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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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Title:
Effect of composted municipal waste on infection of citrus by Phytophthora nicotianae and the infection of citrus roots by Phytophthora spp
Creator:
Widmer, Timothy Lee, 1964-
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English
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vi, 212 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Citrus trees ( jstor )
Diseases ( jstor )
Hyphae ( jstor )
Infections ( jstor )
Municipal waste ( jstor )
Pathogens ( jstor )
Phytophthora ( jstor )
Plant roots ( jstor )
Soil science ( jstor )
Zoospores ( jstor )
Citrus -- Diseases and pests -- Control ( lcsh )
Dissertations, Academic -- Plant Pathology -- UF ( lcsh )
Phytophthora diseases ( lcsh )
Phytophthora nicotianae ( lcsh )
Plant Pathology thesis, Ph. D ( lcsh )
City of Lake Alfred ( local )
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1996.
Bibliography:
Includes bibliographical references (leaves 189-211).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Timothy Lee Widmer.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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36732417 ( OCLC )
ALF8351 ( NOTIS )

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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




EFFECT OF COMPOSTED MUNICIPAL WASTE ON INFECTION OF CITRUS
BY PHYTOPHTHORA N1COT1ANAE 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 Gods 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 Brothers 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 continous support
throughout my life, none of this would have been possible.


TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS ii
ABSTRACT 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
Materials and Methods 32
Results 43
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
Materials and Methods 70
Results 79
Discussion 99
5 THE EFFECT OF COMPOSTED MUNICIPAL WASTE ON
MANAGEMENT OF PHYTOPHTHORA ROOT ROT IN
MATURE CITRUS TREES 103
in


Introduction 103
Materials and Methods 106
Results 110
Discussion 123
6 THE INFECTION OF CITRUS ROOTS BY PHYTOPHTHORA
N1COTIANAE AND P. PALMIVORA AT THE
ULTRASTRUCTURAL LEVEL 126
Introduction 126
Materials and Methods 130
Results 135
Discussion 166
7 CONCLUSION 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
IV


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.
v


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 of P. 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 signficantly
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.
VI


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
1


2
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).
3


4
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 -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


5
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 of Phytophthora 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 soilbome fungi, such as Phytophthora spp. result in root rot, foot rot, brown


6
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 Carne and P. syritigae
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,


7
gummosis, and root rot (Graham, 1990). Phytophthora palmivora causes similar disease
symptoms but is more restricted in distribution (Zitko and Timmer, 1994). Fibrous root
rot is a common problem in citrus nurseries (Sandler 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.
paradisi 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 (Sandler et
al., 1989). Also, isolates of P. 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


8
(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


9
(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


10
filling, with only 10% being recycled (USEPA, 1989a). Biodegradable organics 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 windrows which require turning to enhance natural airflow
and ensure that all sections of a windrow 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 Comette, 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 35 C were reduced in comparison to those at 28 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 organics (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


14
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 Pathosvstems
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).


15
Composts and other organic soil amendments have been shown to suppress
certain soilbome diseases caused by fungi, including those caused by Rhizoctonia solani
Kuhn, Pythium ultimum Trow, Fusarium oxysporum Schlechtend.:Fr f. sp. conglutincms
(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 vitcola (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 (Solatium 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 soilbome diseases. Certain composts have proven effective in
suppressing soilbome diseases caused by Fusarium spp., Phytophthora spp., Pythium
spp., and Rhizoctonia solani on cyclamen, azaleas, poinsettias, and other ornamentals


16
(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. conglutinaos, 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


17
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 100 C, but not at 60 C. They attributed
the suppression to spore-forming microorganisms, such as Bacillus spp., that tolerated
60 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, 1991a; 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


18
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. cinnamonn 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


19
natural soils (Hie 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.,
1981a), perhaps to phenylalanine and glucose (Nesbitt et al., 1981b) 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) Schrt., 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 (Turney 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 hosts response to invasion.


22
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 Hwl from com, which encodes a reductase that deactivates HC-toxin of
Cochliobolus carbonum Nelson. The third type of resistance genes encodes altered


23
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


24
(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


25
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
hosts 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. infestcms 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


26
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).


27
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).
Callse 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


30
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


31
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


32
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 CaC03 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.


33
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
commeal 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 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.
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, '.-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


34
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 18 C, the mycelium was washed on a 38-pm-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 (uncoated, 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) (R1 and R3) and two from Bedminster


35
Corporation (Sevierville, TN) (B2 and B4). Batch R1 was divided into two subbatches,
one maintained as Rl, 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 CaCl2 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
(Corning 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 Peters 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.


37
Infection of Citis Seedlings bv 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. paradisi 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 27 C. Roots were rated as positive for incidence of
infection if any P. nicotianae colonies were detected. The percentage of total root length


38
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.


39
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 27
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 Rl) 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.


40
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 27 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 pL 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-pm 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 200 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 of DB5 (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 (R1 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 (R1 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 25 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 com meal agar
(CMA) containing 2-day-old cultures of P. nicotianae. The plates were incubated at 25
C, and any interaction between the bacterial colony and P. nicotianae was observed.


43
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


Table 3-1. The effect of concentration of batch B4 of Bedminster (Bedminster Corp., Sevierville, TN) composted municipal waste
(CMW) on total plant fresh weight, fresh shoot weight, and dried root weight of 5-week-old sour orange (Citrus aurantium L.)
seedlings grown for 8 weeks.
Experiment lx Experiment 2
Fresh
Fresh
Dried
Fresh
Fresh
Dried
Treatment*
Total wt (g)
Shoot wt (g)
Root wt (g)
Total wt (g)
Shoot wt (g)
Root wt (g)
Control
2.93
1.79
0.25
2.99
1.76
0.31
10% CMW
2.54
1.58
0.18
2.70
1.52
0.22
20% CMW
1.95
1.23
0.15
2.10
1.27
0.18
50% CMW
1.05
0.72
0.07
1.17
0.73
0.09
Contrasts:2
P> F
P> F
Control vs. 10%
0.145
0.188
0.029
0.161
0.055
0.001
Control vs. 20%
0.001
0.001
0.001
0.001
0.001
0.001
Control vs. 50%
0.001
0.001
0.001
0.001
0.001
0.001
10% vs. 20%
0.029
0.033
0.246
0.006
0.003
0.043
10% vs. 50%
0.001
0.001
0.001
0.001
0.001
0.001
20% vs. 50%
0.002
0.002
0.022
0.001
0.003
0.001
'Two independent experiments were conducted to evaluate the effect of treatments on the total plant fresh weight, fresh shoot weight,
and dried root weight of 5-week-old sour orange seedlings.
treatments consisted of untreated soil (control), 10% of the total soil volume amended with CMW (10% CMW), 20% of the total
soil volume amended with CMW (20% CMW), and 50% of the total soil volume amended with CMW (50% CMW).
zContrast analysis of different treatments using paired t-test.


45
Figure 3-1. Effect of percentage of composted municipal waste (CMW) in soil on the
total plant fresh weight (), the plant fresh shoot weight (), and the dried root weight
(A) of sour orange seedlings grown for 8 weeks. The regression lines fit the following
equations: y=2.9-0.04x (rM).98) for total plant weight, y=1.7-0.02x (^=0.96) for total
shoot weight, and y=0.26-0.004x (r=0.94) for dried root weight.


Table 3-2. The effect of composted municipal waste (CMW) on the incidence of infection by Phytophthora nicotianae of roots of
Ridge Pineapple sweet orange seedlings in soil infested with 10 chlamydospores per cm3; no infection occurred in plants grown in
noninfested controls.
Variety1*
Treatment'
Age of
compost"
Number of
seedlings
Incidence of
infection'
Significance'
Ridge Pineapple
Soil alone
-
40
78%
-
Ridge Pineapple
Reuter, Rlaz
0.2
41
37%
**
Ridge Pineapple
Reuter, Rib
0.2
20
55%
NS
Ridge Pineapple
Reuter, R3
3.0
10
90%
NS
Ridge Pineapple
Bedminster, B2
0.2
10
20%
* *
Ridge Pineapple
Bedminster, B2
2.5
10
90%
NS
Ridge Pineapple
Bedminster, B4
0.3
20
30%
**
Five-week-old Ridge Pineapple sweet orange seedlings used as planting material.
'Pasteurized Candler fine sand nonamended or amended with 20% (v/v) CMW batches Rla, Rib, and R3 from Reuter Recycling
(Pembroke Pines, FL) and batches B2 and B4 from Bedminster Bioconversion (Sevierville, TN).
wThe age of the CMW (years) used in the bioassay.
'Percent of total seedlings infected with P. nicoticmae.
yStatistical analysis of comparisons of infested, nonamended treatments (experiments pooled for each variety; data not shown) and
infested, CMW-amended treatements with the same variety of citrus tested; NS = not significant, = significant (P<0.05), ** =
significant (P<0.01).
?Both Rla and Rib were from the same lot; however, batch Rla was stored at 4 C and batch Rib was stored at room temperature.


Table 3-3. The effect of composted municipal waste (CMW) on the incidence of infection by Phytophthora nicotianae of roots of
Citrumelo hybrid seedlings, F80-3 and F80-8, in soil infested with 10 chlamydospores per cm3; no infection occurred in plants grown
in noninfested controls.
Variety"
Treatmentw
Age of
Compost*
Number of
seedlings
Incidence of
infectiony
Significance2
Citrumelo, F80-3
Soil alone
-
35
60%
-
Citrumelo, F80-3
Reuter, R1
0.2
23
43%
NS
Citrumelo, F80-8
Soil alone
-
35
60%
-
Citrumelo, F80-8
Reuter, R1
0.2
23
22%
*
"Five-week-old seedlings of Citrumelo hybrids, F80-3 and F80-8, were used as planting material.
"Pasteurized Candler fine sand nonamended or amended with 20% (v/v) CMW batch R1 from Reuter Recycling (Pembroke Pines,
FL).
The age of the CMW (years) used in the bioassay.
'Percent of total seedlings infected with P. nicotianae.
Statistical analysis of comparisons of infested, nonamended treatments (pooled for each variety; data not shown) and infested,
CMW = amended treatments with the same variety of citrus tested; NS = not significant, = significant (/%0.05).


Table 3-4. The effect of composted municipal waste (CMW) on the incidence of infection by Phytophthora nicotianae of roots of sour
orange, Cleopatra mandarin, and Volkamer lemon seedlings in soil infested with 10 chlamydospores per cm3; no infection occurred in
plants grown in noninfested controls.
Varietyv
Treatment*
Age of
Compost
Sour orange
Soil alone
-
Sour orange
Reuter, R3
3.0
Sour orange
Bedminster, B4
0.3
Cleo. mandarin
Soil alone
-
Cleo. mandarin
Reuter, R1
0.2
Cleo. mandarin
Bedminster, B2
2.5
Volkamer lemon
Soil alone
-
Volkamer lemon
Reuter, R1
0.2
Volkamer lemon
Bedminster, B2
2.5
Number of
seedlings
Incidence of
infectiony
Significance
20
90%
-
20
75%
-
40
35%
**
16
94%
-
8
13%
**
16
100%
NS
20
80%
-
8
0%
**
20
90%
NS
vTest plants included five-week-old sour orange, Cleopatra mandarin, and Volkamer lemon seedlings.
"'Pasteurized Candler fine sand nonamended or amended with 20% (v/v) CMW batches R1 and R3 from Reuter Recycling (Pembroke
Pines, FL) and batches B2 and B4 from Bedminster Bioconversion (Sevierville, TN)-
xThe age of the CMW (years) used in the bioassay.
'Percent of total seedlings infected with P. nicotianae.
'Statistical analysis of comparisons to infested, nonamended treatments (pooled for each variety; data not shown) and infested CMW-
amended treatments with the same variety of citrus tested; NS = not significant, = significant (Pi0.05), ** = significant (PiO.Ol).
oo


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 R1 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%)


Table 3-5. The effect of different mixtures of composted municipal waste (CMW) on suppression of the incidence of infection of roots
of sour orange seedlings by Phytophthora nicotianae.
Varietyv
Treatment"
CMW ratio"
Number of
seedlings
Incidence of
infectiony
Significance2
Sour orange
Soil alone
0%
20
90%
-
Sour orange
Bedminster, B4
10%
20
40%
**
Sour orange
Bedminster, B4
20%
20
30%
**
Tive-week-old sour orange seedlings used as planting material.
"Pasteurized Candler fine sand nonamended or amended with CMW batch B4 from Bedminster Corporation (Sevierville, TN) less
than 0.5 year after completion of composting process.
"The percent of CMW (v/v) of the total volume amended to the pasteurized soil.
^Percent of total seedlings infected with P. nicotianae.
Statistical analysis of comparisons of infested, nonamended treatments (pooled for each variety; data not shown) and infested, CMW
= amended treatments with the same variety of citrus tested; NS = not significant, = significant (P0.05), ** = significant (P0.01)
according to linear regression.


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 R1 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 (PO.OOl 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 H2S04 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 R1 of Reuter CMW were not significantly different than that in the soil extracts,
except that colony diameter was significantly greater in the H2S04 extract than in the
control.


52
Table 3-6. Effect of batch R1 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
Treatment*
Trial #1 Trial #2 Trial #3
Soil alone
1.88 a;
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.
Pasteurized Candler fine sand nonamended or amended with 20% (v/v) CMW.
Tvleans followed by the same letter within a column are not significantly different
(P>0.05) according to the paired student t-test.


53
Table 3-7. Effect of source of composted municipal waste (CMW) on colony diameter of
Phytophthora nicotianae.
Treatment51 Colony area (cm2)y
Soil alone 1.51 az
20% Bedminster (batch B2) 1.27 ab
20% Reuter (batch Rl) 0.48 b
x Average colony areas of Phytophthora nicotianae grown on PARP-H selective medium
66 hours after plating soil dilutions of infested, CMW-amended or nonamended soils.
Pasteurized Candler fine sand nonamended or amended with 20% (v/v) CMW, batch B2
from Bedminster Bioconversion (Sevierville, TN) or batch Rl from Reuter Recycling
(Pembroke Pines, FL).
zMeans 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.
54
Treatment1
Colony area (cm2)5"
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.
*Means followed by the same letter within a column are not significantly different
(P>0.05) according to the paired student t-test.


55
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 be
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 R1 and R3 of Reuter Recycling CMW (Pembroke Pines, FL).
x Average colony areas of P. nicotianae grown on PARP-H selective medium 66 hours
after plating soil dilutions of infested, CMW-amended or nonamended soils.
-'Means 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 Rla was stored at 4
while Rib was stored at room temperature.


