Timmer at the Citrus Research and Education Center in Lake Alfred,

Florida, were maintained on solid, clarified V-8 medium.

Chlamydospores of P. nicotianae and P. palmivora were produced by the method

of Mitchell and Kannswicher-Mitchell (1992). Four agar plugs taken from the edge of a

5-day-old colony of either P. nicotianae or P. palmivora were aseptically transferred to

15 mL of sterile, '/2-strength V-8 broth in 150-mL prescription bottles. The bottles were

stored flat on their sides in an incubator at 250 C. After 44 hours, the prescription bottles

were carefully shaken to break the mycelium into fragments, and placed back on their











sides in the incubator. After 1 week of growth, 100 mL of sterile, distilled, deionized

water were added to each bottle. The bottles were placed upright in an incubator at 18 C

in the dark. After 3 weeks at 180 C, the mycelium was washed on a 38-1m-mesh screen

with water to rinse out any nutrient broth. The mycelium was placed in a sterile blender

cup containing 20 mL of sterile deionized water, and blended on high for 1 minute. The

suspension was poured into a sterile tissue grinder and macerated. The volume was

brought up to 50 mL with sterile deionized water, and the suspension was sonicated in an

ice bath with a Braunosonic 1510 sonicator (B. Braun Melsungen, U.S.A.) for 30 seconds

at 240 watts. After the suspension was cooled in an ice bath for 30 seconds, the

sonication treatment was repeated. The suspension was poured through one layer of

sterile cheesecloth to remove mycelial debris. The chlamydospores were counted using a

hemacytometer.

Candler fine sand uncoatedd, hyperthermic Typic Quartzipsamments) was first

steam sterilized for 5 hours at 120 C at 0.1 Mpa and allowed to cool. The

chlamydospores were mixed into the sterilized sand and maintained for 1 week.

Quantities of P. nicotianae and P. palmivora chlamydospores were checked by diluting 1

g of the infested soil in 40 mL of 0.25% water agar and plating 1 mL on PARP-H

medium plates. The plates were placed in an incubator at 27 C. After 2 days the soil

was washed off the plates and the colonies were counted.

Compost Sources

Four separate batches of CMW were used throughout the experiments: two from

Reuter Recycling (Pembroke Pines, FL) (RI and R3) and two from Bedminster











Corporation (Sevierville, TN) (B2 and B4). Batch RI was divided into two subbatches,

one maintained as R1, which was stored at 4 C, and the other labelled Rib, which was

stored at room temperature. Analyses for all batches are listed in Appendix A.

The hydrogen ion concentrations of CMW and CMW-amended soil were

measured by the method of Peech (1965). Ten grams of soil or medium were mixed with

20 mL of 0.01 M CaCI2 solution, stirred several times for 30 minutes, and then allowed to

stand for 30 minutes. The pH values were measured with a Corning pH meter 240

(Coming Science Products, Coming, NY) and recorded.

The electrical conductivities of CMW and CMW-amended soil were measured by

a modified procedure of Warnke (1988); 400 cm3 of the soil or medium were saturated

with deionized water in a 1-liter beaker to a consistency where the sample flowed, but no

appreciable water accumulated on the surface. After incubation for 1 hour, the sample

was stirred again. Some of the sample was filtered through Whatman 41 filter paper

(Whatman Limited, England) after an additional 30 minutes of incubation. The filtrate

was centrifuged for 30 minutes at 5000 rpm. Conductivity of the supernatant was

measured with an ElectroMark analyzer conductivity meter (Markson Scientific Co.,

Mara, CA).

Effect of CMW on Seedling Growth

Seedlings of sour orange (Citrus aurantium L.) were grown from seed in Metro-

Mix 500 (The Scotts Co., Marysville, OH) under glasshouse conditions. After 5 weeks,

the seedlings were removed for testing.









36

Candler fine sand, acquired near Davenport, FL, was pasteurized by microwaving

1 kg of moist (approximately 7% w/w moisture) for 4 minutes (Ferriss, 1984; Wolf and

Skipper, 1994) in an 800 watt GE microwave oven model JE2810A (General Electric

Co., Louisville, KY). The soil was cooled to room temperature.

Composted municipal waste, batch B4 from Bedminster Corp., was mixed with

the pasteurized soil to final concentrations of 10%, 20%, and 50% (v/v) of the total

volume. Sour orange seedlings (10 per treatment) were planted individually in 250-mL

cone-tainers (Stuewe & Sons, Inc., Corvallis, OR) containing either nonamended soil or

CMW-amended soil in the various concentrations. The seedlings were grown under

greenhouse conditions for 8 weeks, fertilized once a week with Peter's 20-10-20 fertilizer

(United Industries Corp., St. Louis, MO), and watered when necessary.

The seedling roots were carefully washed to remove any soil. The fresh weight of

the entire seedling, the fresh weight of the shoot, and the fresh weight of the roots were

measured. The roots were dried in an incubator at 70 C for 3 days and weighed.

The conductivities of CMW-amended and nonamended soils before planting and

after completion of the experiment were measured by the modified procedure of Warnke

(1988) as described above. The hydrogen ion concentrations of the CMW-amended and

nonamended soils before planting and after completion of the experiment were measured

by the method of Peech (1965), as described above.

The experiment was repeated as described above. Statistical analysis was

performed on the data using the SAS analysis of variance procedure (ANOVA) with

linear regression calculated to distinguish significance.













Infection of Citrus Seedlings by P. nicotianae

Seedlings of 'Ridge Pineapple' sweet orange (C. sinensis [L.] Osbeck), citrumelo

hybrids F80-3 and F80-8 (Poncirus trifoliata [L.] Raf. X C. paradise Macf.), Cleopatra

mandarin (C reshni Hort. ex Tan.), Volkamer lemon (C volkameriana (L.) Burm. f.),

and sour orange were grown from seed in Metro-Mix 500 under glasshouse conditions.

After 5 weeks, the seedlings were removed for testing.

The CMW was mixed with pasteurized Candler fine sand to provide a final

volume of 10% or 20% (v/v) of the total volume. The chlamydospore-infested soil was

added to inoculated treatments for a final population density of 10 chlamydospores per

cm3 of soil. The seedlings were planted in 75-mL cone-tainers (Stuewe & Sons, Inc.,

Corvallis, OR) filled with soil that was either infested with P. nicotianae or noninfested,

and amended with CMW or nonamended. The plants were grown under greenhouse

conditions, watered on a regular schedule, and fertilized once every week with Peters 20-

10-20 fertilizer. After 3 weeks, the plants were removed from the cone-tainers and the

roots rinsed with tap water. Shoots were cut off and the roots were surfaced sterilized in

70% ethanol for 5 seconds and rinsed twice with sterile, deionized water. The roots were

placed on a sterile paper towel and flattened with a glass bottle. Each root system was

plated on an individual petri plate containing PARP-H selective medium. The plates

were incubated for 2 days at 270 C. Roots were rated as positive for incidence of

infection if any P. nicotianae colonies were detected. The percentage of total root length











infected with P. nicotianae was also recorded. All experiments were repeated at least

once and combined, unless otherwise noted.

Effect of CMW on Colony Growth

Samples of Candler fine sand and Wabasso fine sand (siliceous, hyperthermic

Alfic Haplaquod) were pasteurized by microwaving 1 kg of soil for 4 minutes. After the

soil cooled to room temperature, the CMW was added as an amount equal to 20% of the

total volume (v/v).

Infested soil media were prepared by diluting chlamydospore-infested stock soil

to a final density of 10 chlamydospores per cm3 of soil media in either nonamended soil

or CMW-amended soil. The infested soil media were mixed well and maintained at room

temperature in closed, but unsealed, plastic bags for 6 days. Forty grams of infested,

CMW-amended soil or nonamended soil were mixed with 40 mL of 0.25% water agar.

One milliliter was plated onto the PARP-H selective medium, and the plates were

incubated at 27 C for 2 days. The soil was washed off of the plates, and the plates were

placed back into the incubator. After a total of 66 hours from the time that the soil was

first plated onto the medium, the colonies of P. nicotianae were counted.

The colony areas were measured by overlaying the marked colonies with a grid

consisting of points 1 cm apart from each other. The area of the colony was calculated

by adding the number of points within the marked colony and dividing by four to

calculate the colony area in square centimeters.