56
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 a'
63.3 a
58.3 b
Reuter CMW
71.3 a
62.0 a
71.3 a
Bedminster CMW
51.9b
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).
^Extractants used on test media: cold, double distilled water (water), 0.4 N KOH (KOH),
2.0 N H2S04 (H2S04).
'Means followed by the same letter within a column are not significantly different
(i^O.05) according to the paired student t-test.


57
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 R1 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 R1 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


58
Figure 3-2. Effect of acetic acid on colony growth of Phytophthora nicotianae on corn
meal agar plates after 96 hours.


acetic acid standard
10
15
Time (minutes)
Jl
Bedminster, B2
Reuter, R3
Reuter, R1
Soil only
i
20
Figure 3-3. Gas chromatograph of acetic acid in comparison to methanol extractions of batch B2 of Bedminster composted municipal
waste (Bedminster Bioconversion, Sevierville, TN), batches R1 and R3 of Reuter CMW (Reuter Recycling, Pembroke Pines, FL) and
Candler fine sand


Detector
Figure 3-4. Gas chromatograph of acetic acid in comparison to an extract of batch B4 Bedminster composted municipal waste
(Bedminster Bioconversion, Sevierville, TN) using the headspace solid-phase microextraction technique.
Os
O


61
Figure 3-5. Acremonium sp. hyperparasitizing Phytophhora 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.


62
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 4 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 R1 and B2 were effective in suppressing root infection when they were
utilized soon after the composting process was completed. However, R1 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.


64
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,


65
this initial effectiveness may be enough to give seedlings or young trees enough time to
outgrow the pathogens 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
Rl, 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 Rl 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
67


68
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
(iCitrus aurantium L.), Carrizo citrange (C. sinensis (L.) Osbeck X Poncirus trifoliata
(L.) Raf), and Swingle citrumelo (C. paradisi 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


69
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.


70
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 CaC03 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
commeal 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 of P. 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, '/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 18 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 (uncoated, hyperthermic, Tipie Quartzipsamments), collected
near Davenport, FL, was pasteurized by microwaving 1 kg of moist (approximately 7%


72
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 R1 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 tngelo 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-P205-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 R1 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 IF AS for a newly established grove in central Florida (Tucker et al., 1995). Two
years after planting, a layer of Reuter 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.


74
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 CaCl2, stirred
occasionally over a 30-minute period, and allowed to settle for 30 minutes. The pH
values were measured with a Corning 240 pH meter (Corning Corporation, Corning, 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.


75
In the fall of the second year, four leaf samples from the spring flush were
collected from each tree. The leaves were composited from four trees of each treatment
in each block and dried at 65 C. The dried leaves were ground into a fine powder using
a Cyclotec 1093 Sample Mill (Tecator, Inc., Herndon, 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 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 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.


76
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


77
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 IF AS 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 70 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.


78
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 KC1 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


79
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 (/^O.Ol). 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).


80
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 analyis, 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


81
Time after planting (yr)
Figure 4-1. The effect of composted municipal solid waste (CMW) and infestation with
Phytophthora nicotianae on the growth of young Orlando tngelo trees on Cleopatra
mandarin rootstock at site A. No CMW, not infested (); no CMW, infested (o); 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.


82
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 tngelo 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.


83
Table 4-1. Effect of composted municipal solid waste (CMW) or inoculation by
Phytophthora nicoticmae on root density of Orlando tngelo 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 ay
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
XA11 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.
ZA11 plots untreated or treated with CMW were combined according to treatment:
noninfested with P. nicotianae (-); infested with P. nicotianae prior to planting (+).


84
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
tngelo 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 ay
0.16a
1.04 a
CMW (+)
1.99 a
0.16a
1.20 a
Treatment at grove site B
N
P
K
Control2
2.05 b
0.15 a
0.95 a
Incorporated Reuter
2.05 b
0.12b
1.02 a
Incorporated Bedminster
2.13 a
0.13 b
0.77 b
Layered Reuter
2.06 b
0.12b
1.06 a
Layered Bedminster
2.06 b
0.12 b
0.78 b
XA11 plots infested or noninfested with Phytophthora nicotianae were combined
according to treatments: no CMW applied (-); CMW applied (+).
Means for each element followed by the same letter are not significantly diffferent
(P>0.05) according to the paired student t-test.
ZA11 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.


85
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 (/*>().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


Table 4-3. Effect of composted municipal waste (CMW) on the uptake of the following microelements: sodium (Na), aluminum (Al),
copper (Cu), calcium (Ca), magnesium (Mg), manganese (Mn), iron (Fe), lead (Pb), zinc (Zn), and mercury (Hg). Microelement
concentrations are expressed as ppm of leaf tissue collected from Orlando tngelo trees 2 years after planting (grove site A), and
Sunburst tangerine trees 1 year after planting (site B).
Treatment at grove site A
Na
Al
Cu
Microelements (ppm)
Ca Mg
Mn
Fe
Pb
Zn
Hg
CMW (-)x
8.5 ay
1.06 a
0.05 a
183 a
23.7 a
0.21 b
0.31 a
0.06 a
0.08 b
0 a
CMW (+)
8.5 a
1.23 a
0.08 a
190 a
23.0 a
0.26 a
0.41 a
0.04 b
0.13 a
0 a
Treatment at grove site B
Na
Al
Cu
Ca
Mg
Mn
Fe
Pb
Zn
Hg
Control2
9.4 a
1.13 a
0.03 a
211 a
36.0 a
0.07 a
0.23 b
0.08 a
0.12a
0 a
Incorporated Reuter
11.4a
1.20 a
0.03 a
245 a
27.1 d
0.05 b
0.26 b
0.07 a
0.16a
0 a
Incorporated Bedminster
9.6 a
1.22 a
0.03 a
248 a
29.6 be
0.07 a
0.30 a
0.08 a
0.13 a
0 a
Layered Reuter
11.0a
1.18 a
0.03 a
232 a
28.4 cd
0.06 a
0.25 a
0.08 a
0.11 a
0 a
Layered Bedminster
10.1 a
1.16a
0.03 a
248 a
30.2 b
0.06 a
0.25 a
0.07 a
0.11 a
0 a
All plots infested or noninfested with Phytophthora nicotianae were combined according to treatments: no CMW applied (-); CMW
applied (+).
'Means for each element followed by the same letter are not significantly diffferent (P>0.05) according to the student paired t-test.
ZA11 plots infested or noninfested with P. nicotianae were combined according to treatments: control = nontreated; 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.
oo
On


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.
87
Number of Average wt
Treatment
fruit/tree
of fruit (g)
CMW"
P. nicotianaew
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
-I-
-
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.
"Treatments with (+) and without (-) Phytophthora nicotianae.
Number of trees per treatment.
yTime elapsed after planting.
Means within each column followed by the same letter are not significantly different
(P>0.05) according to LSD analysis.


88
Table 4-5. Juice analysis of Orlando tngelos harvested 3 years after planting from four
different treaments at grove site A.
Treatment
CMW1 P. nicotianae
% Juice'
% Acid"
TSSX
TSS/Acidy Solids2
-
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.
TCilograms of solids per box.


89
(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


90
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)
ControP
0.79 dz
Incorporated Bedminster
1.01 a
Layered Bedminster
0.99 ab
Incorporated Reuter
0.91 be
Layered Reuter
0.85 cd
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.
"Means for treatments, followed by the same letter are not significantly different
(P>0.05) according to the paired student t-test.


91
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 (), 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 (), NI/C = noninfested, nonamended plots (o),
NI/IB = noninfested, incorporated with Bedminster CMW plots (), 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.


92
Table 4-7. The effect of infestation with Phytophthora nicotianae on the increase of stem
diameter ofSunburst tangerine trees on Sun Chu Sha rootstocks after 1.5 years of
growth at grove site B,
Increase in
Treatment stem diameter (cm)z
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.
zMeans for treatments, followed by the same letter are not significantly different
(Z^O.05) according to the paired student t-test.


93
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 ofSunburst
tangerine trees on Sun Chu Sha mandarin rootstocks 0.50 and 1.00 years after planting in
Treatment
Root Density (mg/cm3)
0.50 year"
1.00 year
15 cm*
15 cm
30 cm
ControP
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.
XA11 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.
'Means 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.
ZA11 plots untreated or treated with composted municipal waste were combined according
to treatment: noninfested with P. nicotianae (-); infested with P. nicotianae (+) prior
to planting.


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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

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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 i n my work and who let me use equipment in the i r 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 thei r continous support throughout my life none of this would have been possible 11

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TABLE OF CONTENTS ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . ii ABSTRACT ......... . ... ...................................... 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 Materials and Methods . . . . . . . . . . . . . . . . 3 2 Results ............................................ 43 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 Materials and Methods . . . . . . . . . . . . . . . . 70 Results ... .......................................... 79 Discussion . . . . . . . . . . . . . . . . . . . . . 99 5 THE EFFECT OF COMPOSTED MUNICIPAL WASTE ON MANAGEMENT OF PHYTOPHTHORA ROOT ROT IN MATURE CITRUS TREES ................................. 103 111

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Introduction . . . . . . . . . . . . . . . . . . . . 103 Materials and Methods . . . . . . . . . . . . . . . . 106 Results . . . . . . . . . . . . . . . . . . . . . . 110 Discussion . . . . . . . . . . . . . . . . . . . . 123 6 THE INFECTION OF CITRUS ROOTS BY PHYTOPHTHORA NICOTIANAE AND P. PAIMIVORA AT THE ULTRASTRUCTURAL LEVEL . . . . . . . . . . . . . . . 126 Introduction . . . . . . . . . . . . . . . . . . . . 126 Materials and Methods . . . . . . . . . . . . . . . . 130 Results . . . . . . . . . . . . . . . . . . . . . . 13 5 Discussion . . . . . . . . . . . . . . . . . . . . . 166 7 CONCLUSION . . . . . . . . . . . . . . . . . . . . . . 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 PAIMIVORA ............................................ 183 D EFFECT OF ACETIC ACID ON CITRUS ROOT INFECTION . . . 185 E EFFECT OFACREMONIUMSP. ON CITRUS ROOT INFECTION .. 187 REFERENCE LIST 189 BIOGRAPIIlCAL SKETCH . . . . . . . . . . . . . . . . . . . 212 IV

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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 CO:MPOSTED 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 Oto 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 V

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Field trials using I-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 15year-old Valencia orange on Carrizo citrange rootstock and 25-year-old white grapefruit on sour orange rootstock were selected based upon high soil populations of P 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 signficantly 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 preand 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 vi

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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

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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 rnicroflora In some cases this rnicroflora 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 2

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CHAPTER2 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 sporang i a germinat i ng 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) 3

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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 oc-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) 4 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

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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 of Phytophthora 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 ofrainfall 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 5

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rot of fruit reduced fruit quality and yield and under optimum conditions trees may be killed (Timmer et al., 1989) 6 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 (RE. Sm. & E H Sm.) Leonian. Other species that have been reported from citrus in limited geographical areas include P. hibernalis Carne 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

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gummosis and root rot (Graham 1990) Phytophthora palmivora causes similar disease symptoms but is more restricted in distribution (Zitko and Timmer 1994) Fibrous root rot is a common problem in citrus nurseries (Sandler et al., 1989) and about 90% of the field nurseries assayed in Florida are infested with P nicotianae (Fisher 1993) 7 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 trifo/iata [L.] Raf. ) Troyer and Carrizo citranges (C. sinensis [L.] Osbeck X P. trif oliata [L. ] Raf), and Swingle citrumelo (P. trif o/iata [L. ] Raf X C. paradisi 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 (Sandler et al., 1989) Also isolates of P 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 Phvtophthora 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

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(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) 8 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

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(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 l 992 ~ 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 9

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10 filling with only 10% being recycled (USEP A 1989a) Biodegradable organics 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 (USEP A, 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 (lobar 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

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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 windrows which require turning to enhance natural airflow and ensure that all sections of a windrow 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 hurnic 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)

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12 Although it is impractical to incorporate compost into the so i l 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., 196 I). 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

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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-Co x 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 35 C were reduced in comparison to those at 28 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 organics (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

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14 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)

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15 Composts and other organic soil amendments have been shown to suppress certain soilborne diseases caused by fungi including those caused by Rhiz octonia solani Kuhn Pythium ult imum Trow, F u s arium 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 Pla s mopara viticola (Berk. & M .A. Curtis ex de Bary) Berl. & de Toni and powdery mildew caused by Uncinula necator (Schwein ) Burr, on grape (Viti s vinifera L.) ; late blight of potato (Solanum tub e rosum L. cv. Grata ) and tomatoes (L y copersicon e s culentum Miller cv Rheinglut ) caused by Phytophthora infes tans ; powdery mildew of barley (Hord e um vulgare L. emend. Bowden cv Gerbel ) caused by Blumeria gramini s (DC) Speer f sp hordei Em. Marchal ; and white mold caused by Botryti s 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 F u sa ri u m spp Phytophthora spp. Pythium spp and Rhizoctonia solani on cyclamen azaleas poinsettias and other ornamentals

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16 (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 Su1mression The exact mechanisms involved in suppression of plant diseases with composts are unclear. However the chemical and physical properties of compost and the biology

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17 of microorganisms coloniz i ng 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 100 C but not at 60 C They attributed the suppression to spore-forming microorganisms such as Bacillus spp. that tolerated 60 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 Bacillu s spp., Ente robacter spp., Flavobact e rium balustinum Harrison Pseudomonas spp ., Streptomyce s spp Tri chod e rma spp., and Gliocladium v ir ens Miller Giddens & Foster (Chung and Hoitink 1990 ; Hardy and Sivasithamparam 1991a ; 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

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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) 18 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 soilbome 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 ofless than 7 days (Tsao 1969) Lysis of mycelium occurs rapidly in

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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-Kahana 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. 19 (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., 1981a) perhaps to phenylalanine and glucose (Nesbitt et al., 1981b) 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) Schrot. 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

PAGE 26

20 culture of Bacillus cereus Frank and Frank reduced mortality of alfalfa (Medicago saliva 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 rnicrographs 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)

PAGE 27

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

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22 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 of400 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 RC-toxin of Cochliobolus carbonum Nelson The third type of resistance genes encodes altered

PAGE 29

targets for pathogen-derived molecules required for pathogenicity The fourth type of resistance genes encodes structural or constitutive biochemical barriers to the pathogen 23 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. i nfestans

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24 (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

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term different pathogens infect host tissue in various ways that can result in a different response by the host. 25 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

PAGE 32

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. 26 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 endoderrnis 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)

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27 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

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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

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CHAPTER3 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 (USEP A, 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

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30 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 88130 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

PAGE 37

31 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

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32 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 nicotiana e 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 ofV-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 ofBacto agar.

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33 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 (P ARP-H) was prepared as a modification of the following procedure by Mitchell and Kannwischer-Mitchell (1992) Seventeen grams of Difeo cornmeal agar (Difeo Laboratories Detroit, MI) were added to 1 L of deionized water and autoclaved for 15 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 ofhymexazol {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 -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

PAGE 40

34 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 18 C the mycelium was washed on a 38-m-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 (uncoated hyperthermic Typic Quartzipsarnments) was first steam sterilized for 5 hours at 120 Cat 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. palmi vora 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) (R 1 and R3) and two from Bedminster

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Corporation (Sevierville TN) (B2 and B4) Batch RI was divided into two subbatches one maintained as RI, 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 35 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 of0.01 M CaCl2 solution stirred several times for 30 minutes and then allowed to stand for 30 minutes The pH values were measured with a Coming 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 I-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

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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 (ANO VA) with linear regression calculated to distinguish significance

PAGE 43

Infection of Citrus Seedlings by P. nicotiana e Seedlings of 'Ridge Pineapple' sweet orange (C. sin e nsis [L.] Osbeck) citrumelo hybrids F80-3 and F80-8 (Poncirus trifoliata [L.] Raf. X C. paradisi Macf.), Cleopatra mandarin (C. re s hni 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 37 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 IO 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 2010-20 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 P ARP-H selective medium. The plates were incubated for 2 days at 27 C Roots were rated as positive for incidence of infection if any P. nicotianae colonies were detected The percentage of total root length

PAGE 44

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 38 Samples of Candler fine sand and Wabasso fine sand (siliceous hyperthermic Alfie Haplaquod) were pasteurized by microwav i ng 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 of0.25% 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. nicotiana e 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

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39 Effect of Acetic Acid on Colony Growth Com meal agar (Difeo Laboratories Detroit MI) was prepared by mixing 3 9 0 g in I 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. nicotiana e grown on V-8 agar was placed in the middle of each plate and the plates were i ncubated at 27 C. After 96 hours the diameters of the colonies of P n i co ti ana e 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 H2SO4 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-m membrane One hundred milliliters of each sterile filtrate were added to the individual flasks of900 mL of liquid CMA supplemented with I mL oftergitol 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 H2SO4

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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 nicotiana e grown on V-8 medium was placed in the middle of each plate, the plates were incubated at 27 C The colony diameters were measured over time Chromatography 40 One gram of Reuter (RI 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 Lor 0 5 L 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-m 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

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41 through the septum of the vial and a plunger was depressed to expose a 1-cm-long 100m-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 200 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-m 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 3 5 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 I 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) (Difeo 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

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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 (Rl and R3) and Bedminster (B2) were diluted I : 30,000 by adding 0 .33 g (dry weight) of the CMW to I 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 I 00 mg of cycloheximide One milliliter of each dilution was pipetted into each of IO sterile petri plates. Approximately 16 mL of cooled (50 C) 10% Tryptic Soy Broth (TSB) medium (1.5 g ofTSB 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 25 C Individual colonies were transferred aseptically to petri plates of TSB amended with I 00 mg of cycloheximide per liter of medium Individual colonies of fungal isolates were transferred to solidified com meal agar (CMA) containing 2-day-old cultures of P. nicotianae. The plates were incubated at 25 C and any interaction between the bacterial colony and P. nicotianae was observed

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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 ofCMW. 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 nicotiana e 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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Table 3-1 The effect of concentrati o n of batch B4 of Bedminster (Bedminster Corp., Sevierville TN) composted municipal waste (CMW) on total plant fresh weight fresh shoot weight and dried root weight of 5-week-old sour orange (Citros aurantium L.) seedlings grown for 8 weeks Experiment 1 x Experiment 2 Fresh Fresh Dried Fresh Fresh Dried Treatment Y Total wt (g) Shoot wt (g) Root wt (g) Total wt (g) Shoot wt (g) Root wt (g) Control 2 .93 1.79 0 25 2 99 1.76 0.31 10%CMW 2 54 1.5 8 0 .18 2 70 1.52 0 22 20%CMW 1.95 1.23 0 .15 2 .10 1.27 0 .18 50%CMW 1.05 0.72 0 07 1.17 0 .73 0.09 C o ntrasts :2 P>F P>F Control vs 10% 0 145 0 188 0 029 0 .161 0 055 0 .001 Control vs 20% 0 001 0 001 0 .001 0 .001 0 .001 0 .001 Control vs 50% 0 .001 0.001 0.001 0 .001 0 .001 0 .001 10% vs 20% 0 029 0 033 0 246 0 006 0 003 0 043 10% vs 50% 0 .001 0 .001 0 .001 0 .001 0 .001 0 .001 20% vs 50% 0 002 0 002 0 022 0 .001 0 003 0 .001 x Two independent experiments were conducted to evaluate the effect of treatments on the total plant fresh weight fresh shoot weight and dried root weight of 5-week-old sour orange seedlings Y Treatments consisted of untreated soil (control), 10% of the total soil volume amended with CMW (10% CMW) 20% of the total soil volume amended with CMW (20% CMW) and 50% of the total soil volume amended with CMW (50% CMW) 2Contrast analysis of different treatments using paired t-test.

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3~-------------------------, --Total plant w eight ---Total shoot weight -Dried root weight 2 -..------0-+----------r---------r----------i 0 20 40 60 Percent CMW mi x ture Figure 3-1 Effect of percentage of composted municipal waste (CMW) in soil on the total plant fresh weight(), the plant fresh shoot weight(), and the dried root weight (A) of sour orange seedlings grown for 8 weeks The regression lines fit the following equations : y=2 9-0 04x (r2=0 98) for total plant weight y=l.7-0.02x (r2=0 96) for total shoot weight and y=0 26-0 004x (r2=0 94) for dried root weight. 45

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Table 3-2 The effect of composted municipal waste (CMW) on the incidence of infection by Phytophthora nicotianae of roots of 'Ridge Pineapple' sweet orange seedlings in soil infested with 10 chlamydospores per cm3; no infection occurred in plants grown in noninfested controls Varietyu Treatmentv --Ridge Pineapple Soil alone Ridge Pineapple Reuter Rlaz Ridge Pineapple Reuter Rlb Ridge Pineapple Reuter R3 Ridge Pineapple Bedminster B2 Ridge Pineapple Bedminster B2 Ridge Pineapple Bedminster B4 Age of compost w 0 2 0 2 3 0 0 2 2 5 0 3 Number of seedlings -40 41 20 10 10 10 20 uFive-week-old 'Ridge Pineapple sweet orange seedlings used as planting material. Incidence of infectionx 78% 37% 55% 90% 20% 90% 30% Significanc&' ** NS NS ** NS ** vpasteurized Candler fine sand nonamended or amended with 20% (v/v) CMW batches Rla, Rib, and R3 from Reuter Recycling (Pembroke Pines FL) and batches B2 and B4 from Bedminster Bioconversion (Sevierville, TN). wThe age of the CMW (years) used in the bioassay xpercent of total seedlings infected with P. nicotianae Y Statistical analysis of comparisons of infested nonamended treatments ( experiments pooled for each variety ; data not shown) and infested CMW-amended treatements with the same variety of citrus tested ; NS = not significant = significant (P ~ 0 05) ** = significant (P 0 01) Z:Soth Rla and Rlb were from the same lot ; however batch Rla was stored at 4 C and batch Rlb was stored at room temperature 0\

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Table 3-3 The effect of composted municipal waste (CMW) on the incidence of infection by Phytophthora nicotianae of roots of Citrumelo hybrid seedlings F80-3 and F80-8 in soil infested with IO chlamydospores per cm3 ; no infection occurred in plants grown in noninfested controls Variety v --Citrumelo F80-3 Citrumelo F80-3 Citrumelo F80-8 Citrumelo F80-8 Treatmentw Soil alone Reuter, RI Soil alone Reuter, RI Age of Compost' 0.2 0 2 Number of Incidence of seedlings infection Y -35 60% 23 43% 35 60% 23 22% five-week-old seedlings of Citrumelo hybrids F80-3 and F80-8 were used as planting material. Significance2 NS wpasteurized Candler fine sand nonamended or amended with 20% (v/v) CMW batch RI from Reuter Recycling (Pembroke Pines FL). x The age of the CMW (years) used in the bioassay YFercent of total seedlings infected with P nicotianae 2Statistical analysis of comparisons of infested nonamended treatments (pooled for each variety ; data not shown) and infested CMW = amended treatments with the same variety of citrus tested ; NS= not significant, = significant (P:::50 05) -..J

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Table 3-4 The effect of composted municipal waste (CMW) on the incidence of infection by Phytophthora nicotianae of roots of sour orange, Cleopatra mandarin, and Volkamer lemon seedlings in soil infested with 10 chlamydospores per cm3 ; no infection occurred in plants grown in noninfested controls Varietyv Treatmentw --Sour orange Soil alone Sour orange Reuter, R3 Sour orange Bedminster, B4 Cleo mandarin Soil alone Cleo. mandarin Reuter, Rl Cleo mandarin Bedminster, B2 Volkamer lemon Soil alone Volkamer lemon Reuter, Rl Volkamer lemon Bedminster, B2 Age of Compostx 3 0 0 3 0 2 2.5 0 2 2 5 Number of seedlings -20 20 40 16 8 16 20 8 20 rest plants included five-week-old sour orange Cleopatra mandarin and Volkamer lemon seedlings Incidence of infectionY 90% 75% 35% 94% 13% 100% 80% 0% 90% wi>asteurized Candler fine sand nonamended or amended with 20% (v / v) CMW batches RI and R3 from Reuter Recycling (Pembroke Pines FL) and batches B2 and B4 from Bedmin s ter Bioconversion (Sevierville TN) rhe age of the CMW (years) used in the bioassa y >'Percent of total seedlings infected with P nicotianae Statistical analysis of comparisons to infested nonamended treatment s (pooled for each variety ; data not shown) and infested CMW amended treatments with the same variety of citrus tes ted ; NS= not significant = significant (P:S0 05) = significant (P:S0 01) Significance2 ** ** NS ** NS 00

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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 5year-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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Table 3-5 The effect of different mixtures of composted municipal waste (CMW) on suppression of the incidence of infection of roots of sour orange seedlings by Phytophthora nicot i anae Number of Incidence of Varietyv Treatment w CMW ratio x seedlings infection Y Significance z Sour orange Soil alone 0% 20 90% Sour orange Bedminster B4 10% 20 40% ** Sour orange Bedminster B4 20% 20 30% * five-week-old sour orange seedlings used as planting material. WJ>asteurized Candler fine sand nonamended or amended with CMW batch B4 from Bedminster Corporation (Sevierville TN) less than 0 5 year after completion of composting process x The percent of CMW (v/v) of the total volume amended to the pasteurized soil. YJ>ercent of total seedlings infected with P. nicotianae z statistical analysis of comparisons of infested nonamended treatments (pooled for each variety ; data not shown) and infested CMW = amended treatments with the same variety of citrus tested ; NS = not significant = significant (P~ 0 05) **=significant (P~ 0 01) according to linear regression Vl 0

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(P= 0 002) Populations of P. nicotiana e 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 51 Soil amended with batch RI from Reuter Recycling significantly reduced the colony area of P. nicotiana e 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 CM.A, 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.

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52 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 nicotiana e Treatment Y Soil alone CMW-amended soil Trial #1 1 .88 a2 0 90 b Colony area ( cm2Y Trial # 2 1.55 a 0 52 b Trial # 3 1. 00 a 0.43 b x Average 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. 2Means followed by the same letter within a column are not significantly different (P>0.05) according to the paired student t-test.

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53 Table 3-7 Effect of source of composted municipal waste (CMW) on colony diameter of Phytophthora nicotianae Treatment' Colony area ( cm2)Y Soil alone I 5 I a z 20% Bedminster (batch B2) I 27 ab 20% Reuter (batch RI) 0 48 b x Average colony areas of Phytophthora nicotianae grown on P ARP-H selective medium 66 hours after plating soil dilutions of infested CMW-amended or nonamended soils YJ>asteurized 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) ~eans followed by the same letter are not significantly different (P>0 05) according to the paired student t-test.

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Table 3-8 Effect of proportion of composted municipal waste (CMW) in soil on colony diameter of Phytophthora nicotianae Treatment x Soil alone 10% Bedminster B4 20% Bedminster, B4 Colony area ( cm2)Y 1.39 a2 0 .83 b 0 80 b 54 "Pasteurized Candler fine sand nonamended or amended with either 10% or 20% (v/v) of batch B4 of Bedminster Bioconversion CMW (Sevierville TN) Y Average colony areas of P nicotianae grown on PARP-H selective medium 66 hours after plating soil dilutions of infested, CMW-amended or nonamended soils 2Means followed by the same letter within a column are not significantly different (P>0 05) according to the paired student t-test.

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Table 3-9 Effect of composted municipal waste (CMW) and soil type on the colony growth of Phytophthora nicotianae Soil type v CMW treatment w Colony area ( cm2Y Candler soil alone 1.51 a Y Candler Bedminster B2 0 .73 C Candler Reuter Rla2 0 .71 C Candler Reuter Rlb 1.11 b Candler Reuter R3 1.11 b Wabasso soil alone 1.37 a Wabasso Bedminster B2 0 77 be Wabasso Reuter Rla 0 .55 C Wabasso Reuter Rlb 0 97 b Wabasso Reuter R3 1.05 b vrwo different soil types collected near Davenport FL (Candler fine sand) and Fort Pierce FL (Wabasso fine sand) w soil treatments consisted of batch I of Bedminster Bioconversion CMW (Sevierville TN) and batches RI and R3 of Reuter Recycling CMW (Pembroke Pines FL) x Average colony areas of P. nicotianae grown on PARP-H selective medium 66 hours after plating soil dilutions of infested CMW-amended or nonamended soils >Means followed by the same letter within a column are not significantly different (P>0 05) according to the paired student t-test. 2Rla and Rlb are from the same batch ofReuter CMW except Rla was stored at 4 while Rlb was stored at room temperature. 55

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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 com meal agar. Colony diameter (rnmt Treatment' Water y KOH H 2 SO 4 --Soil 70 9 a2 63. 3 a 58 3 b ReuterCMW 71. 3 a 62 0 a 71.3 a Bedminster CMW 51.9 b 54 7 b 50 7 C w Average colony diameters of P. nicotianae grown on com meal agar medium x soil 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) 56 YExtractants used on test media : cold double distilled water (water), 0.4 N KOH (KOH), 2.0 N H2SO4 CH2SO4). 2Means followed by the same letter within a column are not significantly different (P>0 05) according to the paired student t-test.