Effect of Acetic Acid on Colony Growth

Corn meal agar (Difco Laboratories, Detroit, MI) was prepared by mixing 39.0 g

in 1 L of water and autoclaving for 20 minutes at 0.1 MPa. The agar was cooled to 45 C

in a water bath. Glacial acetic acid was added to the liquid medium to final

concentrations of 0, 5, 10, 25, 50, 100 and 160 ppm. The pH values of the media were

measured but did not change. The medium was poured into petri plates (100 X 15 cm)

and allowed to solidify. A 5-mm disk from a 5-day-old culture of P. nicotianae grown

on V-8 agar was placed in the middle of each plate, and the plates were incubated at 270

C. After 96 hours, the diameters of the colonies of P. nicotianae were measured. The

impact of acetic acid on colony areas was analyzed by SAS using quadratic regression

analysis.

Effect of Sterile CMW Extracts on Colony Growth

One hundred grams of Candler fine sand, 20 g of Bedminster CMW (Batch B2) or

20 g of Reuter CMW (Batch RI) were mixed with 100 mL of either 0.4 N KOH, 2 N

H2S04, or sterile, deionized water in 250-mL flasks for 6 hours. The suspension was

filtered through cheesecloth and Whatman 50 filter paper. The filtrate was sterilized by

filtration through a 0.2-pm membrane.

One hundred milliliters of each sterile filtrate were added to the individual flasks

of 900 mL of liquid CMA supplemented with 1 mL of tergitol NP-10 (Sigma Chemical

Co., St. Louis, MO), 0.1 g of streptomycin sulfate (Sigma Chemical Co., St. Louis, MO),

and 0.05 g of chlortetracycline (Sigma Chemical Co., St. Louis, MO). The pH was

determined and adjusted to 5.5 to 6.0 with either 10 N KOH or concentrated H2S04.











Fifteen milliliters were poured into each of 10 petri plates (100 X 15 cm) and allowed to

solidify. A 5-mm disk from a 5-day-old culture of P. nicotianae grown on V-8 medium

was placed in the middle of each plate, the plates were incubated at 270 C. The colony

diameters were measured over time.

Chromatography

One gram of Reuter (R1 and R3) or Bedminster (B2) CMW and 4 grams of

Candler fine sand were placed in separate 150-mL Erlenmeyer flasks. Ten milliliters of

optima grade methanol (Sigma Chemical Co., St. Louis, MO) were added to the samples

and stirred with a magnetic stir bar for 15 minutes. The extract was filtered through a

fretted glass filter by vacuum. The filtrate was stored in a closed glass vial.

Either 2.0 pL or 0.5 gL of the filtrate sample were injected into a Hewlett Packard

5890A gas chromatograph. The compounds were separated using a 30-m X 0.32-mm

column with a 1.0-gm film of Stabilwax (carbowax) (Restek Corporation, Bellefonte,

PA). Helium, the carrier gas, was set at a flow rate of 2 mL per minute. The initial

temperature of the column, 40' C, was maintained for 3 minutes, and temperature was

then increased at a rate of 8 C per minute to a final temperature of 200 C. The total run

time was 23.0 minutes. Standards of acetic acid, methanol, and isopropanol were also

tested.

Bedminster batch B4 was also tested using a modified headspace, solid-phase

microextraction technique (Steffen and Pawliszyn, 1996; Zhang and Pawliszyn, 1993). A

15-cm3 sample of batch B4 CMW was placed in a 50-mL glass vial with a Teflon septum

lid. The sample was stored at room temperature for 24 hours. A needle was pierced









41
through the septum of the vial, and a plunger was depressed to expose a 1-cm-long, 100-

pm-thick, poly(dimethylsiloxane)-coated fiber (Supelco, Inc., Bellefonte, PA) into the

headspace for 30 minutes. The fiber was withdrawn into the needle and transferred to the

injection port of the gas chromatograph (GC). The needle penetrated the septum of the

GC inlet and the fiber was exposed in the 2000 C injection port on a Hewlett Packard

5890A gas chromatograph. The compounds were separated using 30-m X 0.32-mm

column with a 0.5-pm film ofDB5 (Resteck Corp.). Helium, the carrier gas, was set at a

flow rate of 2 mL per minute. The initial temperature of the column was set at 35 C, and

temperatures were increased to 275 C at a rate of 6 C per minute. An acetic acid

standard was set up and analyzed by the same technique by placing one drop of glacial

acetic acid (Fisher Scientific Co., Pittsburgh, PA) in a 50-mL, closed glass vial.

Isolation of Microbial Antagonists

Composted municipal wastes from Reuter (RI and R3) and Bedminster (B2) were

diluted 1:3000 by adding 0.33 g (dry weight) of the CMW to 1 liter of sterile water

amended with 0.05% tergitol NP-10 (Sigma Chemical Co., St. Louis, MO), 100 mg of

streptomycin sulfate (Sigma Chemical Co., St. Louis, MO), and 50 mg of

chlortetracycline-HCl (Sigma Chemical Co., St. Louis, MO). One milliliter of each

dilution was pipetted into each of 10 sterile petri plates (100 X 15 mm). Approximately

16 mL of cooled (50* C) potato dextrose agar (PDA) (Difco Laboratories, Detroit, MI),

amended with 100 ppm streptomycin sulfate plus 50 ppm chlortetracycline-HCl per liter

of medium and 0.05% tergitol NP-10, were poured into each of the petri plates containing

the sample. The plates were gently mixed and allowed to solidify. The plates were









42

stored under continuous light at room temperature. Individual colonies were transferred

aseptically and grown on solidified PDA amended with 100 mg of streptomycin sulfate

and 50 mg of chlortetracycline-HCL per liter of medium.

Individual colonies of fungal isolates were transferred to solidified CMA

containing 2-day-old cultures of P. nicotianae. The plates were incubated at 25 C, and

any interaction between the fungal colony and P. nicotianae was observed.

Composted municipal wastes from Reuter (RI and R3) and Bedminster (B2) were

diluted 1:30,000 by adding 0.33 g (dry weight) of the CMW to 1 liter of sterile water

amended with 100 mg of cycloheximide (Sigma Chemical Co., St. Louis, MO). Ten

milliliters of the dilution were added to 990 mL of sterile water amended with 100 mg of

cycloheximide. One milliliter of each dilution was pipetted into each of 10 sterile petri

plates. Approximately 16 mL of cooled (50 C), 10% Tryptic Soy Broth (TSB) medium

(1.5 g of TSB and 7.5 g of agar in 500 mL of water) amended with 100 mg of

cycloheximide were poured into each of the petri plates containing the sample. The

plates were gently mixed and allowed to solidify. The plates were stored in the dark in

an incubator at 250 C. Individual colonies were transferred aseptically to petri plates of

TSB amended with 100 mg of cycloheximide per liter of medium.

Individual colonies of fungal isolates were transferred to solidified corn meal agar

(CMA) containing 2-day-old cultures of P. nicotianae. The plates were incubated at 250

C, and any interaction between the bacterial colony and P. nicotianae was observed.











Results



Effect of CMW on Seedling Growth

The dried root weight of the controls was significantly higher than the root

weights of the compost-amended treatments (Table 3-1). The total plant fresh weight and

shoot weight of the nonamended control were not different than those in the 10% CMW-

amended treatments. Linear regression analyses showed a decrease in total plant fresh

weight, shoot weight, and root weight with an increasing proportion of CMW (P<0.001)

(Figure 3-1). The conductivities of the amended soils were 0.11, 2.01, 3.45, and 5.91 mS

for volumes of 0%, 10%, 20%, and 50% CMW, respectively. The initial pH values were

5.67, 6.70, 6.69, and 7.37, respectively, for the same volumes of CMW. After 8 weeks,

the conductivities were 0.14, 0.28, 0.35, and 0.53 mS, respectively. The pH values after

8 weeks (5.55, 6.24, 6.55, and 7.13, respectively), were not significantly different from

the initial values.

Infection of Citrus Seedlings by P. nicotianae

There was no infection by P. nicotianae in the noninfested controls in any

treatment. Because percentages of infected root systems in the seedling bioassay of

infested, nonamended treatments, regardless of the compost age, gave similar results, the

results were combined for analyses, but not presented in the tables (Tables 3-2, 3-3, 3-4).

The results from all treatments with CMW amendments were combined if the CMW was

stored for less than 6 months or reported separately if the CMW was stored for a longer























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49

period of time. Susceptible varieties, Ridge Pineapple, sour orange, Cleopatra mandarin,

and Volkamer lemon, had 78%, 90%, 94%, and 80% infection, respectively, in

nonamended soil infested with P. nicotianae (Tables 3-2 and 3-4). Tolerant varieties,

citrumelo hybrids F80-3 and F80-8, had 60% and 60% infection, respectively, in

nonamended, infested soil (Table 3-3).