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57 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 Rl 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 Rl 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

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70 65 y = 66.8 -0 .lx -3.lx2 60 r2 = 0 .995 i 55 li 50 0 8 45 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 ni c otiana e on com meal agar plates after 96 hours 58

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l M .9 8 0 s 10 l 15 20 Time (minutes) acetic acid standard -Bedminster, B2 Reuter,R3 Reuter RI Soil only Figure 3-3 Gas chromatograph of acetic acid in comparison to methanol extractions of batch B2 of Bedminster composted municipal waste (Bedminster Bioconversion Sevierville TN) batches RI and R3 of Reuter CMW (Reuter Recycling Pembroke Pines FL) and Candler fine sand Vl \0

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i g __________________ A\_ r Bedminster,B4 0 1 2 3 Time (minutes) 4 5 Figure 3-4 Gas chromatograph of acetic acid in comparison to an extract of batch B4 Bedminster composted municipal waste (Bedminster Bioconversion Sevierville TN) using the headspace solid-phase microextraction technique 0

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61 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 nicotiana e hypha (x 1800) p = P nicotianae hyphae a= Acremonium sp. hyphae bar = 10 m.

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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 62 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 4 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

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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 Rl and B2 were effective in suppressing root infection when they were utilized soon after the composting process was completed However Rl 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. nicotiana e Even at a concentration of only 10% (v/v) CMW suppressed incidence of infection regardless of the citrus variety tested

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64 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

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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 (De Vleeschauwer 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 65 R 1, 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 RI CMW but not from plates with soil dilutions from the nonsuppressive batch R3 or batch B2 When grown together Acre monium 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 com 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, Spha e rotheca fuliginea (Malathrakis 1985) The suppression of infection caused by CMW may result

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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

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CHAPTER4 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 need& 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 67

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68 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. paradisi 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

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69 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 Rhi z octonia s olani Pythium ultim u m 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

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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 70 Clarified V-8 medium was prepared by mixing 2 29 grams of CaCO3 with 163 mL ofV-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 Difeo cornmeal agar (Difeo 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

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71 A strain of P 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 ofa 5-day-old culture of P. nicotianae on V-8 agar were aseptically transferred to 15 mL of sterile, -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 18 C the mycelium was washed on a 38-m-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 (uncoated hyperthermic Tipic Quartzipsamments) collected near Davenport FL was pasteurized by microwaving 1 kg of moist ( approximately 7%

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72 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 ofCMW 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 Appendi x A. Grove Site A Plant infestation One year-old Orlando tangelo trees (Citrus re ticulata 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 1000cm 3 citripots and fertilized with slow release 17-7-10 (N-P205-K20) Osmocote fertilizer and Micromax micronutrients (The Scotts Co., Marysville, OH) One month before planting approximately 3000 chlamydospores of P. nicotiana e 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

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73 infection by P. nicotiana e 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 nicot i ana e were very low if not absent. Approximately 3 months before planting batch R 1 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 ex periment 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 I-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 of Reuter 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

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74 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-cm 3 (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 of0. 25% water agar One milliliter of the soil solution was spread over each of 10 plates of the P ARP-H selective medium After 2 days in an incubator at 27 C 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 of0.01 M CaCl2 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

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75 In the fall of the second year four leaf samples from the spring flush were collected from each tree The leaves were composited from four trees of each treatment in each block and dried at 65 C The dried leaves were ground into a fine powder using a Cyclotec 1093 Sample Mill (Tecator Inc Herndon 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 500 C for 4 hours and resuspended in I M HCI. 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 0Brix, 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 at-test to separate the means

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Grove Site B Plant infestation 76 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 177-10 (N-E205-K20) Osmocote fertilizer and Micromax micronutrients (The Scotts Co., Marysville OH) Approximately I month before planting the trees were carefully removed from the citripots and 3 7 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 IO cm on top of the soil after

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77 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 I-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 IF AS 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 70 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 of0.25% 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 ofN and Pin 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 HCI.

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78 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 (NO3-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 KCl 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 ofNO3-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

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79 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 of3.44 and 3.46 cm respectively Stem diameters of the two CMW-amended treatments after 2 7 5 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)

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80 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 analyis 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 4 7% 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 Kin CMW-amended trees in comparison to the controls (Table 4-2) There were no

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81 6 s a a 4 b C a C j 3 J 2 0-------------------------0 0 o s 1.0 u 2 0 2 5 3. 0 Time after planting (y r) Figure 4-1 The effect of composted municipal solid waste (CMW) and infestation with Phytophthora nicotianae on the growth of young Orlando tangelo trees on Cleopatra mandarin rootstock at site A No CMW, not infested (D) ; no CMW, infested (o); CMW, not infested (O) ; and CMW, infested (t>). Curves followed by similar letters are not significantly different (P>0. 05) using the GLM procedure for repeated measures analysis of variance

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70 .("' 60 El (,) 50 40 -f 30 "'0 g ::s -a 20 ,::i.. 10 0 0 5 1.0 1.5 2 0 Time after planting (yr) 1- -CMW -A-NoCMW 2 5 3 0 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. 82

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83 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. Treatment CMW(-Y CMW(+) P. nicotianae (-Y P nicotianae ( + ) 0 .75 years 2 96 a Y 2 .51 a 3 60 a 2.42 b Fresh root weight (mg/cm3 soil) 1.25 years 8 82 a 9.21 a 11.08 a 8.27 b 2 00 years 5 67 b 9 .76 a 8 .75 a 7 34 a 2 .75 years 4 08 a 3 96 a 4 57 a 3 82 a x All 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 ( + )

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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 CMW (-Y 1 .93 a Y CMW (+) 1.99 a Treatment at grove site B N Control2 2 05 b Incorporated Reuter 2.05 b Incorporated Bedminster 2 .13 a Layered Reuter 2 06 b Layered Bedminster 2 06 b p 0.16 a 0 16 a p 0 .15 a 0 12 b 0 .13 b 0. 12 b 0 12 b K 1.04 a 1.20 a K 0 95 a 1 02 a 0 77 b 1 .06 a 0 78 b x All 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 diflferent (P>0. 05) according to the paired student t-test. 2All plots infested or noninfested with Phytophthora nicotianae were combined 84 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 of20% (v/v) of the total backfill ; layered Reuter= CMW from Reuter Recycling applied as a top-dressing 5 cm thick around the tree.

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significant differences in the rnicroelements 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. 85 The CMW treatment alone (CMW treatments with soil infested or noninfested with P nicotianae were combined) significantly increased the average fiuit 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>O. l). There was no significant interaction between CMW and P. nicotianae on any of the fiuit measurements After 3 years the CMW treatment in noninfested soil had significantly more fiuit 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 fiuit weight (P>O. l), but there was a significant interaction on the number offiuit 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 4-3 Effect of composted municipal waste (CMW) on the uptake of the following microelements: sodium (Na), aluminum (Al) copper (Cu), calcium (Ca), magnesium (Mg), manganese (Mn) iron (Fe) lead (Pb) zinc (Zn) and mercury (Hg) Microelement concentrations are expressed as ppm of leaf tissue collected from Orlando tangelo trees 2 years after planting (grove site A) and Sunburst tangerine trees 1 year after planting (site B) Microelements (m;~m) Treatment at grove site A Na Al Cu Ca Mg Mn Fe Pb Zn Hg CMW(-Y 8 5 a Y 1 06 a 0 .05 a 183 a 23. 7 a 0 .2 1 b 0 .31 a 0 06 a 0 08 b 0a CMW(+) 8 5 a 1 .23 a 0 08 a 190 a 23. 0 a 0 26 a 0.41 a 0 04 b 0 .13 a 0a Treatment at grove site B Na Al Cu Ca Mg Mn Fe Pb Zn Hg Control2 9.4 a 1.13 a 0 .03 a 211 a 36 0 a 0 07 a 0 .23 b 0 08 a 0 .12 a 0a Incorporated Reuter 11.4 a 1 20 a 0 .03 a 245 a 27 1 d 0.05 b 0 26 b 0 07 a 0 16 a 0a Incorporated Bedminster 9 6 a 1.22 a 0 .03 a 248 a 29 6 be 0 07 a 0 30 a 0 08 a 0.13 a 0a Layered Reuter 11.0 a 1.18 a 0 .03 a 232 a 28.4 cd 0 06 a 0 25 a 0 08 a 0 .11 a 0a Layered Bedminster 10 1 a 1.16 a 0 .03 a 248 a 30 2 b 0.06 a 0.25 a 0 07 a 0 .11 a 0a -'All plots infested or noninfested with Phytophthora nicotianae were combined according to treatments : no CMW applied(-) ; CMW applied( + ) l'Means for each element followed by the same letter are not significantly diflferent (P>0 05) accor ding to the studen t paired t-test. 'All plots infested or noninfested with P nicotiana e were combine d according to treatments : control = nontreated ; incorporated Bedminster = CMW from Bedminster Corp (Sevierville TN) incorporated into the soil at a level of20% (v / v) of the total backfill ; layered Bedminster = CMW from Bedminster Corp applied a s a top-dre ss ing 5 cm thick around the tree ; incorporated Reuter = CMW from Reuter Recycling (Pembroke Pines FL) incorporated into the soil at a level of20% (v /v) of the total backfill ; layered Reuter = CMW from Reuter Recycling applied as a top-dres s ing 5 cm thick around the tree 00

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Table 4-4 Effect of composted municipal solid waste (CMW) and infection by Phytophthora nicotianae on the number of fiuit per tree and the average weight of individual fruit 2 and 3 years after planting in the field at site A Number of Treatment fiuit/tree CMW P nicotianaew No x 2 years Y 3 years 12 2 5 b2 3 8 b + 20 4 7 ab 5 9 b + 5 5 6 ab 19. 2 a + + 27 9 9 a 3 3 b V'freatments with(+) and without(-) CMW w Treatments with(+) and without(-) Phytophthora nicotianae ~umber of trees per treatment. Y Time elapsed after planting Average wt of fiuit (g) 2 years 3 years 92 b 139 a 135 ab 156 a 221 a 215 a 173 a 121 a 2Means within each column followed by the same letter are not significantly different (P>0 05) according to LSD analysis 87

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88 Table 4-5. Juice analysis of Orlando tangelos harvested 3 years after planting from four different treaments at grove site A. Treatment CMW P nicotianaeu % Juice v % Acidw TSS x 63. 9 0 60 9 64 + 64 7 0 .63 9 54 + 65 2 0 .63 10 08 + + 64 8 0 65 9 89 'Plots treated(+ ) or untreated(-) with CMW IIJ>lots infested(+ ) or noninfested (-) with Phytophthora nicotianae vi>ercentage of juice per fruit on a weight basis wi>ercent titratable acid x Total soluble solids expressed as 0Brix >Ratio of Total Brix/Acid 21<.ilograms of solids per box TSS/ Acid Y Solids2 16 07 2 52 15. 14 2 .51 16 00 2 69 15. 22 2 62

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89 (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 nicotiana e 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

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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 Treatment Control Y Incorporated Bedminster Layered Bedminster Incorporated Reuter Layered Reuter Increase in Stem Diameter (cm) 0 .79 d2 1 .01 a 0 99 ab 0 .91 be 0 .85 cd Y All plots infested or noninfested with Phytophthora nicotianae were combined according to treatments : control= nontreated plots ; incorporated Bedminster= 90 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 2Means for treatments followed by the same letter are not significantly different (P>0 05) according to the paired student t-test.

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26 --r------------------------, 24 22 ,,...._ 20 i J 18 16 14 12 1/C --1/IB ---AI/IR -+I/LB .... ,.. . I/LR -o-NI/C -a-NI/IB NI/IR -0 05) using the GLM procedure for repeated measures analysis of variance.

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92 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 Treatment Y P. nicotianae (-) P. nicotianae ( +) Increase in stem diameter (cm)2 0 94 a 0 88 b Y All 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.

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93 Table 4-8 Effect of composted municipal solid waste (CMW) and infection by Phytophthora ni c otianae on root density 15 and 3 0 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 v 1 00 year Treatment 15 cm w 15 cm Controix 0 32 a Y 0 .75 b Incorporated Bedminster 0.48 a 1 .31 a Incorporated Reuter 0 .51 a 0 96 ab Layered Bedminster 0 38 a 0 76 b Layered R~uter 0 39 a 0 74 b P. nicot i anae ()2 0.48 a 1.83 a P nicot i anae ( +) 0 35 b 0 74 b 'Time after planting into field WOistance of root baskets from the base of the tree x All plots infested or noninfested with Phytophthora nicotiana e were combined according to treatments : control = nontreated plots ; incorporated Bedminster = 30 cm 0 22 a 0 32 a 0 16 a 0 38 a 0 34 a 0 29 a 0 28 a 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 of20% (v / v) of the total backfill ; layered Reuter= CMW from Reuter Recycling applied as a top-dressing 5 cm thick around the tree >Means within the columns for CMW or P nicotiana e treatments followed by the same letter are not significantly different (P>0 05) according to the paired student t-test. 2 All plots untreated or treated with composted municipal waste were combined according to treatment: noninfested with P. nicotiana e () ; infested with P. ni cotianae ( +) prior to planting

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94 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 Shoot Root Root fresh wt fresh wt fresh wt dried wt Treatment Y {grams} {grams} {grams} {grams} Control 900 C2 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 be 380 b 660 be 39.4 ab Y All 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 Means 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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95 Bedminster treatments Shoot fresh weights of plants grown in layered or incorporated Bedminster were greater than that of the control. There was a correlation between the total fibrous roots and both the stem diameter (r=0 320) and the total weight of the tree (r=0 720) Phytophthora nicotianae significantly decreased total plant weight (P < 0 01) shoot weight (P<0 01) total fresh root weight (P< 0 01) and the total dried fibrous root weight (P< 0 01) (Table 4-10) There was not a significant interaction in the total fresh root weight (P=0.42) and the total dried fibrous root weight (P= 0 69) between the CMW treatments and P nicotianae. Nitrogen concentrations in leaves were 2 .05 -2 06% of the total leaf dry weight except in leaves of plants in the incorporated Bedminster CMW treatment which had a significantly higher nitrogen concentration of 2 13% (Table 4-2) Phosphorus levels were significantly lower in the CMW-amended treatments than in the control and potassium levels were significantly reduced in plants grown in soil with layered or incorporated Bedminster CMW There were no significant differences in the microelements except in Mg where the control treatment was significantly higher and in Mn and Fe where the incorporated Reuter treatment was significantly lower (Table 4-3) The NH4-N le v els of the soil around the root zone did not differ significantly between the nontreated control and the treatments with either the source of compost (Bedminster or Reuter) or the application (top-dressing or incorporation) (Tables 4-11 and 4-12) However there was a significant interaction between the source and the

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96 Table 4-10 Effect of infestation by Phytophthora nicotiana e 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. Treatment Y P. nicotianae (-) P nicotianae ( +) Total fresh wt (grams) 1290 a z 1060 b Shoot fresh wt (grams) 475 a 390 b Root fresh wt. (grams) 815 a 670 b Root dried wt. (grams) 44 6 a 35.4 b Y All plots untreated or treated with composted municipal waste were combined according to treatment: noninfested with P. nicotiana e () ; infested with P. ni c otiana e ( + ) prior to planting 2Means within the same column for P. nicotianae treatments followed by the same letter are not significantly different (P>0 05) according to the paired student t-test.

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Table 4 .11. The effect of the source of composted municipal waste (CMW) on the extractable levels of soil NO3-N and NH4-N sampled from grove site B. SourceY NoCMW Bedminster Reuter Nitrogen Concentrations (ppm) 2 70 a2 2 10 a 2 .10 a NH4-N 5 20 a 4 90 a 4 .80 a Y All plots infested or noninfested with Phytophthora nicotianae and untreated or 97 treated with composted municipal waste (CMW), either as a top-layer or incorporated into the backfill, were combined according to the source of CMW: no CMW = untreated controls ; Bedminster = Bedminster Corp (Sevierville TN) ; Reuter = Reuter Recycling (Pembroke Pines FL.) 2Means within each column followed by the same letter are not significantly different (P>0 05) according to paired t-test.

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98 Table 4 .12. The effect of the application method of composted municipal waste (CMW) on the extractable levels of soil NO3-N and NH4-N sampled from grove site B. Application NoCMW Incorporated Layered Nitrogen Concentrations (ppm) 2 70 a2 3 30 a 0 90 b NH4-N 5 20 a 5 00 a 4 70 a Y All plots infested or noninfested with Phytophthora nicotianae and treated or untreated with composted municipal waste (CMW) either from Bedminster Corp (Sevierville TN) or Reuter Recycling (Pembroke Pines FL) were combined according to the application treatment: no CMW = untreated controls ; incorporated = incorporated into the backfill as 20% (v / v) ; layered = top-dressing 5-cm thick. 2Means within the same column followed by the same letter are not significantly different (P>0.05) according to the paired student t-test.

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application method (P=0 0278) The N03-N levels of the soil around the root zone did not differ significantly with the source The layered treatments had significantly lower N03-N levels than either the control or the incorporated treatments. There was a significant interaction between the source and the application in N03-N (P=0 0179) One year after planting the differences in the soil pH values were not significantly different ; soil pH ranged from 5 6 -6 6 Discussion 99 Phytophthora nicotianae has been shown to reduce fibrous root density in mature trees (Sandler et al., 1989) and cause a general tree decline (Lutz and Menge 1986) At both sites in the present study the presence of P. nicotianae significantly reduced tree growth tree size and the root system. Composted municipal waste has been demonstrated to suppress Phytophthora root rot of azalea under containerized greenhouse conditions (Ownley and Benson 1992) However one obstacle with the use of composts and other biocontrol agents for disease suppression is the lack of effectiveness demonstrated in the field. At grove site B populations of P nicotianae were not detected in a majority of the samples and thus no useful data were obtained Perhaps because the experiment was terminated early due to freeze damage P. nicotianae did not have time to increase populations in the areas sampled (15 and 30 cm from the base of the tree)

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100 At grove site A, populations of P nicotiana e were not reduced by the presence of CMW At two sampling dates populations were significantly higher in the CMW amended plots than in the nonamended plots A possible explanation for this increase in P n i cot i anae populat i ons is an increase in water retention in the soil from the incorporation of CMW Duncan et al. (1993) found that soil temperature and moisture were the two most highly predictable factors of population dynamics of P. n i cotiana e on citrus roots in Florida Composted organic matter has been shown to increase the water holding capacity in so ils with low organic matter (Rose 1991 ; Turner et al., 1994) The CMW by increasing soil water retention may be conducive for polycyclic production of sporangia release of numerous motile zoospores, and root infection Since the increase in tree growth did not appear to be related to a reduction in pathogen populations other explanations such as a nutritional advantage from CMW for the tree were considered Humates that comprise a large portion of organic matter in the soi] have a large e x change capacity and greatly affect the availability of nutrients to plant roots (Schnitzer and Poapst 1967) Nitrogen is a key element found in composts which is released slowly into the soil over time becoming available for plant utilization (Dick and McCoy, 1993 ; Gallardo-Lara and Nogales 1987) Nitrogen is the most important nutrient for optimal growth of citrus and yield (Tucker et al., 1995) The leaf analysis for nitrogen showed levels at approximately 2 0-2 1 % of the total dried leaf weight which are slightly below the optimum range of2. 5-2 7% (Koo 1984) ; however leaves did not show visible nitrogen deficiency symptoms

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101 Other properties affected by humates may also be important. In a study done on beans humates increased root initiation (Schnitzer and Poapst 1967) Bloomfield et al. (1996) reported a correlation between increased humates and increased root length and stem diameter although it does vary with plant species The stem cross-sectional area of young citrus trees increased when humates were added to a soil (Webb et al. 1988) Castle and Krezdom (1974) did not find a correlation in Citrus spp. between the total feeder root weight and the tree height. Also in this study no correlation was found between the root densities taken from core samples and the growth of the citrus trees at either site A or site B. If humates as influenced by compost amendments affect citrus root formation sampling may not have been intensive enough to measure the early responses at site A or within the root baskets at site B Major roots of small trees are often unevenly distributed and fibrous roots may occur in patches (Castle 1987) Most of the trees were sampled with only one core of soil per tree to avoid damage to the root system ; perhaps multiple smaller core samples would have allowed detection of differences in root densities This became apparent at site B where the whole tree was destructively sampled There was a correlation between the total fibrous root weight and the stem diameter or the total weight of the tree Other explanations for the increase in tree growth are that the physical and chemical properties of the soil were altered by the addition of compost. Organic matter has a fundamental effect on the total porosity of various soils (Dick and McCoy 1993) and on the exchange and buffering properties of citrus soils (Obreza 1995 ; Webb et al.,

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102 1988) Citrus grows best in soil with a pH range of 5.5 to 6 5 (Jackson 1991) At both sites the pH values of the treatments were within this range and thus hydrogen ion concentration does not appear to have been a factor for the increase in growth in the various CMW treatments. Compost also has been shown to affect water movement or drainage and therefore may make water more available to the plant (Lutz et al., 1986 ; Lyda 1982 ; Obreza 1995) Young citrus trees suboptimally irrigated did not grow to the same size as those optimally supplied with water (Goell et al., 1981) Improved water holding capacity due to CMW may be the most important factor in the yield increase. There have been numerous studies which show that composts improve the water holding capacity (Epstein 1976 ; Mays et al., 1973 ; Rose 1991) In a study of integrated crop management of citrus in California irrigation was the factor having the most impact on yield responses (Menge et al. 1990) Graser and Allen ( 198 7) also showed that an increase in water increased yields and growth of citrus in Florida One of the hypotheses in this study was that with the addition of CMW and therefore an increase in water holding capacity a yield response would be observed However, a good correlation between the fruit yields and CMW treatments was not seen at site A, probably because of the young age of the trees Fruit yield is highly variable because young trees do not consistently start bearing fruit until after they are 5 years old (Jackson, 1991) Although nitrogen is the key fertilizer element affecting early yield response (Tucker et al., 1995) available nitrogen nutrition does not appear to be increased by treatments with the sources of CMW used (Eichelberger 1994).

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CHAPTERS THE EFFECT OF COMPOSTED MUNICIPAL WASTE ON MANAGEMENT OF PHYTOPHTHORA ROOT ROT IN MATURE CITRUS TREES Introduction Citrus was introduced into Florida around 1565 with the establishment of the Spanish colony at St. Augustine (Jackson 1991 ) The first big expansion of citrus occurred in the 1870s in north-central Florida Several severe freezes in the 1890s caused the citrus industry to move south Since the early 1900s production of citrus has changed from a crop of secondary interest to the grower to a large scale commercial operation. Over this time improved horticultural practices and new technologies have helped the citrus industry to grow. Today citrus is planted in Florida on over 273 000 hectares One of the more important advances for citrus production was the propagation of citrus trees on rootstocks rather than as seedlings or as cuttings Rootstocks provide certain advantages such as reduction in juvenility environmental adaptation and improved horticultural performance that are beneficial to the tree (Castle et al. 1993) For hundreds of years citrus trees were grown primarily as seedlings (Jackson 1991) When the market expanded in the early 1800s interest developed for improved horticultural practices In 1830 the first use of budded trees to raise citrus trees for the commercial fresh fruit market was developed (Castle et al. 1993) In a short time 103

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104 budding sweet oranges onto seedlings of sour orange from the wild groves became widespread Sour orange rough lemon and Cleopatra mandarin were the most widely used rootstocks prior to 1970 After this time rootstocks became a more critical issue because of higher incidences of citrus blight and tristeza and the frequency of freezes Although new rootstocks were developed to overcome these problems and others, not all of them were tolerant to Phytophthora root rot. Phytophthora root rot of citrus caused by Phytophthora nicotianae Breda de Haan (synonym = P. parasitica Dastur) results in a slow decline that includes foliage yellowing and twig dieback (Lutz and Menge 1986) If the disease is severe larger limbs may also dieback. When fibrous roots are destroyed faster than they are replaced the tree declines and growth and fruit production are reduced Before the development of fosetyl-Al and metalaxyl no highly effective fungicides were available to control root rot, and therefore the impact of the disease on tree performance and fruit yield could not be determined In the field applications of these fungicides increased fibrous root density over that in the nontreated controls (Sandler et al. 1989 ; Timmer et al. 1989) Tree appearance was improved in some of the groves treated and yields and fruit sizes were also increased at some sites (Sandler et al., 1988 ; Sandler et al., 1989). Ponds et al. (1984) showed an increase in yields of37-59% when metalaxyl was applied and of20-88% when fosetyl-Al was applied Tree response is variable and fungicide applications may not be economically feasible when populations of P. nicotianae are below 10-15 propagules per gram of soil (Sandler et al., 1989) Menge (1986) calculated that P

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105 nicotianae populations of 15-20 propagules per gram of soil detected during the growth phase are necessary to reduce yield by 20% Phytophthora nicotianae also causes foot rot and gummosis (Whiteside et al., 1988) Foot rot is usually controlled by budding the susceptible scion cultivars on resistant rootstocks 15 cm above the soil line and keeping the trunk of the tree dry (Grimm and Timmer 1981; Whiteside 1971 ) Fungicide applications can also control above ground foot rot and gummosis (Sandler et al., 1989) However rootstocks resistant to bark infection may be susceptible to fibrous root infection Fungicides have been effective in reducing population numbers (Ponds et al., 1984 ; Sandler et al., 1989 ; Timmer et al., 1989) but the pathogen is not erradicated When conditions become favorable for pathogen growth, the populations can be damaging again (Menge, 1986) Other management strategies can also be implemented These include improving soil drainage altering irrigation practices or replanting susceptible trees with tolerant rootstocks Another potential management strategy is to apply organic amendments The use of composted organic materials as amendments to suppress soilbome diseases has been evaluated in numerous studies Composted bark when added as a soil amendment has been shown to suppress soilborne diseases caused by Rhizoctonia so/ani Pythium ultimum and Fusarium oxysporum f.sp. conglutinans (Davis 1982 ; Nelson and Hoitink, 1982 ; Trillas-Gay et al., 1986) There also has been success in managing diseases caused by Phytophthora spp Root rot of avocado, caused by P. cinnamomi was effectively

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controlled with intensive mulching and applications of gypsum (Broadbent and Baker 1974) 106 The purpose of this field study was to examine the effects of composted municipal waste (CMW) applied as a mulch layer on top of the soil surface on soil populations of P. nicotianae and on root densisties and fruit yields of mature citrus trees growing on marginal soil types with damaging soil populations of the pathogen Materials and Methods Composted Municipal Waste Composted municipal wastes from two different sources were used in these trials Batch R2 of CMW from Reuter Recycling (Pembroke Pines FL) was derived from household garbage and contained relatively large pieces of broken glass and plastic The other source of CMW delivered as batches B2 and B4 from Bedminster Bioconversion (Sevierville TN) was a combination of composted garbage (approximately 90%) and composted sewage sludge (approximately 10%) and contained almost no visible inert material. Analyses for both sources are listed in Appendix A. Preparation of Field Plot A plot (Val-LA) of 10-year-old Valencia sweet orange trees (Citrus sinensis (L. ) Osb ) on Carrizo citrange (Poncirus trifoliata (L.) Raf X C. sinensis) rootstock at the University of Florida Citrus Research and Education Center Lake Alfred FL, was selected for study because of decline-like symptoms of the trees characteristic of citrus blight. Trees showed marked symptoms of zinc deficiency and attenuated size of recent

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leaf flushes Twenty trees in one row were selected for this study The trees were 4 7 meters apart (trunk to trunk) with variable canopy sizes 107 In plot Val-LA, IO alternate trees in a single row were selected for treatment with CMW A 5-cm layer (180 metric tons per hectare) of batch R2 of CMW was applied underneath the canopy of the trees The remaining 10 trees were maintained as untreated controls Three additional sites were chosen in a commercial citrus grove near Vero Beach Florida Site one (Val-VB) consisted of 6-year-old Valencia sweet orange trees on Carrizo citrange rootstock in a calcareous soil. Sites two and three (GF-1 and GF-11) consisted of 25-year-old grapefruit (C. paradisi Macf) trees on sour orange (C. aurantium L.) rootstock growing in very coarse sandy soil The trees in these areas were stunted compared to trees on adjacent finer-textured sandy soils. Phytophthora nicotianae was present at all three sites, and the trees were showing symptoms of decline from Phytophthora root rot. There were three rows per bed in the Val-VB plot. One hundred trees from the middle rows were selected in blocks of five trees each based upon tree uniformity with a total of20 blocks The trees within the rows were 3 6 meters apart (trunk to trunk) with 3 0-meter canopies In plot Val-VB IO blocks were selected randomly for treatment with CMW. The other 10 blocks were maintained as untreated controls Batch R2 of CMW was applied under the canopies of the treated trees using a modified New Holland spreader (New Holland Corp New Holland, MI) with a side chute Several passes by the spreader were