Batch RI of Reuter CMW, when less than 6 months old, suppressed infection by

P. nicotianae in all varieties tested, except the tolerant citrumelo hybrid (F80-3)

and Ridge Pineapple grown in CMW previously stored at room temperature (Tables 3-1,

3-2, and 3-3). Batch R3 was not suppressive with any variety tested when the CMW was

3 years old (Tables 3-2 and 3-4). Batch B2, when less than 6 months old, suppressed

infection of Ridge Pineapple, but infection of other varieties was not reduced with 2.5-

year-old CMW (Tables 3-2 and 3-4). Batch B4 suppressed infection of both varieties

tested, Ridge Pineapple and sour orange; batch B4 also suppressed infection by P.

palmivora (Appendix C). The infested, 10% CMW mixture significantly lowered

incidence of infection in comparison to the infested, nonamended control, but there was

no difference among the infested, 20% CMW mixture and the control (P<0.01) (Table 3-

5).

The infected roots were rated according to the percentage of the total root system

infected with P. nicotianae. Root systems grown in soil amended with batch B4 of

Bedminster CMW had 24% of the root length infected with P. nicotianae, which was

significantly less than the infected root systems grown in nonamended soil (51.4%)












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51

(P=0.002). Populations of P. nicotianae recovered after the completion of the seedling

bioassay experiments ranged from 9 to 120 propagules per cm3, but they were not

significantly different in the CMW-amended, infested treatments than in the

nonamended, infested controls (P=0.49). Furthermore, no differences were detected

among the citrus varieties tested (P=0.995).

Effect of CMW on Colony Growth

Soil amended with batch RI from Reuter Recycling significantly reduced the

colony area of P. nicotianae in comparison to the area formed with the infested,

nonamended soil (P=-0.02) (Tables 3-6 and 3-7). Batch B2 from Bedminster

Bioconversion did not significantly reduce the colony area (P=0.5) (Table 3-7), but Batch

B4 from Bedminster Bioconversion significantly reduced the colony area when amended

at 10% and 20% of the total volume (P<0.001 and P<0.001, respectively) (Table 3-8).

The soil type did not have a significant effect on the colony area (P=0.11) (Table 3-9).

Effect of Sterile CMW Extracts on Colony Growth

The cold water, KOH, and H2SO4 extracts of batch B2 of the Bedminster CMW,

when added to CMA, significantly reduced the colony diameters of P. nicotianae in

comparison to those in nonamended soil extracts (Table 3-10). Growth in extracts of

batch RI of Reuter CMW were not significantly different than that in the soil extracts,

except that colony diameter was significantly greater in the H2SO4 extract than in the

control.











Table 3-6. Effect of batch RI of composted municipal waste (CMW) from Reuter
Recycling (Pembroke Pines, FL), when added as a soil amendment, on the colony growth
of Phytophthora nicotianae.
Colony area (cm2)x
Treatmenty Trial #1 Trial #2 Trial #3
Soil alone 1.88 az 1.55 a 1.00 a

CMW-amended soil 0.90 b 0.52 b 0.43 b

xAverage colony areas of P. nicotianae grown on PARP-H selective medium 66 hours
after plating soil dilutions of infested, CMW-amended or nonamended soils.
YPasteurized Candler fine sand nonamended or amended with 20% (v/v) CMW.
zMeans followed by the same letter within a column are not significantly different
(P>0.05) according to the paired student t-test.











Table 3-7. Effect of source of composted municipal waste (CMW) on colony diameter of
Phytophthora nicotianae.
Treatmentx Colony area (cm2y
Soil alone 1.51 aW

20% Bedminster (batch B2) 1.27 ab

20% Reuter (batch RI) 0.48 b

xAverage colony areas of Phytophthora nicotianae grown on PARP-H selective medium
66 hours after plating soil dilutions of infested, CMW-amended or nonamended soils.
YPasteurized Candler fine sand nonamended or amended with 20% (v/v) CMW, batch B2
from Bedminster Bioconversion (Sevierville, TN) or batch RI from Reuter Recycling
(Pembroke Pines, FL).
'Means followed by the same letter are not significantly different (P>0.05) according to
the paired student t-test.











Table 3-8. Effect of proportion of composted municipal waste (CMW) in soil on colony
diameter of Phytophthora nicotianae.
Treatments Colony area (cm2)y
Soil alone 1.39 az

10% Bedminster, B4 0.83 b

20% Bedminster, B4 0.80 b

xPasteurized Candler fine sand nonamended or amended with either 10% or 20% (v/v) of
batch B4 of Bedminster Bioconversion CMW (Sevierville, TN).
YAverage colony areas of P. nicotianae grown on PARP-H selective medium 66 hours
after plating soil dilutions of infested, CMW-amended or nonamended soils.
zMeans followed by the same letter within a column are not significantly different
(P>0.05) according to the paired student t-test.











Table 3-9. Effect of composted municipal waste (CMW) and soil type on the colony
growth of Phytophthora nicotianae.


Soil typev CMW treatment Colony area (cm2)x
Candler soil alone 1.51 ay
Candler Bedminster, B2 0.73 c
Candler Reuter, Rlaz 0.71 c
Candler Reuter, Rib 1.11 b
Candler Reuter, R3 1.11 b

Wabasso soil alone 1.37 a
Wabasso Bedminster, B2 0.77 bc
Wabasso Reuter, Rla 0.55 c
Wabasso Reuter, Rib 0.97 b
Wabasso Reuter, R3 1.05 b

'Two different soil types collected near Davenport, FL (Candler fine sand) and Fort
Pierce, FL (Wabasso fine sand).
wSoil treatments consisted of batch 1 of Bedminster Bioconversion CMW (Sevierville,
TN) and batches RI and R3 of Reuter Recycling CMW (Pembroke Pines, FL).
xAverage colony areas of P. nicotianae grown on PARP-H selective medium 66 hours
after plating soil dilutions of infested, CMW-amended or nonamended soils.
YMeans followed by the same letter within a column are not significantly different
(P>0.05) according to the paired student t-test.
zRla and Rib are from the same batch of Reuter CMW, except Ria was stored at 40
while Rlb was stored at room temperature.











Table 3-10. Effect of sterile medium extract on colony growth of Phytophthora
nicotianae 88 hours after plating dilutions of composted municipal waste (CMW)-
amended or nonamended soil on corn meal agar.
Colony diameter (mm)"
Treatment" Watery KOH H2SO4
Soil 70.9 az 63.3 a 58.3 b

Reuter CMW 71.3 a 62.0 a 71.3 a

Bedminster CMW 51.9 b 54.7 b 50.7 c
"Average colony diameters of P. nicotianae grown on corn meal agar medium.
xSoil dilutions were prepared from soil (100 g per 100 mL extractant) or CMW from
Reuter Recycling (Pembroke Pines, FL) and Bedminster Bioconversion (Sevierville,
TN) (20 g per 100 mL extractant).
YExtractants used on test media: cold, double distilled water (water), 0.4 N KOH (KOH),
2.0 N H2S04 (H2SO4).
Means followed by the same letter within a column are not significantly different
(P>0.05) according to the paired student t-test.












Effect of Acetic Acid on Colony Growth

When CMA was supplemented with acetic acid, the colony growth of P.

nicotianae was reduced in a quadratic regression as the concentration of acetic acid

increased (Figure 3-2). There was not a significant reduction in percentage of infected

root systems of sour orange seedlings when acetic acid was supplemented to pasteurized

soil at final concentrations of 50 and 150 ppm (Appendix D).

Chromatography

A single peak was observed in the extract of batch B2 of Bedminster CMW 17.4

minutes after injection (Figure 3-3). This peak was also observed in the extracts of

batches RI and R3 of the Reuter CMW, but it was not as intense. This peak matched the

peak of the acetic acid standard. A single peak was also observed in the chromatograph

of the extract of batch B4 of Bedminster CMW 2.8 minutes after injection (Figure 3-4).

This peak matched that of the acetic acid standard.

Isolation of Microbial Antagonists

Bright orange, gelatinous colonies grew on plates of PDA plus antibiotics after

incubation of dilutions of soil amended with the suppressive batch RI of Reuter CMW;

these colonies were not observed in dilutions of soil amended with any of the other

CMWs. When cultured together with P. nicotianae, this fungus appeared to coil around

and penetrate the P. nicotianae hyphae (Figure 3-5). The fungus was identified as an

Acremonium sp. and its identity was confirmed by Dr. James Kimbrough (University of

Florida, Gainesville, FL). No other bacteria or fungi that grew on the medium showed


































40 -


35 -


30-
0 20 40 60 80 100 120 140 160 180 200
Acetic acid concentration (ppm)




Figure 3-2. Effect of acetic acid on colony growth of Phytophthora nicotianae on corn
meal agar plates after 96 hours.

















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61





























copp'

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Figure 3-5. Acremonium sp. hyperparasitizing Phytophthora nicotianae. A. Hypha of
Acremonium sp. coiling around hypha of P. nicotianae (x 1100). B.Hypha of
Acremonium sp. penetrated into P. nicotianae hypha (x 1800). p = P. nicotianae hyphae,
a = Acremonium sp. hyphae, bar = 10 pm.











any suppressive or antagonistic behavior towards P. nicotianae. There was not a

significant reduction in percentage of infected root systems of sour orange seedlings

when pasteurized soil was supplemented with the Acremonium sp. (Appendix E).



Discussion



Composted municipal waste was effective, when added as a soil amendment, in

suppressing infection of citrus seedlings by P. nicotianae in the greenhouse. Both

incidence of infection and the percentage of the root system infected were reduced. The

results from this study show that it is important to use fresh CMW, as the CMW lost its

suppressive behavior over time, even when stored at 40 C. It is also important not to

apply an excess of CMW, which can be detrimental to seedling growth and development.

Addition of CMW, especially immature CMW, to agricultural soil may result in

phytotoxicity to the plants if too much is applied. However, if too little is applied there

may be little or no benefit and the cost of application will be wasted. Before any extra

benefits of disease suppression are examined, it must first be determined if properly aged

CMW can be applied and how much can be applied without causing phytotoxicity to

citrus plants.

The lower root weights of plants grown in the CMW-amended treatments may be

due to the higher salt concentrations of the growth media, as measured by the

conductivity. The acceptable recommended range for agricultural use is 0.75 to 3.5 mS









63

(Warnke, 1988). Most of the CMW used was at the higher limit and exceeded this level

at the higher concentrations. Younger roots may be more sensitive to high salt

concentrations, which may cause physiological maladies as well as increased

susceptibility to pathogen infection. Vavrina (1994) found that different sources of

municipal solid waste at different mixture ratios suppressed tomato seed germination and

reduced root and shoot dry weights. Perhaps if the experiments in the present study had

been allowed to continue for another 8 weeks, there would have been no harmful effects

from the salts. Other studies have shown that, as time passes, the plant overcomes the

initial growth suppression (Obreza, 1995; Obreza and Reeder, 1994).

Although there was a phytotoxic effect at a 20% (v/v) level, the main objective of

this study was to evaluate suppression of infection. Since plants in the bioassays were

grown for only 3 weeks, it was decided to amend the soil with a mixture of 20% CMW

(v/v). Batches RI and B2 were effective in suppressing root infection when they were

utilized soon after the composting process was completed. However, RI lost its

suppressiveness when it was stored for over 6 months at room temperature and then

utilized. Batch B2 also lost its suppressiveness over time after being stored at 4' C for

over 2 years. When CMW was utilized immediately or within 6 months, if stored at 4 C,

it was consistent in suppressing infection of citrus roots by P. nicotianae. Even at a

concentration of only 10% (v/v), CMW suppressed incidence of infection, regardless of

the citrus variety tested.











There were no differences observed in the percent incidence of infection or

percentage of root systems infected among citrus varieties tested. Most of the varieties

tested ('Ridge Pineapple' sweet orange, sour orange, and Cleopatra mandarin) are rated

as susceptible (Graham, 1990), but even a 5-week-old 'tolerant' citrus seedling is rated as

susceptible (Carpenter and Furr, 1962), which was indicated by the high incidence of

infection in the infested, nonamended controls.

Pathogen suppression has been reviewed in detail in other studies (Hoitink and

Fahy, 1986; Hoitink and Grebus, 1994). Possible mechanisms include impact on

pathogen growth, pathogen survival, and infection (resistance, competition for infection

sites) of the host. The ability of CMW to induce resistance of the host against

Phytophthora root rot was not examined in this study. However, the effect on the

pathogen, P. nicotianae, was studied. Addition of fresh CMW significantly reduced the

colony growth of P. nicotianae. Colony growth was even suppressed by soil amended

with 10% (v/v) CMW. This was consistent within two different soil types sampled from

two citrus-growing regions. Compost amendments, however, did not reduce the

populations of P. nicotianae recovered after the completion of the test. Thus, although

CMW appears to be fungistatic to P. nicotianae, it does not appear to be fungicidal. This

may be important if seedlings produced with CMW amendments are to be transferred to

the field. Although the seedlings may appear to be healthy, the pathogen may still be

present and active in sufficient quantities to do serious damage in the field. In contrast,











this initial effectiveness may be enough to give seedlings or young trees enough time to

"outgrow" the pathogen's effects.

The mechanisms involved in suppression of infection in this system appear to be

two different processes, depending upon the CMW and probably on the environmental

conditions. A chemical mechanism was first indicated by the suppression of colony

growth of P. nicotianae in sterile extracts of batch B2 added to the growing medium.

Acetic acid was discovered in some of the CMWs that showed suppressive behavior.

Acetic acid is a natural product of the composting procedure (DeVleeschauwer et al.,

1981), especially in immature composts, and it has been shown to suppress growth of P.

cactorum (Utkhede and Gaunce, 1983).

A biological explanation for suppression was also indicated, especially in batch

RI, which showed suppressive behavior in the seedling bioassay but not in the growth of

P. nicotianae in sterile extracts. Acremonium sp. was isolated from the culture plates

with dilutions of the suppressive R1 CMW, but not from plates with soil dilutions from

the nonsuppressive batch R3 or batch B2. When grown together, Acremonium sp. coiled

around and penetrated some P. nicotianae hyphae; however, colony suppression of P.

nicotianae was not convincingly evident when the two organisms were grown together

on corn meal agar. Perhaps the isolate of Acremonium sp. recovered from the general

medium is a weaker hyperparasite than other isolates. Acremonium alternatum was

shown to severely damage the cucurbit powdery mildew pathogen, Sphaerotheca

fuliginea (Malathrakis, 1985). The suppression of infection caused by CMW may result









66

from a combination of factors. There may have been more antagonists, such as bacteria,

in the composts that were not detected, and they may have had a synergistic effect against

P. nicotianae when several were present together. Also, the low concentrations of acetic

acid found in these batches of CMW may have influenced the lack of suppression.














CHAPTER 4
THE EFFECT OF COMPOSTED MUNICIPAL WASTE AS A SOIL AMENDMENT
ON THE GROWTH OF YOUNG CITRUS TREES AND PHYTOPHTHORA
NICOTIANAE


Introduction


Citrus is one of the most economically important crops in Florida, with continuing

increases in production. As an example, 103.7 million trees were planted from 1992 to

1994 around the state (Florida Agricultural Statistics Service, 1996). Commercial,

independent nurseries provide most of the citrus trees to growers (D.P.H. Tucker, 1996,

personal communication). Desirable scions, depending upon the needs of the grower, are

grafted onto various rootstocks. The two main techniques for citrus tree production are

container-grown trees and field-grown trees that either are sold as bareroot plants or are

dug, potted, and sold in containers (Jackson, 1991). Trees are usually ready to be planted

in the grove after 1 year in containers. Ideally, the nursery trees would be planted in the

same soil as that in which the trees will be planted. However, this is not always possible,

and differences in potting medium and production soil can lead to plant stress when trees

are transplanted (Schoeneweiss, 1975).

Availability of water and good drainage are the two most important factors in

considering a site for citrus planting (Jackson, 1991). Other site factors include water-

holding capacity, nutrient supply and soil pH. Most of the 273,000 hectares of citrus in











Florida are planted on sandy soils with low organic matter and poor residual fertility.

These soils have a low cation exchange capacity and retain only small amounts of applied

plant nutrients after the leaching action of rainfall and irrigation (Tucker et al., 1995).

Common horticultural practices include routine chemical fertilization to increase plant

growth and production. However, even as chemicals are applied over time, land becomes

depleted of its natural resources and growers become more dependent upon chemicals.

Excessive applications of fertilizers can also lead to various environmental problems,

such as groundwater contamination (Calvert and Phung, 1972; Embleton et al., 1978).