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made to apply a 10-cm layer ofCMW (360 metric tons per hectare) The CMW was spread evenly with a rake and pushed away from the trunks After approximately 2 years, batch B4 CMW was applied as a 5-cm layer (180 metric tons per hectare) under the canopies of the treated trees as described above 108 There were two rows of trees per bed in plots GF-I and GF-11 that were 6 7 meters apart (trunk to trunk) The trees within the rows were 5 5 meters apart (trunk to trunk) and had 4 0-meter canopies Adjacent uniform trees were selected for treatment in the two rows for each bed The GF-I plot consisted of 10 blocks of six trees each, and the GF-11 plot consisted of eight blocks of five trees each In plots GF-I and GF-11, one block within each bed was randomly selected for treatment with CMW The other block within the bed was maintained as untreated controls Batch B2 of CMW was applied as a 5-cm top-dressing (180 metric tons per hectare) under the canopies of the treated trees using a modified New Holland spreader with a side chute The CMW was spread evenly with a rake and pushed away from the trunks After approximately 1 5 years batch B4 of CMW was applied as a 5-cm layer (180 metric tons per hectare) under the canopies of the treated trees as described above Sampling and Analysis Soil samples were taken prior to the application of the CMW using a 980-cm3 (7 5-cm diameter) volume auger to a depth of 15 cm Soil was collected from under the canopy approximately 0 5 meter from the trunk within the wetted zone of the microsprinkler irrigation system In plot ValVB two cores from different trees within each block were combined for a total of 10 replications per treatment. In plot GF-I three

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109 trees were sampled in each block for a total of 15 replications per treatment while in plot GF-II two trees per block were sampled for a total of eight replications per treatment In plots GF-1 and GF-11 the cores were not combined The roots were separated by sifting the soil through a 2-mm-mesh screen A subsample of approximately 250 cm3 of soil was collected in a plastic bag The roots were dried in an oven at 65 C and weighed Population densities of P nicotianae were estimated by the modified procedure 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 dilution was plated on a medium (P ARP-H) selective for the isolation of Phytophthora spp (Mitchell and Kannwischer-Mitchell 1992 ; Chapter 3) After 2 days in an incubator at 27 C the soil was washed off of 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 of0.01 M CaCl2 solution stirred occasionally over a 30-minute period and then allowed to stand for 30 minutes The pH values were measured with a Corning pH meter 240 and recorded The soil moisture was measured by weighing approximately 20 g of soil in an aluminum weigh pan The soil was dried for 2 days in an oven at 80 C The percent moisture was calculated by subtracting the dried weight from the wet weight dividing by the wet weight and multiplying by 100 The bulk density (grams per cm3 ) was measured by weighing 10 cm3 of the dried soil and dividing by 10

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110 Fruit production was evaluated for the second harvest after the initial CMW application at the Val-LA, Val-VB and GF-1 plots Yields were recorded as number of fruit boxes (0 075 m3 ) per tree Fruit and rind sizes were determined by randomly selecting 50 fruit from each treatment, cutting the fruit in half, and measuring the fruit diameter and rind thickness with a Mitutoyo Digimatic Caliper (Mitutoyo Corporation Japan) Juice from the fruit was analyzed by the Florida Department of Citrus Lake Alfred FL (10 replications per treatment) Data over the total time period were analyzed with a general linear model procedure using repeated measures analysis The means were separated and tested for significance by using least significant differences (LSD) or paired t-tests with SAS statistical software (SAS Institute Carey NC) Results Effect on Phytophthora nicotianae Populations The population densities of P. nicotianae in the Val-VB plot were not significantly different in relation to the treatments over time (P=0.43) based on repeated measures analysis (Figure 5-1 ) There were no significant differences at any individual sampling times in the Val-VB plot. In the GF-1 and GF-11 plots, population densities of P nicotianae were significantly higher over time in the CMW-treated blocks than in the nontreated blocks (P=0 043 and P=0 035 respectively) based on repeated measures analysis (Figures 5-2 and 5-3) The only significant difference at an individual sampling

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111 80 70 1--oNo CMW --0CMW .f" 60 5 50 'I 40 \". -i 'q_ "' ij 30 ~ 20 10 o----------~-----------.----~ 0 2 Time after planting (yr) Figure 5-1. The effect of composted municipal waste (CMW), in comparison to nontreated trees (No CMW), on population densities of Phytophthora nicotianae ( colony forming units [CFU] per cm3 of soil). Samples were collected over time after application ofCMW to 6-year-old 'Val encia oranges on Carrizo citrange rootstocks in plot Val-VB

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140 -------------------------, 120 40 1---0No CMW --o-CMW .....a.....__ I // .................. -................_ I ---0 I I I I I I I I p 20 -------------------------4 0 2 Time after planting (yr) Figure 5-2. The effect of composted municipal waste (CMW) in comparison to 112 nontreated trees (No CMW) on population densities of Phytophthora nicotianae ( colony forming units [CFU] per cm3 of soil). Samples were collected over time after application of CMW to 25-year-old grapefruit trees on sour orange rootstocks in plot GF-1. Points followed by an indicate significant differences (P~0 05) at the specified sampling time based on the paired t-test.

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113 250 -,------------------------, ---
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114 time between the CMW-treated and nontreated plots was 1 .25 years after application (sampled in December) for both of the GF-1 and GF-11 plots (P<0.05) Soil populations of P. nicotianae were not different depending upon the season sampled in GF-11 (P=0 125) and ValVB (P=0. 795) plots, but populations were significantly higher in the spring in the GF-1 plot (P=0 01) Effect on Root Densities Root density was significantly higher in plot ValVB at the initial sampling time (0.4 year after application) for the nontreated blocks (P=0 044) However, 1.2 years after application the CMW-treated blocks were significantly higher (P<0 001) No significant differences were observed at the remaining sampling times. However second order regression analysis over 2 .25 years showed root densities were significantly higher in the Val-VB plot for CMW-treated trees (P<0 01) (Figure 5-4) Root densities were significantly lower for trees in plot GF-1 treated with CMW than for nontreated trees (P=0 014) when evaluated over 1 .75 years by comparing second order regression analyses (Figure 5-5) There was no significant difference between the CMW and nontreated trees over time in plot GF-11 (P=0 39) (Figure 5-6) At individual sample dates in plots GF-1 and GF-11, root densities were significantly higher in the nontreated blocks except at 1.75 years after application (the last sampling time) Effect on Soil Characteristics The percent moisture of samples collected from the ValVB plot 1 0 year after CMW application was significantly higher (P=0 002) in CMW-treated blocks (19 8%) than in nontreated blocks (10 9%) In the GF-1 plot the percent moisture 0 .75 year after

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115 1.2 1--NoCMW /-.......... --CMW 1.0 / "' I \ I \ -. \ := * I 0 UI 0 .8 \ a (.) I \ bO ,g I \ ] 0 6 I \ ... I \ i \ 0.4 \ 0.2 0 2 Time elapsed (years) Figure 5-4 The effect of composted municipal waste (CMW) on the root densities over time after initial application of CMW to 6-year-old 'Valencia orange trees on Carrizo citrange rootstocks in plot Val-VB Points followed by an* indicate significant differences (P~0.05) at the specified sampling time based on the paired t-test. Points fit curves for second order regressions: y=-0 19+2 03x-0 81x2 (R2=0 99) for CMW treatment ; y=0.91-0 .23x+O.Olx2 (R2=0 76) for nonamended treatment.

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5 4 0 0 ---...--. /' .......... V '-. Time elapsed (years) 116 2 Figure 5-5 The effect of composted municipal waste (CMW) on root densities over time after initial application of CMW to 25-year-old grapefruit trees on sour orange rootstocks in plot GF-1. Points followed by an* indicate significant differences (P~0.05) at the specified sampling time based on the paired t-test. Points fit the curves for second order regressions : y=l. 7+ l.9x-l.3x2 (R2= 0 84) for CMW treatment ; y=2 3+4 3x-2 6x2 (R2=0.45) for nonamended treatment.

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6 5 S' 0 Ul <:' 4 s u tll) g -~ 0 3 "'O 2 0 ------- ...._ ........ .......... ' ' Time elapsed (years) 117 2 Figure 5-6. The effect of composted municipal waste (CMW) on the root densities over time after initial application of CMW to 25-year-old grapefruit trees on sour orange rootstocks in plot GF-Il Points followed by an indicate significant differences (P~0 05) at the specified sampling time based on the paired t-test. Points fit the curves for second order regressions: y=2 9+0.8x-0 9x2 (R2= 0 59) for CMW treatment ; y=4 1 +2 2x-1.8x2 (R2= 0.52) for nonamended treatment.

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118 CMW application was not significantly different (P=0 173) in the CMW-treated blocks (32 9%) than in the nontreated blocks (16 7%) After 2 years in the Val-VB plots the pH of the soil in the blocks treated with CMW was significantly higher than in the nontreated blocks (P< 0 01) (Table 5-1) There were no significant differences in the hydrogen ion concentrations in the GF-1 plot (Table 5-2) The bulk densities in the Val-VB plot 1.2 and 1 5 years after application were significantly lower (P = 0 005 and P = 0 035 respectively) in the CMW-treated blocks but no differences were detected after 2 0 years (P=0 235) (Table 5-1) In the GF-1 plot the bulk density was only significantly lower in CMW-treated trees 1.25 years after application (P= 0 01) (Table 5-2) Effect on Yields and Juice Quality There was no significant difference in yields between the treatments for any of the plots harvested (Table 5-3) The plots treated with CMW had significantly larger fruit in the GF-1 and Val-VB plots (P< 0 .01 and P < 0 .01, respectively) but there was not a significant difference in rind thickness (P=0 94 and P=0.17, respectively) There were not significant differences in fruit size (P=0 06) or rind thickness (P=0 08) in plot ValLA In the GF-1 plot the percentage of juice per fruit and the total soluble solids to acid ratio were significantly lower in the blocks treated with CMW (P= 0 018) while the total soluble solids were higher (P= 0 007) (Table 5-4) In plot Val-LA there were no significant differences in any of the analyses and in plot ValVB the total soluble solids

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Table 5-1. Effect of composted municipal waste (CMW) over time on the pH and bulk density of soil under Valencia orange trees on Carrizo citrange rootstocks in plot ValVB Elapsed time v 0 0 year 1.2 years 1.5 years 2 0 years Treatment w pW Bulk density Y pH Bulk density pH Bulk density pH Bulk density NoCMW 6 75 a z 1 72 a 1.75 a 7 .05 a 1 37 a 6.48 b 1.25 a CMW 6 76 a 1.74 a 1.59 b 6 90 a 1.26 b 7 02 a 1 07 a v sampling times after initial application of CMW wcMW = Trees treated with CMW from Reuter Recycling Pembroke Pines FL (360 metric tons per hectare) ; no CMW = Trees untreated. x soil pH measured in a suspension of I part soil : 2 parts 0 .01 M CaCl2 YBulk density of dried soil (grams per cm3 ) ~eans within a column followed by the similar letter are not significantly different (P< 0 05) according to paired t-test. --\0

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Table 5-2 Effect of composted municipal waste (CMW) over time on the pH and bulk density of soil under grapefruit trees on sour orange rootstocks in plot GF-1 Elapsed time v 0 .75 year 1 25 year 1.75 years 120 Treatmentw pH' Bulk densityY pH Bulk density pH Bulk density NoCMW 1.60 a 6 04 a 1 30 a 6.92 a 1. 23 a CMW 1.42 a 6 .21 a 1 09 b 7 06 a 0 .93 a v sampling times after initial application of CMW wcMW = Trees treated with CMW from Reuter Recycling Pembroke Pines FL (360 metric tons per hectare) ; no CMW = Trees untreated x soil pH measured in a suspension of 1 part soil:2 parts 0 .01 M CaC12 YBulk density of dried soil (grams per cm3 ) Means within a column followed by the similar letter are not significantly d i fferent (P>0 05) according to the paired student t-test.

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Table 5-3 The effect of composted municipal waste (CMW) 2 years after the initial application of CMW on fruit size, rind size and yields collected in three different field plots Yield Fruit Rind Plot x Treatment Y (Boxes/Tree) diameter (mm) thickness (mm) GF-1 NoCMW 3.37 a2 104 6 b 8 77 a GF-1 CMW 2 59 a 113. 1 a 8 72 a Val-VB NoCMW 2 67 a 76 6 b 4 .13 a Val-VB CMW 2 76 a 81.6 a 4 32 a Val-LA NoCMW 1.13 a 75.6 a 3.73 a Val-LA CMW 1 30 a 77 5 a 3 99 a x Type of fruit and location of plot: Grapefruit located near Vero Beach FL (GF-1); Valencia oranges located near Vero Beach FL (Val-VB) ; Valencia oranges located in Lake Alfred FL (Val-LA) Y Treatments applied to trees within a block : no CMW = no composted municipal waste applied ; CMW = composted municipal waste applied 2Means listed in columns within each plot, followed by the same letter are not significantly different (P>0 05) according to the paired student t-test. 121

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Table 5-4 Juice analysis from fruit collected 2 years after initial application of composted municipal waste (CMW) Fruit Typer Plot Treatment' % Juiceu % Acid v TSSW TSS/Acid x Solids Y Grapefruit GF-1 NoCMW 55 .78a2 1.17 a 10.77 b 9 20 a 6.00 a Grapefruit GF-1 CMW 51.17b 1.24 a 11.07 a 8 95 b 5 66 a Valencia oranges Val-VB NoCMW 61.90 a 0 64 a 12.44 a 19.53 a 7 .71 a Valencia oranges Val-VB CMW 60 30 a 0 64 a 11.90 b 18. 72 a 7 .17 b Valencia oranges Val-LA NoCMW 60 10 a 0 .73 a 11. 32 a 15. 77 a 6 .81 a Valencia oranges Val-LA CMW 61.40 a 0 69 a 11.14 a 16. 10 a 6 .85 a 7ype of fruit sampled for juice analysis Location of field plot : GF-1 = near Vero Beach FL ; Val-VB= near Vero Beach FL ; Val-LA= Lake Alfred FL. 'Treatments applied to trees within block : no CMW = no composted municipal waste applied ; CMW = composted municipal waste applied uPercentage of juice per fruit on a weight basis vpercent titratable acid wTotal soluble solids expressed as 0Brix 'Ratio of Total Brix/Acid YK.ilograms of solids per box. ~eans listed in columns within each plot followed by the same letter are not significantly different (P>0 05) according to the paired student t-test. -N N

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(P=0 05) and kilograms of solids per box (P= 0.03) were significantly lower in trees treated with CMW than in the control. Discussion 123 Although population densities of P. nicotianae recovered in all of the field plots fluctuated over time the populations were not reduced by treatment with CMW. When the sampling time (spring versus autumn) was considered as an independent factor the season did not have an effect on propagule densities in the GF-II and ValVB plots but populations were higher in the spring in the GF-1 plot. Timmer et al. (1989b) found that seasonal sampling was not significant in recovery of P. nicotiana e populations in four citrus groves over a 3-year study In both grapefruit plots (GF-1 and GF-11), root densities were less in the blocks treated with CMW than in the nontreated blocks This difference may be explained by the technique used for sampling Although there are some differences among rootstocks the majority of fibrous roots in citrus grow in the top 15 cm of soil (Castle et al 1993) Fibrous roots were detected in the CMW layer half a year after application but the samples collected by the soil auger may not have been accurate representations After a 5-to 10-cm layer of CMW had been applied only the top 5 to 10 cm of base soil were collected by the soil auger and therefore fewer roots may have been detected

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The bulk densities of the soil treated with CMW were significantly lower than those of the nontreated soil at the earlier time periods after application in the Val-VB 124 plot. The bulk density of the soil treated with CMW in the GF-I plot was only significantly lower than that in the untreated control at one time period Turner et al. (1994) found bulk densities in soil to vary significantly with a linear relation to the amount of CMW applied In that study the CMW was incorporated into the soil where it would have had a more direct and immediate impact on the bulk density In the present study it may have taken more time for the bulk density of the soil to be affected because the CMW was applied as a top-dressing. The hydrogen ion concentration of the soil can have a significant impact on the growth of citrus trees (Aldrich et al., 1955 ; Anderson and Martin 1969 ; Guest and Chapman 1944) However the pH values measured in all of the samples were within the range for optimum citrus tree growth In the study by Aldrich et al. (1955) citrus vegetative growth was not affected in the soil pH range of six to seven and in some soils as low as five Thus, the application of CMW did not appear to have a detrimental effect on tree health The trees in the blocks treated with CMW looked greener than the nontreated trees. During periods of water stress due to low amounts of rainfall the leaves of the trees treated with CMW in contrast to the leaves of the nontreated trees were not wilted or flaccid Compost added to soil has been demonstrated to increase both the water

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125 holding capacity (Mays et al., 1973) and water available to the plant (Epstein et al. 1976) Despite differences in appearance yields were not significantly different between the treatments Yields could only be collected for one harvest in the GF-I and ValVB plots because the grower picked all plots the first year after application before samples and yield data could be taken In the GF-II plot the fruit was harvested both years before samples could be collected Fruit sizes were larger in the GF-I and Val-VB plots amended with CMW than in nonamended plots but not in the Val-LA plot Blight proteins were detected in all of the trees in plot Val-LA, which may account for the smaller fruit and the lack of effect of CMW on fruit size as observed in the other plots In a study by Nemec and Lee (1992) blight-affected trees were not influenced by the addition of soil amendments

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CHAPTER6 THE INFECTION OF CITRUS ROOTS BY PHYTOPHTHORA NICOTIANAE AND P. PALMIVORA AT THE ULTRASTRUCTURAL LEVEL Introduction Species in the genus Phytophthora are known to cause some of the most damaging diseases worldwide on a wide range of hosts (Zentmeyer 1983) Typically categorized as soilbome pathogens of root systems and crowns of plants, some species also cause extensive damage to aerial plant parts Phytophthora spp proliferate under moist conditions and depending mainly upon environmental conditions produce various structures Probably the most important structure formed in regard to infection and spread of the pathogen in most species is the sporangium Sporangia are asexual structures produced in high humidity when nutrients are low and oxygen is abundant (Ribeiro 1983) Sporangia can either germinate directly or cleave internally into motile zoospores which are released into free water (MacDonald and Duniway 1978) The zoospores of Phytophthora spp are chemotactically attracted to root exudates (Morris and Ward 1992 ; Schwab et al., 1984) near the surface of root tips where they encyst and germinate Depending upon the species of Ph y tophthora and the host penetration of host cells occurs by different mechanisms Phytophthora spp can invade host tissue through natural openings or penetrate the host cells by mechanical pressure or enzymatic activity 126

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127 Phytophthora infestans (Mont.) de Bary usually penetrates the host via leaf stomata (Hohl and Suter 1976) Since most Phytophthora spp are root pathogens it is believed that they can enter through wounds or natural openings in the root ; however this has not yet been observed Zoospores of P. citrophthora (Smith & Smith) Leonian were shown to be attracted toward wounds in resistant and susceptible citrus (Broadbent, 1969) Other fungal species have been observed ingressing through points where lateral roots emerge or from natural openings (Sadasivan and Subramanian 1960 ; Nemec et al., 1986) After contact with the host is made most Phytophthora spp form germ tubes that can penetrate through the periclinal wall of the epidermal cell or through the middle lamella of the anticlinal walls of epidermal cells Most pathosystems studied have shown Phytophthora spp to gain ingress by penetration through the middle lamella of the anticlinal walls (Hinch et al. 1985 ; Philips, 1993 ; Tippet et al., 1977) Stossel et al. (1981) compared two races of P. sojae Kaufmann & Gerdemann (synonym = P. megasperma Drechs var sojae Hildeb ) and observed that penetration of soybean (Glycine max [L. ] Merr.) between anticlinal walls or the periclinal walls was a characteristic of each race and not influenced by the compatibility of the interaction In another study P. sojae (synonym = P megasperma f. sp glycinea Kuan & Erwin) penetrated the anticlinal walls of soybean roots 94 % of the time (Beagle-Ristaino and Rissler 1983) Alterations of the middle lamella matrices without major deformation of penetrated primary cell walls supports the idea that root invasion by some species is

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128 achieved primarily by enzymatic means rather than by mechanical means (Benhamou and Cote 1992 ; Tippett et al., 1976) Once zoospores encyst, initial penetration into the host by Phytophthora spp is very rapid Germ tubes are formed within 30 to 60 minutes after encystment (Cahill et al., 1989 ; Hinch et al., 1985), and initial penetration into the epidermal cells occurs within 0 5 to 2 hours (Cahill and Weste, 1983; Hanchey and Wheeler, 1971 ; Hinch et al., 1985; Ward et al., 1989) Broadbent (1969) observed that zoospores of P citrophthora encysted and produced germ tubes within 3 hours after inoculation Phytophthora spp infect over 2000 plant species, some of which are non-host plants and do not display symptoms (e g Gramineae) Host response to penetration can vary depending upon the pathosystem A hypersensitive response has been observed in several pathosystems involving Phytophthora spp., including P. infestans on potato (Solanum tuberosum L.) (Goodman and Novacky, 1994), P. nicotianae Breda de Haan on tobacco (Nicotianae tabacum L.) (Yu 1995) P. sojae on soybean (Graham and Graham, 1994 ) and P. cryptogea Pethybridge & Lafferty on tobacco (Devergne et al., 1992) Other species such as P cinnamomi Rands on pine (Pinus taeda L.) (Jang and Tainter 1990) and P capsici Leonian on pepper (Capsicumfrutescens L. var grossum) (Jones et al., 1974), did not elicit a hypersensitive response in incompatible reactions. One common observation in incompatible interactions which did not elicit a hypersensitive reaction is the deposition of callose at the site of penetration or contact (Cahill et al., 1989 ; Cahill and Weste 1983 ; Slusher et al., 1974) The deposition was also detected in compatible interactions but not as frequently (Cahill et al., 1989) Hinch

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129 et al. (1985) detected callose on the anticlinal wall 2 hours after penetration of epidermal cells of Zea mays L. by P c innamomi hyphae It was also observed that the callose did not completely surround the hypha Ward et al. (1989) found that in incompatible interactions of P. soja e on soybean all epidermal cells in contact with hyphae were dead within 2 to 3 hours following inoculation and dead cells surrounded the hyphae Compatible interactions showed less damage Slusher et al. (1974) however, observed no evidence of soybean cell necrosis i n advance of P. soja e hyphae Once within the host the hyphae can spread rapidly Colonization was established in avocado cortical cells by P cinnamomi within 2-4 hours and endodermis was colonized after 16 hours (Philips 1993 ) Ward et al. ( 1989) observed that P. s ojae hyphae had penetrated to the third layer of soybean cortical cells within 3 hours and to the eighth layer within 7 hours In an inoculation study of tobacco roots w i th zoospores of P nicotianae (Hanchey and Wheeler 1970) drastic effects on susceptible host cells were observed as early as 3 hours after inoculation The most common changes observed in infected cells were a separation of the plasmalemma from the cell wall rudimentary dictyosomes dilated endoplasmic r eticulum and a decreased electron density of the cytoplasm (Hanchey and Wheeler 1971) After 6 hours the pathogen s effects were observed three to four cells from the pathogen Similar results of protoplasmic shrinkage were also observed 24 hours after eucalypts were infected with P cinnamomi (Tippett et al., 1970) com infected with P. c innamomi (Wetherbee et al., 1985) soybean with P sojae (Ward et al., 1989) and susceptible potato cultivars inoculated with P. infestans (Hohl and Suter 1976)

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130 In Florida P. nicotianae a common and destructive pathogen of citrus roots causes a fibrous root rot. AJthough this pathosystem has been studied extensively, there is no information on the infection process Phytophthora palmivora (Butler) Butler also has been isolated in Florida citrus groves and found to be highly pathogenic on the root system (Zitko et al., 1991) In comparison to P nicotianae P. palmivora can be a more aggressive and damaging pathogen of certain Citrus spp. (Zitko and Timmer 1994). The infection processes of P nicotianae and P palmivora on both a susceptible and tolerant citrus host were elucidated in this study Initial penetration and colonization throughout root tissue of the host were studied using light and electron microscopy Materials and Methods Media and Soil Preparation Clarified V-8 medium was prepared by mixing 2 29 grams of CaC03 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 6000 rpm 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 ofBacto 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 Difeo cornmeal agar (Difeo Laboratories Detroit MI) were added to 1 L of deionized water

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131 and the medium was 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 ofhymexazol (Tachigaren 99 5% a i., Sankyo Co., Ltd Tokyo Japan) were mixed into the medium Fifteen-milliliters of medium were poured into a sterile petri plate (100 X 15 mm) and the plates were stored in the dark until ready for use The strains of P. nicotianae (Duda) and P. palmivora (Shaw) used in this study were isolated from citrus roots by L.W Timmer at the Citrus Research and Education Center in Lake Alfred FL They were maintained on solid clarified V-8 medium Candler fine sand (uncoated 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 (Ferriss 1984 ; Wolf and Skipper 1994) (General Electric Co., Louisville KY) The soil was cooled to room temperature Zoospore Production Zoospores of P nicotianae and P palmivora were produced by a procedure modified from a technique described by Mitchell and Kannswicher-Mitchell (1992) Four 5-mm disks from V-8 agar cultures of P. nicotianae or P. palmivora were dispensed into each of three petri plates containing 15 mL of sterile half-strength V-8 broth The

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132 cultures were grown for 3 days in the dark in an incubator at 25 C. The liquid medium was aseptically removed with a pipet and the mycelium was washed three times with sterile distilled water The cultures were resuspended in sterile, distilled water and placed under direct continuous light for 3 days at room temperature to induce sporangium formation After chilling for 30 minutes at 4 C the cultures were placed at room temperature until the sporangia released their zoospores The zoospores were carefully separated from the mycelium by slowly pouring the suspension into a beaker and an equal volume of sterile distilled water was added to the zoospore suspension The active zoospores were counted by pipetting 1 L onto a glass slide and observing them under a microscope Plant Inoculations Five-week-old sour orange ( C itrus aurantium) seedlings which are susceptible to Phytophthora root rot and trifoliate 50-7 (Poncirus trifoliata) seedlings which are tolerant to the disease were carefu11y removed from Metro-Mix 500 growth medium (The Scotts Co. Marysville OH) Seedlings with white healthy root tips were laid on top of a layer of pasteurized soil i n a large petri dish (150 X 15 cm) Small parafilm wells were placed under the root tips Sterile water was pipetted into the wells to cover approximately 10 mm of the root tip Approximately 1000 active zoospores were pipetted into each well The remaining root system which was not inoculated was covered with pasteurized soil. The petri plates were covered and placed in an incubator at 27 C in the dark.

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133 Light and Electron Microscopy After 1 2 4 24 48 72 and 168 hours inoculated roots were excised from the root system just above the point where the zoospore solution covered the root. Approximately three root tips were sampled for each time period for each host and for each Phytophthora spp The samples were fixed in 3% glutaraldehyde (Electron Microscopy Sciences Fort Washington PA) in 0 .05 M cacodylate buffer (Ted Pella Inc Redding CA) at pH 7 2 overnight at 4 C. The samples were washed two times in 0 .05 M cacodylate buffer at pH 7 2 The samples were treated in 2% OsO4 (Electron Microscopy Sciences) for 3 hours at room temperature After washing two times in the buffer the samples were dehydrated in a graded series of acetone (10 20 30 ... 100%) for 10 minutes each wash The samples were washed a total of three times in 100% acetone A fresh solution of Spurr s resin was prepared (Dawes 1994) Ten grams of vinyl cyclohexene dioxide (ERL 4206 ; Ted Pella Inc ) were poured into a 50-mL plastic disposable beaker. Twenty-six grams of nonenyl succinic anhydride (NSA ; Ted Pella Inc ) were added and mixed To this mixture, 6 0 g of diglycidyl ether of propylene glycol (DER 736 ; Electron Microscopy Sciences Fort Washington PA) and then 0.4 g of dimethylaminoethanol (DMAE ; Ted Pella Inc ) were added and mixed The root samples were infiltrated with plastic by maintaining them for 1 hour at room temperature in a mixture of30% plastic and 70% acetone for 4 hours in a mixture of 50% plastic and 50% acetone and then overnight in a mixture of 70% plastic and 30% acetone After an additional 8 hours in 100% plastic the samples were placed in molds covered with fresh plastic and placed in an oven at 70 C overnight.