Disease pressure is also a factor for consideration in site selection. Phytophthora

nicotianae Breda de Haan (synonym = P. parasistica Dastur) (Hall, 1993) causes a rot of

fibrous citrus roots, and Phytophthora root rot is a common problem in citrus nurseries

(Zitko et al., 1987). In 1993, over 90% of the nurseries surveyed in Florida were infested

with P. nicotianae (Fisher, 1993). Commonly used rootstocks, such as sour orange

(Citrus aurantium L.), Carrizo citrange (C. sinensis (L.) Osbeck X Poncirus trifoliata

(L.) Raf), and Swingle citrumelo (C. paradise Macf. X P. trifoliata), range from

susceptible to tolerant to Phytophthora root rot (Agostini et al., 1991; Graham, 1990).

However, because of citrus blight, viral diseases and other pest problems and

horticultural preferences, use of rootstocks tolerant to Phytophthora root rot may not

always be feasible (Graham, 1995). In infested nurseries, even tolerant rootstocks suffer

serious root rot damage when over-watered (Zitko, et al. 1987). When transplanted to the

field, initially low populations of P. nicotianae can increase quickly under the favorable

environmental conditions often found in Florida. These high populations can cause tree











decline and reduce yields in mature trees. Fungicides are usually applied in nurseries to

control P. nicotianae; however, resistance to the important fungicide, metalaxyl, has been

found in some citrus nurseries (Fisher, 1993).

Because of the continuing disease pressure and concern with the environmental

impact of high applications of chemicals, new strategies for alternative methods for

management of Phytophthora root rot of citrus are being examined. Other agricultural

systems, particularly those developed in the floriculture industry, have been successful in

disease management when potting media are amended with composted organic materials

(Hoitink et al., 1991). When added as a soil amendment, certain composts, such as tree

bark, have been shown to suppress soilborne diseases caused by Rhizoctonia solani,

Pythium ultimum, and Fusarium oxysporum f.sp. conglutinans (Davis, 1982; Nelson and

Hoitink, 1982; Trillas-Gay et al., 1986). There also has been some success with the

addition of organic amendments in field trials (Broadbent and Baker, 1974b; Cook and

Baker, 1983; Galindo et al., 1983; Sun and Huang, 1985).

Composted municipal waste (CMW) is a potentially valuable source of organic

amendments. Landfills are near capacity across the United States, and incineration of

waste material is environmentally unsound. The application of CMW to agricultural

areas can help alleviate this problem. Although CMW has been shown to promote plant

health and increase yields in some field crops (Bryan et al., 1985; Obreza, 1995), limited

information is available on the effectiveness of CMW for disease management in the

field, especially for tree crops.











The purpose of this study was to evaluate the influence of CMW on populations

of P. nicotianae and on general tree health in a newly planted citrus grove.


Materials and Methods


Clarified V-8 medium was prepared by mixing 2.29 grams of CaCO3 with 163 mL

of V-8 juice (Campbell Soup Co., Camden, NJ) for 20 minutes with a magnetic stirrer.

The suspension was centrifuged for 15 minutes at 2500 x g. The supernatant was

carefully decanted and saved. Solid V-8 agar medium was prepared by mixing 200 mL

of the clarified V-8 medium with 800 mL of water and adding 17.0 grams of Bacto agar.

Half-strength V-8 broth was prepared by mixing 110 mL of the clarified V-8 medium

with 890 mL of water. The media were autoclaved for 20 minutes at 0.1 MPa.

A selective medium (PARP-H) was prepared as a modification of the following

procedure by Mitchell and Kannwischer-Mitchell (1992). Seventeen grams of Difco

cornmeal agar (Difco Laboratories, Detroit, MI) were added to 1 L of deionized water,

and autoclaved for 20 minutes at 0.1 MPa. After cooling to 50 C, 5.0 mg of pimaricin

(Delvocid, 50% active ingredient (a.i.), Gist-Brocades N.V., Delft, Holland), 250 mg of

ampicillin (98% a.i., Sigma Chemical Co., St. Louis, MO), 10.0 mg of rifampicin (100%

a.i., Sigma Chemical Co., St. Louis, MO), 100 mg of pentachloronitrobenzene (Terraclor,

75% a.i., Olin Mathieson Chemical Corp., Little Rock, AR), and 50 mg of hymexazol

(Tachigaren, 99.5% a.i., Sankyo Co., Ltd., Tokyo, Japan) were mixed into the medium.

Fifteen-milliliters were poured into a sterile petri plate (100 X 15 mm), and the plates

were stored in the dark until ready for use.









71

A strain ofP. nicotianae (Duda), isolated from citrus roots by L.W. Timmer at the

University of Florida Citrus Research and Education Center in Lake Alfred, FL, was used

for soil infestation. The isolate was stored in an incubator at 18 C. Chlamydospores of

P. nicotianae were produced by the method of Mitchell and Kannwischer-Mitchell

(1992). Four 5-mm disks from the edge of a 5-day-old culture of P. nicotianae on V-8

agar were aseptically transferred to 15 mL of sterile, 1/2-strength V-8 broth in 150-mL

prescription bottles. The bottles were stored flat on their sides in an incubator at 25 C.

After 44 hours, the prescription bottles were carefully shaken to break the mycelium into

fragments, and placed back on their sides in the incubator. After 1 week of growth, 100

mL of sterile, distilled, deionized water were added to each bottle. The bottles were

placed upright in an incubator at 18 C in the dark. After 3 weeks at 180 C, the mycelium

was washed on a 38-pm-mesh screen with water to rinse out any nutrient broth. After the

mycelium was placed in a sterile blender cup containing 20 mL of sterile deionized water

and blended on high for 1 minute, the suspension was poured into a sterile tissue grinder

and macerated. The volume was brought up to 50 mL with sterile deionized water, and

the suspension was sonicated in an ice bath with a Braunosonic 1510 sonicator (B. Braun

Melsungen, U.S.A.) for 30 seconds at 240 watts. After the suspension was cooled in an

ice bath for 30 seconds, the sonication treatment was repeated. The suspension was

poured through one layer of sterile cheesecloth to remove mycelial debris.

Chlamydospores were counted using a hemacytometer.

Candler fine sand uncoatedd, hyperthermic, Tipic Quartzipsamments), collected

near Davenport, FL, was pasteurized by microwaving 1 kg of moist (approximately 7%











w/w moisture) soil for 4 minutes in an 800 watt GE microwave oven (General Electric

Co., Louisville, KY) (Ferriss, 1984; Wolf and Skipper, 1994). The microwaved soil was

infested with P. nicotianae chlamydospores at a final density of 70 chlamydospores per

cm3 of soil.

Composted municipal waste from two different sources was used in this

experiment. Batches RI and R3 of CMW from Reuter Recycling (Pembroke Pines, FL)

were derived from household garbage and contained relatively large pieces of broken

glass and plastic. The other source of CMW, batch B4 from Bedminster Bioconversion

(Sevierville, TN), was a combination of composted garbage and sewage sludge

(approximately 10%) that contained almost no visible inert material. Analysis for both

sources are listed in Appendix A.


Grove Site A

Plant infestation

One-year-old Orlando tangelo trees (Citrus reticulata Blanco X C. paradisi) on

Cleopatra mandarin rootstock (C. reticulata) were acquired from a commercial nursery;

the trees had been grown in Metro-Mix 500 (The Scotts Co., Marysville, OH) in 1000-

cm3 citripots and fertilized with slow release 17-7-10 (N-P20s-K20) Osmocote fertilizer

and Micromax micronutrients (The Scotts Co., Marysville, OH). One month before

planting, approximately 3000 chlamydospores of P. nicotianae were applied in a 0.5-cm

layer of infested soil on top of the potting medium in each citripot treated with the

pathogen. The drainage holes of the citripot were taped shut and the plants were flooded

above the level of the medium in the pot for 7 days to promote zoospore release and









73

infection by P. nicotianae. Inoculated trees were moved to a screenhouse 1 week before

planting to allow adaptation to field conditions.

Field plot

The field experiment was conducted on Candler fine sand at the Citrus Research

and Education Center of the Institute of Food and Agricultural Sciences, University of

Florida, in Lake Alfred, Florida. The field site was fallow for 2 years before the

experiment, so it was assumed that endemic populations of P. nicotianae were very low

if not absent. Approximately 3 months before planting, batch RI of Reuter CMW was

broadcast onto the field plot at a rate of 100 metric tons per hectare. The compost was

incorporated into the soil by disking to a depth of 15 to 30 centimeters.

The experiment was a split-plot design with four-tree plots of CMW and no

CMW in each of eight blocks of eight trees; alternating noninfested and infested trees

were planted in each block. Additional Reuter CMW was incorporated into the backfill

of the compost-amended plots at a level of approximately 20% of the total volume. A

plastic mesh with 1-mm-square holes was placed around the roots of each tree to

delineate the zone where new roots emerged from the root ball. Each tree was planted in

the center of the hole, which was then backfilled and watered well to pack soil around the

roots and eliminate air pockets. The trees were fertilized under procedures recommended

by IFAS for a newly established grove in central Florida (Tucker et al., 1995). Two

years after planting, a layer ofReuter CMW batch R3 was applied to the compost-

amended plots as a 5-cm-thick top dressing (140 metric tons per hectare) extending just

beyond the drip line.











Tree growth and sample analysis

Soil samples were initially taken in late fall (3 months after planting), then in the

spring and fall of each subsequent year, with either a 100-cm3 (2.5-cm diameter) or 980-

cm3 (7.5-cm diameter) auger to a depth of 15 cm. Soil was collected from under the

canopy, approximately halfway between the trunk and drip line. Citrus roots were sieved

from the soil with a 2-mm-mesh screen and weighed. Population densities of P.

nicotianae were estimated by the procedure modified from that of Timmer et al. (1988).

The sieved soil was placed in Styrofoam cups with drainage holes and watered to field

capacity. After 3 days, approximately 10 grams of soil were mixed with 40 mL of 0.25%

water agar. One milliliter of the soil solution was spread over each of 10 plates of the

PARP-H selective medium. After 2 days in an incubator at 27C, the soil was washed off

the plates and the colonies were counted.

The hydrogen ion concentration of the soil medium was measured by the method

of Peech (1965). Ten grams of soil were mixed with 20 mL of 0.01 M CaCI2, stirred

occasionally over a 30-minute period, and allowed to settle for 30 minutes. The pH

values were measured with a Coming 240 pH meter (Corning Corporation, Coming, NY)

and recorded.

The soil was analyzed 4 months after planting by the soil testing laboratory at the

University of Florida (Gainesville) for the carbon:nitrogen ratio using the Walkley-Black

procedure (Allison, 1965) for determination of organic carbon and a total Kjeldahl

nitrogen procedure (Bremner, 1965) for determination of nitrogen.











In the fall of the second year, four leaf samples from the spring flush were

collected from each tree. The leaves were composite from four trees of each treatment

in each block and dried at 650 C. The dried leaves were ground into a fine powder using

a Cyclotec 1093 Sample Mill (Tecator, Inc., Hemdon, VA). The concentration of total N

was determined by a Kjeldahl method using a Buchi 322 distillation unit (Buchi

Laboratories, Switzerland) with a Brinkman automatic titration unit (Brinkman

Instruments, Switzerland). The concentration of phosphorus in leaf tissue was

determined by ICPES analysis (Plasma 40, Perkin-Elmer Corp., Norwalk, CT) after the

samples were ashed at 5000 C for 4 hours and resuspended in 1 M HC1.

The effect of the treatments on tree growth was evaluated by measuring the stem

diameter of the tree 28 cm above the soil line in the spring and the fall of each year. Fruit

was harvested the second and third year after planting, and the weight of fruit per tree

was recorded. The juice from the fruit picked after the third year was analyzed by the

Florida Department of Citrus (Lake Alfred, FL) for percentage juice per fruit on a weight

basis, total soluble solids (TSS) as 'Brix, titratable acid, and kg of solids per box.

Data for each evaluation were analyzed with SAS statistical software (SAS

Institute, Carey, NC) for variance and any interactions between the treatments using the

General Linear Models procedure (GLM) with repeated measures analysis, least

significant differences (LSD), or paired in a t-test to separate the means.











Grove Site B

Plant infestation

One-year-old 'Sunburst' tangerine trees (C. reticulata hybrid) on Sun Chu Sha

mandarin (C. reticulata) rootstock were acquired from a commercial nursery; the trees

had been grown in Metro-Mix 500 (The Scotts Co., Marysville, OH) in 1000-cm3

citripots and fertilized with slow release 17-7-10 (N-P205-K20) Osmocote fertilizer and

Micromax micronutrients (The Scotts Co., Marysville, OH). Approximately 1 month

before planting, the trees were carefully removed from the citripots and 37 g of infested

soil (approximately 7000 chlamydospores per tree) were spread along one side of the root

system. Trees not inoculated with P. nicotianae received noninfested soil. The trees

were then carefully placed back into the citripots, placed in a screenhouse to allow

adaptation to field conditions, and watered every day to maintain adequate soil moisture

for chlamydospore germination.

Field plot

The field experiment was conducted on Candler fine sand at the Citrus Research

and Education Center of the Institute of Food and Agricultural Sciences, University of

Florida, in Lake Alfred, Florida. The field site was fallow for more than 5 years before

the experiment, and no P. nicotianae was detected in sampled field soil.

The experiment was a completely randomized, split-plot design with five-tree

plots infested with P. nicotianae or non-infested with P. nicotianae. Within each block,

trees were randomly chosen for the following treatments: i) no CMW added (control), ii)

CMW from either the Reuter or Bedminster source layered 10 cm on top of the soil after











planting, or iii) CMW from either the Reuter or Bedminster source incorporated into the

backfill at a level of 20% of the total volume (v/v). Three cylinders (3.0 cm in diameter

and 26.0 cm in length) made from 1-mm-mesh plastic screen were filled with soil and

buried in the soil at each distance of 15, 30, and 45 cm from the base of the tree at the

time of planting. The trees were fertilized under procedures recommended by IFAS for a

newly established grove in central Florida (Tucker et al., 1995).

Tree growth and sample analysis

Soil samples were taken in the late spring, 9 months after planting, by carefully

removing one cylinder 15 cm from the tree. Soil samples were also taken in the late fall,

15 months after planting, by carefully removing one cylinder 15 cm and one cylinder 30

cm from the tree. Citrus roots were sieved from the soil with a 2-mm-mesh screen, dried

in an oven at 700 C for 3 days, and weighed. Population densities of P. nicotianae were

estimated by the procedure modified from that of Timmer et al. (1988). The sieved soil

was placed in Styrofoam cups with drainage holes and watered to field capacity. After 3

days, approximately 40 grams of soil were mixed with 40 mL of 0.25% water agar. One

mL of the soil suspension was plated on the PARP-H selective medium. After 2 days at

27C, the soil was washed off the agar and the colonies were counted.

The pH of the soil medium and the concentrations of N and P in leaf tissue were

determined as described for Grove site A. In addition, the concentrations of K, Na, Al,

Cu, Ca, Mg, Mn, Fe, Pb, Zn, and Hg in leaf tissue were determined by ICPES analysis

(Plasma 40, Perkin-Elmer Corp., Norwalk, CT) after the samples were ashed at 500 C

for 4 hours and resuspended in 1 M HC1.











The effect of the treatments on tree growth was evaluated by measuring the stem

diameter of the tree at 28 cm above the soil line in the spring and the fall of each year.

After several hard freezes damaged the trees (18 months after planting), the stem

diameters were measured once more and the trees were removed. The soil was washed

off the roots. The fresh weight of the complete tree, the fresh shoot weight, and the

weight of the complete fresh root system were determined. The fibrous roots were

removed from the root system, dried at 70 C for 5 days, and weighed.

Soil samples were collected from around the root zone after the trees were

removed. Nitrate-nitrogen (N03-N) and ammonium-nitrogen (NH4-N) were analyzed by

the procedure of Maynard and Kalra (1993). Two grams of dried soil were weighed for

each sample (five replicates per treatment), and each sample was placed into a 50-mL

centrifuge tube. Twenty milliliters of 2 M KCI were added to each tube and shaken for

30 minutes. The suspension was allowed to settle for 30 minutes and was then filtered

through Whatman No. 42 filter paper (Whatman Limited, England). A Rapid Flow

Analyzer was used to measure the concentrations of N03-N and NH4-N in the soil

extract.

Soil temperature and moisture analysis

Soil temperature and moisture data were collected using a Campbell 21X

micrologger (Campbell Scientific, Inc., Logan, UT). Five Campbell 227 gypsum soil

moisture blocks (Campbell Scientific, Inc.) were buried 7.5 cm below the soil level,

approximately 15 cm from the trunk of each tree, in one repetition of each of the

following five treatments: control, top-dressed with Reuter CMW, top-dressed with











Bedminster CMW, incorporated with Reuter CMW, and incorporated with Bedminster

CMW. Three Campbell 107B soil/water temperature probes (Campbell Scientific, Inc.)

were buried 7.5 cm below the soil level, approximately 15 cm from the trunk of each tree,

in each of the following three treatments: control, top-dressed with Reuter CMW, and

incorporated with Reuter CMW. The probe leads were connected to the micrologger, and

a program was written to record measurements every 6 hours from an average composite

of measurements taken every 30 minutes (Appendix B). Data for all treatments were

analyzed as described for Grove site A.


Results


Grove Site A

Treatment with CMW significantly increased the size of the trees and the rate of

growth over a 2.75-year period, regardless of whether the trees were infested with P.

nicotianae or not. After 2.75 years, the noninfested, CMW-amended trees had an

average stem diameter of 5.36 cm, and the infested, CMW-amended trees had an average

stem diameter of 3.89 cm. In contrast the noninfested, nonamended trees and infested,

nonamended trees, had stem diameters of 3.44 and 3.46 cm, respectively. Stem diameters

of the two CMW-amended treatments after 2.75 years were significantly higher than the

nonamended treatments (P<0.01). Overall, CMW increased the stem diameter 20% over

nonamended trees. There was a significant interaction between the presence of CMW

and P. nicotianae (P<0.05).











Comparisons of the growth curves using repeated measures analysis showed a

significantly greater growth increase in the two CMW-amended treatments in comparison

to the nonamended treatments (Figure 4-1). The CMW treatment significantly increased

tree growth (P<0.01), while the P. nicotianae treatment significantly decreased tree

growth (P<0.01). A significant interaction between the CMW and P. nicotianae was

detected in the analysis, which is evident by the greater growth increases in the trees,

regardless of the presence of the pathogen (P<0.01).

Phytophthora nicotianae was detected in the roots of 47% of the trees not

inoculated at the beginning of the study. Either the trees were infected in the nursery

before the experiment, or there was residual inoculum in the field before planting.

Statistical analysis was performed using GLM because of uneven sample numbers within

the treatments.

The CMW did not have a significant suppressive effect on P. nicotianae. In fact,

populations of P. nicotianae in soil containing citrus roots were as great or greater in the

CMW-amended plots as in the nonamended plots at each of the sampling times (Figure

4-2).

Composted municipal waste did not significantly affect root density, except at 2

years after planting (Table 4-1). Root density was significantly reduced on trees exposed

to P. nicotianae only during the first half of the experiment. There was not a statistical

correlation between the sampled root density and the growth of the trees (r=0.093).

Leaf analysis did not show a significant increase in leaf concentrations of N, P, or

K in CMW-amended trees, in comparison to the controls (Table 4-2). There were no

















6

















0.0 0.5 1.0 1.5 2.0 2.5 3.0
Time after planting (yr)




Figure 4-1. The effect of composted municipal solid waste (CMW) and infestation with
3-


2-














Phytophthora nicotianae on the growth of young Orlando tangelo trees on Cleopatra
mandarin rootstock at site A. No CMW, not infested (0); no CMW, infested (0); CMW,
not infested (0); and CMW, infested (A). Curves followed by similar letters are not
significantly different (P>0.05) using the GLM procedure for repeated measures analysis
of variance.
















70 -

60 /
60 / -*- CMW
0/ \ --- No CMW
-50 / \N
S40 /
/ V


30 / N



0
120 \N
10 10



0.5 1.0 1.5 2.0 2.5 3.0
Time after planting (yr)




Figure 4-2. The effect of composted municipal solid waste (CMW) on the recovery of
soil populations of Phytophthora nicotianae (estimated as colony forming units [CFU])
from rhizospheres of Orlando tangelo trees on Cleopatra mandarin rootstocks at site A.
Points followed by an indicate significant differences (P>0.05) at the specified
sampling time based on the paired student t-test.











Table 4-1. Effect of composted municipal solid waste (CMW) or inoculation by
Phytophthora nicotianae on root density of Orlando tangelo trees on Cleopatra mandarin
rootstock, 0.75 to 2.75 years after planting in the field at site A.


Fresh root weight (mg/cm3 soil)
Treatment 0.75 years 1.25 years 2.00 years 2.75 years

CMW (-)x 2.96 ae 8.82 a 5.67 b 4.08 a

CMW(+) 2.51 a 9.21 a 9.76 a 3.96 a


P. nicotianae (-)z 3.60 a 11.08 a 8.75 a 4.57 a

P. nicotianae (+) 2.42 b 8.27 b 7.34 a 3.82 a

XAll plots infested or noninfested with Phytophthora nicotianae were combined
according to treatments: no CMW applied (-); CMW applied (+).
YMeans in the same column for CMW or P. nicotianae treatments followed by the same
letter are not significantly different (P>0.05) according to paired t-test.
ZAll plots untreated or treated with CMW were combined according to treatment:
noninfested with P. nicotianae (-); infested with P. nicotianae prior to planting (+).













Table 4-2. Effect of composted municipal waste (CMW) on the uptake of
macroelements: nitrogen (N), phosphorous (P), and potassium (K). Macroelement
concentrations are expressed as percent dry weight of leaf tissue collected from Orlando
tangelo trees 2 years after planting (grove site A) and from 'Sunburst' tangerine trees 1
year after planting (grove site B).

Macroelements (% dry wt)
Treatment at grove site A N P K

CMW ()x 1.93 ae 0.16 a 1.04 a

CMW(+) 1.99 a 0.16 a 1.20 a



Treatment at grove site B N P K

Control 2.05 b 0.15 a 0.95 a

Incorporated Reuter 2.05 b 0.12 b 1.02 a

Incorporated Bedminster 2.13 a 0.13 b 0.77 b

Layered Reuter 2.06 b 0.12 b 1.06 a

Layered Bedminster 2.06 b 0.12 b 0.78 b
XAll plots infested or noninfested with Phytophthora nicotianae were combined
according to treatments: no CMW applied (-); CMW applied (+).
YMeans for each element followed by the same letter are not significantly different
(P>0.05) according to the paired student t-test.
ZAll plots infested or noninfested with Phytophthora nicotianae were combined
according to treatments: control = nontreated plots; incorporated Bedminster =
composted municipal waste (CMW) from Bedminster Corp. (Sevierville, TN)
incorporated into the soil at a level of 20% (v/v) of the total backfill; layered
Bedminster = CMW from Bedminster Corp. applied as a top-dressing 5 cm thick around
the tree; incorporated Reuter = CMW from Reuter Recycling (Pembroke Pines, FL)
incorporated into the soil at a level of 20% (v/v) of the total backfill; layered Reuter =
CMW from Reuter Recycling applied as a top-dressing 5 cm thick around the tree.













significant differences in the microelements, except for Mn, Pb, and Zn (Table 4-3). The

carbon:nitrogen ratios of the soil, 16.1 and 15.5 for CMW-amended plots and

nonamended plots, respectively, were not significantly different. Three years after

planting, there was no significant difference in the soil pH, which averaged 5.5 for non-

amended treatments and 6.1 for CMW-amended treatments.

The CMW treatment alone (CMW treatments with soil infested or noninfested

with P. nicotianae were combined) significantly increased the average fruit weight

(P=0.02) and yield (P=-0.03) 2 years after planting, whereas the effect of P. nicotianae

alone (pathogen treatments with or without CMW were combined) was not significant

(P>0.1). There was no significant interaction between CMW and P. nicotianae on any of

the fruit measurements. After 3 years, the CMW treatment in noninfested soil had

significantly more fruit per tree than the other treatments (P<0.01), but the average

weights were not significantly different (P>0.05) (Table 4-4). There was no significant

interaction between the CMW treatment and the presence of P. nicotianae on the average

fruit weight (P>0.1), but there was a significant interaction on the number of fruit per tree

(P<0.01). The juice yields from the trees were not great enough to allow statistical

comparisons with each other (Table 4-5).

Grove Site B

After 1.5 years from the time of planting, all trees with CMW (data from the P.

nicotianae infested and noninfested plots were combined), except the layered Reuter

treatment, had significantly greater increases in stem diameters than the untreated control













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Table 4-4. Effect of composted municipal solid waste (CMW) and infection by
Phytophthora nicotianae on the number of fruit per tree and the average weight of
individual fruit 2 and 3 years after planting in the field at site A.


Number of Average wt
Treatment fruit/tree of fruit (g)
CMW1 P. nicotianae" No.x 2 yearsy 3 years 2 years 3 years
12 2.5 bz 3.8 b 92 b 139 a

+ 20 4.7 ab 5.9 b 135 ab 156 a

+ 5 5.6 ab 19.2 a 221 a 215 a

+ + 27 9.9 a 3.3 b 173 a 121 a

'Treatments with (+) and without (-) CMW.
wTreatments with (+) and without (-) Phytophthora nicotianae.
"Number of trees per treatment.
YTime elapsed after planting.
zMeans within each column followed by the same letter are not significantly different
(P>0.05) according to LSD analysis.













Table 4-5. Juice analysis of Orlando tangelos harvested 3 years after planting from four
different treatments at grove site A.

Treatment
CMWt P. nicotianae" % Juicev % Acid" TSSx TSS/Acidy Solidsz
63.9 0.60 9.64 16.07 2.52
+ 64.7 0.63 9.54 15.14 2.51
+ 65.2 0.63 10.08 16.00 2.69
+ + 64.8 0.65 9.89 15.22 2.62

'Plots treated (+) or untreated (-) with CMW.
"Plots infested (+) or noninfested (-) with Phytophthora nicotianae.
'Percentage of juice per fruit on a weight basis.
"Percent titratable acid.
XTotal soluble solids expressed as 'Brix.
'Ratio of Total Brix/Acid.
zKilograms of solids per box.













(P<0.01) (Table 4-6). The growth rates of the trees were also significantly affected by

the compost treatments (Figure 4-3). Phytophthora nicotianae was difficult to recover

from soil but did significantly decrease the stem diameters after 1.5 years (Table 4-7).

There was not a significant interaction between the CMW treatment and P. nicotianae

treatment on stem diameter (P=0.87).

No significant differences were detected in the fibrous root densities between the

nontreated control and the CMW-amended treatments 15 cm from the base of the tree

after 0.5 year (Table 4-8). One year after planting root density was greater than the

control only in the incorporated Bedminster treatment 15 cm from the tree. No

significant differences occurred between the treatments 30 cm from the tree after 1 year.

There was not a statistical correlation between the root density measured 15 cm or 30 cm

away from the trunk of the tree after 1 year and the stem diameter (r=0.205 and r=-0.077,

respectively) or total root weight (r=0.228 and r=0.029, respectively). Phytophthora

nicotianae significantly reduced the production of new roots during the first year when

sampled 15 cm from the tree.

After 1.5 years, the total plant fresh weight and the fresh root weight of the

controls were significantly lower than those of the CMW treatments, except for the

layered Reuter treatment (Table 4-9). The dried fibrous root weights were not

significantly different than the controls, except for the incorporated Reuter and layered













Table 4-6. The effect of source and application of composted municipal waste (CMW)
on the increase of stem diameter after 1.5 years of growth at grove site B.

Increase in
Treatment Stem Diameter (cm)

Control 0.79 dz
Incorporated Bedminster 1.01 a
Layered Bedminster 0.99 ab
Incorporated Reuter 0.91 bc
Layered Reuter 0.85 cd

YAl plots infested or noninfested with Phytophthora nicotianae were combined
according to treatments: control = nontreated plots; incorporated Bedminster =
CMW from Bedminster Corp. (Sevierville, TN) incorporated into the soil at a level of
20% (v/v) of the total backfill; layered Bedminster = CMW from Bedminster Corp.
applied as a top-dressing 5 cm thick around the tree; incorporated Reuter = CMW from
Reuter Recycling (Pembroke Pines, FL) incorporated into the soil at a level of 20%
(v/v) of the total backfill; layered Reuter = CMW from Reuter Recycling applied as a
top-dressing 5 cm thick around the tree.
zMeans for treatments, followed by the same letter are not significantly different
(P>0.05) according to the paired student t-test.
















26 -


24- t oa




Sd20 -e
20 -- ...... d
0.. .......... ......




18 I/B
1I1

S16 -- I/LB
.. I/LR
NI/C
14- -0- NI/B
( -.-- NI/IR
--7- NI/LR
12 --- NI/LB


10 II IIIII
0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6
Time after planting (yr)



Figure 4-3. The effect of composted municipal solid waste (CMW) and infestation with
Phytophthora nicotianae on the growth of young 'Sunburst' tangerine trees on Sun Chu
Sha mandarin rootstocks. I/C = infested, nonamended plots (*), I/IB = infested,
incorporated with Bedminster CMW plots (U), I/IR = infested, incorporated with Reuter
CMW plots (A), I/LB = infested, layered with Bedminster CMW plots (*), I/LR =
infested, layered with Reuter CMW plots (v), NI/C = noninfested, nonamended plots (0),
NI/IB = noninfested, incorporated with Bedminster CMW plots (0), NI/IR = noninfested,
incorporated with Reuter CMW plots (A), NI/LB = noninfested, layered with Bedminster
CMW plots (0), and NI/LR = noninfested, layered with Reuter CMW plots (v). Lines
followed by similar letters are not significantly different (P>0.05) using the GLM
procedure for repeated measures analysis of variance.











Table 4-7. The effect of infestation with Phytophthora nicotianae on the increase of stem
diameter of'Sunburst' tangerine trees on Sun Chu Sha rootstocks after 1.5 years of
growth at grove site B.

Increase in
Treatmenty stem diameter (cm)"

P. nicotianae (-) 0.94 a
P. nicotianae (+) 0.88 b

YAll plots untreated or treated with composted municipal waste were combined according
to treatment: noninfested with P. nicotianae (-); infested with P. nicotianae (+) prior
to planting.
'Means for treatments, followed by the same letter are not significantly different
(P>0.05) according to the paired student t-test.











Table 4-8. Effect of composted municipal solid waste (CMW) and infection by
Phytophthora nicotianae on root density 15 and 30 cm from the base of 'Sunburst'
tangerine trees on Sun Chu Sha mandarin rootstocks 0.50 and 1.00 years after planting in
the field at grove site B.

Root Density (mg/cm3)
0.50 year 1.00 year
Treatment 15 cmw 15 cm 30 cm

Control 0.32 ay 0.75 b 0.22 a
Incorporated Bedminster 0.48 a 1.31 a 0.32 a
Incorporated Reuter 0.51 a 0.96 ab 0.16 a
Layered Bedminster 0.38 a 0.76 b 0.38 a
Layered Reuter 0.39 a 0.74 b 0.34 a

P. nicotianae (-)z 0.48 a 1.83 a 0.29 a
P. nicotianae (+) 0.35 b 0.74 b 0.28 a

'Time after planting into field.
"Distance of root baskets from the base of the tree.
XAll plots infested or noninfested with Phytophthora nicotianae were combined
according to treatments: control = nontreated plots; incorporated Bedminster =
CMW from Bedminster Corp. (Sevierville, TN) incorporated into the soil at a level of
20% (v/v) of the total backfill; layered Bedminster = CMW from Bedminster Corp.
applied as a top-dressing 5 cm thick around the tree; incorporated Reuter = CMW from
Reuter Recycling (Pembroke Pines, FL) incorporated into the soil at a level of 20%
(v/v) of the total backfill; layered Reuter = CMW from Reuter Recycling applied as a
top-dressing 5 cm thick around the tree.
YMeans within the columns for CMW or P. nicotianae treatments followed by the same
letter are not significantly different (P>0.05) according to the paired student t-test.
ZAll plots untreated or treated with composted municipal waste were combined according
to treatment: noninfested with P. nicotianae (-); infested with P. nicotianae (+) prior
to planting.











Table 4-9. Effect of composted municipal waste (CMW) on total plant, shoot, and root
weights of'Sunburst' tangerine trees on Sun Chu Sha mandarin rootstocks after 1.5 years
of growth at grove site B.


Total
fresh wt.


Shoot
fresh wt.


Root
fresh wt.


Root
dried wt.


Treatmenty (grams) (grams) (grams) (grams)
Control 900 cz 340 b 560 c 31.0 b
Incorporated Bedminster 1370 a 510 a 860 a 39.8 ab
Incorporated Reuter 1170 b 405 b 765 ab 42.9 a
Layered Bedminster 1390 a 525 a 870 a 47.0 a
Layered Reuter 1050 bc 380 b 660 bc 39.4 ab

YAll plots infested or noninfested with Phytophthora nicotianae were combined
according to treatments: control = nontreated plots; incorporated Bedminster =
CMW from Bedminster Corp. (Sevierville, TN) incorporated into the soil at a level of
20% (v/v) of the total backfill; layered Bedminster = CMW from Bedminster Corp.
applied as a top-dressing 5 cm thick around the tree; incorporated Reuter = CMW from
Reuter Recycling (Pembroke Pines, FL) incorporated into the soil at a level of 20%
(v/v) of the total backfill; layered Reuter = CMW from Reuter Recycling applied as a
top-dressing 5 cm thick around the tree.
zMeans within the same column for CMW treatments followed by the same letter are not
significantly different (P>0.05) according to the paired student t-test.




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