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134 Thick sections (1 m) were cut with a glass knife mounted in an ultra-microtome (LKB Instruments Sweden) The sections were mounted on a glass slide by heating stained with a solution of methylene blue-azure A (0 13 g of Methylene blue [J.T. Baker Chemical Co Phillipsburg NJ] 0 020 g of Azure A [Eastman Kodak Co. Rochester NY], 10 g of glycerol 10 mL of methanol, 30 mL of phosphate buffer at pH 6 9 and 50 mL of distilled water) and counter stained with a solution of basic fuchsin (0 10 g of Basic fuchsin [Fisher Scientific Co., Fair Lawn, NJ] and 10 mL of 50% ethanol) for light microscopy Ultrathin sections (gold colored, 90-150 nm; Dawes, 1994) were cut with a glass knife using an ultra-microtome mounted on a 200-mesh formvar-coated copper grid and stained with uranyl acetate (Polysciences Inc., Warrington, PA; Stempak and Ward 1964) and lead citrate (Ted Pella Inc.; Reynolds, 1963) Thick sections were observed using a Nikon binocular light microscope (Nikon Tokyo Japan) Ultrathin sections were observed with a Philips 201 transmission electron microscope (Philips Scientific the Netherlands) Colonization of roots was measured by observing thick (1 m) cross-sections of stained roots at approximately 5 mm from the root tip. Three sections from each of three different roots were examined Colonization was rated on a zero to nine scale A rating of 0 was given for zero hyphae per section (hps) ; 1 for 1-10 hps ; 2 for 11-25 hps ; 3 for 26-40 hps ; 4 for 41-50 hps ; 5 for 51-75 hps; 6 for 76-90 hps ; 7 for 91-125 hps ; 8 for 126175 hps ; and 9 for more than 175 hps.

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135 Electrolyte Leakage Experiments were set up following a modified procedure by Zilberstein and Pinkas (1987) Healthy white root tips of 5-week-old sour orange (susceptible) and trifoliate orange (tolerant) seedlings were cut 15 mm in length The root tips were washed three times in sterile double distilled water to remove any residual electrolytes Ten root tips of each citrus variety were placed in a single petri plate (60 X 15 mm) containing 10 mL of sterile double distilled water. One thousand zoospores of either P. nicotianae or P palm iv ora were added to each of five petri plates Controls were also prepared with no zoospore inoculations The root tips were placed in an incubator at 27 C and the conductivities of the solution were measured every 12 hours using an ElectroMark analyzer (Markson Science Inc Mara CA). After the last measurement the root tips were plated on the PARP-H selective medium to confirm infection Ten healthy white-tipped roots from either sour orange or trifoliate varieties were cut 15 mm in length weighed and placed in each of three test tubes The root tips were washed three times in sterile double distilled water After the final rinse 10 mL of double distilled water were added The suspension was transferred to a tissue grinder and the roots were macerated until no visible segments were observed The electrical conductivities of the suspensions were measured The experiment was repeated once Results Light and Electron Microsco12y Penetration of roots of both susceptible and tolerant citrus hosts occurred within 1 hour after inoculation with zoospores of either P. nicotianae or P. palmi vora Light

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136 microscopy revealed that in all treatments the encysted zoospores formed germ tubes that appeared swollen at the sites of wall penetration (Figure 6-1 ) Penetration through the periclinal wall of the hypodermal cell or through the middle lamella of the anticlinal wall was observed in all treatments and was not dependent upon the host variety or Phytophthora spp Electron micrographs of initial penetration into the susceptible variety showed a slight swelling of the germ tube at the point of penetration (Figure 6-2) Zoospores were also attracted to regions where natural wounding appeared on the root (Figure 6-3) Electron microscopy showed germ tubes entering wound sites after 2 hours At 24 hours light microscopy revealed hyphae of P nicotianae and P. palmivora colonizing the cortical cells as deep as the third layer (Figure 6-4) Hyphae of Phytophthora palmivora were even observed to colonize the stele in the susceptible variety Phytophthora palmivora had a significantly higher colonization rating in the susceptible variety than in the tolerant variety (Table 6-1 ) The colonization ratings of the other pathosystems were not significantly different from each other at any of the times of sampling In the tolerant variety the hyphae of both P. nicotianae and P. palmivora were limited to the intercellular spaces (Table 6-2) In the susceptible variety 14% of the total number of P. palmivora hyphae observed, and 1 % of the P. nicotianae hyphae were in the intracellular spaces At 24 hours the cells of the susceptible variety sour orange, adjacent to the intercellular P. palmivora hyphae were disrupted (Figure 6-Sa). The plasma membrane collapsed and no organelles were present. The middle lamella was more pronounced and

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Figure 6-1. Prepenetration and initial penetration stages 1 to 4 hours after inoculation of root. A Germ tube initial from encysted zoospore of Phytophthora palmivora penetrating through periclinal wall of hypodermis of Citrus aurantium (x 160) Bar = 50 m B. Germ tube initial from encysted zoospore of P. nicotianae penetrating through middle lamella ofhypodermis of C. aurantium (x 160) Bar = 50 m C. Germ tube initial from encysted zoospore of P. palmivora penetrating through the middle lamella and periclinal wallofhypodermis of Poncirus trifoliata Note swelling of germ tube (g) near the host surface (x 160) Bar= 50 m D Germ tube initial from encysted zoospore of P nicotianae penetrating through periclinal walls of hypodermis of P trifoliata host (x 800) Bar= 20 m

PAGE 144

138 B

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Figure 6-2 Electron micrographs of initial penetration of Phytophthora spp on sour orange Citrus aurantium and trifoliate, Poncirus trifoliata, 1 to 4 hours after inoculation with zoospores A Germ tube initial from encysted zoospore of P. palmivora penetrating through periclinal wall of C. aurantium (x 5760) B Germ tube initial from encysted zoospore of Phytophthora nicotianae penetrating through middle lamella of C. aurantium (x 8210) C. Germ tube initial from encysted zoospore of P. palmivora penetrating through the periclinal wall of P. trifoliata (x 12960) D Germ tube initial from encysted zoospore of P nicotianae penetrating through periclinal walls of P. trifoliata (x 8210) g = germ tube h = host cell bar = 2 m

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140 C

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Figure 6-3 Accumulation and germination of encysted zoospores of Phytophthora palmivora at a natural wound site on a root of Ponciru s trif oliata 2 hours after inoculation A Light micrograph showing accumulation and encystment of zoospores at the wound site (x 160) Bar = 100 m. B Electron micrograph showing germ tube from an encysted zoospore entering wound site (x 3700) c = encysted zoospore g = germ tube h = host cell w = wound site bar = 2 m

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142

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Figure 6-4 Colonization of citrus host roots by Phytophthora spp 24 hours after inoculation A Phytophthora palmivora on Citrus aurantium (x 400) B P nicotianae on C. aurantium (x 400) C. P. palmivora on Poncirus trifo/iata (x 400) D P nicotianae on P. trifo/iata (x 400) Arrows indicate hyphae of Phytophthora spp bar = 20 m

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144

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145 Table 6-1 Root colonization by hyphae of Phytophthora spp 24 48 and 72 hours after inoculation of susceptible and tolerant citrus varieties Colonization rating v Phytophthora sp w Species x 24 hours Y 48 hours 72 hours P. palmivora sour orange 2 8 a2 8 2 a 7 9 a P. nicotianae sour orange 1 5 ab 4 2 b 6.4 b P palmivora trifoliate orange 1.0 b 4 7 b 6 7 b P. nicotianae trifoliate orange 0.4 b 3 0 b 4 5 C v Average rating of three sections from each of three different roots based upon the following scale : 0 = 0 hyphae per section (hps) ; 1 = 1-10 hps ; 2 = 11-25 hps ; 3 = 26-40 hps ; 4 = 41-50 hps ; 5 = 51-75 hps ; 6 = 76-90 hps ; 7 = 91-125 hps ; 8 = 126-175 hps; and 9 = > 175 hps ~oots were inoculated with zoospores of P. palmivora and P. nicotianae x species of host inoculated : sour orange (Citrus aurantium ; susceptible) and trifoliate orange (Poncirus trifoliata ; tolerant) Y Time sampled after inoculation with Phytophthora spp. zoospores Means within the same column followed by the same letter are not significantly different (P ~ 0 05) according to least significant differences

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146 Table 6-2 Percentage of total number ofhyphae of Phytophthora spp in host tissue that were intracellular Percent intracellular v Phytophthora sp.w Species x 24 hours Y 48 hours 72 hours P. palmivora sour orange 14. 2 a2 21.7 a 44.0 a P. nicotianae sour orange 0 9 b 24.4 a 27 9 b P. palmivora trifoliate orange 0 0 b 15. 1 ab 18. 1 b P. nicotianae trifoliate orange 0 0 b 8 5 b 18. 3 b ~ean of the percentage of total number of hyphae observed that were intracellular. ~oots were inoculated with zoospores of P. palmivora and P. nicotianae x variety of host inoculated : sour orange (Citrus aurantium ; susceptible) and trifoliate (Poncirus trifoliata ; tolerant). Y Time sampled after inoculation with Phytophthora spp. zoospores 7Means within the same column followed by the same letter are not significantly different (P ~ 0 05) according to least significant differences

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Figure 6-5 Electron micrographs of intercellular hyphae of Phytophthora spp and the effect on adjacent cortical cells of the host 24 hours after inoculation A Phytophthora palmivora on C itru s aurantium (x 12960) B. P. nicot i anae on C. aurantium (x 12960) C. P. palmivora on Poncirus trifoliata (x 8210) D. P. nicotianae on P. trifoliata (x 5760) c = cortical cell h = hypha ml = middle lamella pm = plasma membrane w = host wall bar = 2 m.

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148 I .;" .... ,<

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149 darker stained At the same time after inoculation, the cells of the susceptible and tolerant varieties adjacent to the intercellular P nicotianae hyphae and the cells of the tolerant variety adjacent to the intercellular P. palmivora hyphae were intact (Figures 6-Sb, c d) After 48 hours hyphae had grown into the stele in all treatments (Figure 6-6) The colonization rating of P palmivora on the susceptible variety sour orange was significantly higher than those of any of the other treatments (Table 6-1 ) The percentage of intracellular hyphae significantly increased over time in all treatments and was significantly higher in the susceptible variety colonized by P. nicotianae than in the tolerant variety (Table 6-2) There was no significant difference between the two varieties in colonization by P. palmi v ora Electron rnicrographs revealed cell disruption adjacent to hyphae of P nicotianae and P. palmivora in the intercellular spaces at 48 hours after inoculation (Figure 67) This was observed in all treatments There was widespread colonization by the pathogens throughout the host in all treatments 72 hours after inoculation (Figure 6-8) The colonization ratings of both Phytophthora spp examined separately was significantly higher in the susceptible variety than in the tolerant variety (Table 6-1 ) Phytophthora palmivora had the highest colonization rating of any of the treatments. There was a significantly higher amount of intracellular hyphae in the susceptible variety inoculated with P. palmivora than in the other treatments (Table 6-2) Electron rnicrographs at 72 hours after inoculation showed

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Figure 6-6 Light micrographs of the colonization of citrus roots by Phytophthora spp 48 hours after inoculation A Phytophthora palmivora on C itrus aurantium (x 200) B. P nicotianae on C aurantium (x 200) C. P. palmivora on Poncirus trifoliata (x 200) D. P nicotianae on P. trifoliata (x 400) Arrows indicate Phytophthora spp hyphae bar = 50 m.

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151 D

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Figure 6-7. Electron rnicrographs of intercellular hyphae of Phytophthora spp and the effect on adjacent cortical cells of the host 48 hours after inoculation A Phytophthora palmivora on Citrus aurantium (x 12960) B. P. nicotianae on C. aurantium (x 8210) C. P palmivora on Poncirus trifoliata (x 12960) D P. nicotianae on P trifoliata (x 5760) c = cortical cell h = hypha, pm = plasma membrane w = host wall, bar = 2 m

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153

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Figure 6-8 Light micrographs of the colonization of citrus roots by Phytophthora spp 72 hours after inoculation A Phytophthora palmivora on C itrus aurantium (x 200) B P. nicotianae on C. aurantium (x 200) C. P. palmivora on Ponciru s trifoliata (x 200) D P nicot i anae on P trifoliata (x 160) Arrows indicate hyphae of Phytophthora spp bar= 50 m

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cell disruption in all cells adjacent to P. nicotianae and P. palmivora hyphae in the intercellular spaces in both varieties (Figure 6-9) 156 The cell walls of the cortical cells were very faint in both the light and electron micrographs 168 hours after inoculation (Figures 6-10 and 6-11 ) Electron micrographs showed complete disruption of all host cells in the vicinity of hyphae Colonization of the susceptible sour orange by the Phytophthora spp ( data combined) was significantly greater than the colonization of the tolerant trifoliate at 24 and 72 hours after inoculation (Table 6-3) There were also higher percentages of intracellular hyphae in sour orange than in trifoliate at all times measured Phytophthora palmivora had a higher colonization rating than P nicotiana e after 48 and 72 hours when ratings for both varieties were combined (Table 6-4) Phytophthora palmivora also had a significantly higher percentage of intracellular hyphae at 24 and 72 hours after inoculation Electrolyte Leakage There was no electrolyte leakage detected from the infected roots until 12 hours after inoculation in any of the treatments (Figure 6-12) After 24 hours only the susceptible variety infected with P. palmi v ora had a significantly higher level of electrical conductivity per 0 1 gram of roots than the noninoculated controls No measurable electrolyte leakage occurred in the tolerant variety inoculated with P nicotianae until after 48 hours After 108 hours the electrolyte leakage of susceptible or tolerant varieties inoculated with P. palmi v ora were significantly higher than the same varieties inoculated with P. ni c otianae. The rate of electrolyte leakage was significantly

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Figure 6-9 Electron rnicrographs of intercellular hyphae of Phytophthora spp and the effect on adjacent cortical cells of the host 72 hours after inoculation A Phytophthora palmivora on Citrus aurantium (x 8210) B. P nicotianae on C. aurantium (x 8210) C. P. palmivora on Poncirus trifoliata (x 12960) D P. nicotianae on P. trifoliata (x 12960) c = cortical cell h = hypha, bar = 1 m.

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158

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Figure 6-10 Light rnicrographs of the colonization of citrus roots by Phytophthora spp 168 hours after inoculation A Phytophthora palmivora on Poncirus trifoliata (x 160) B P. nicotianae on P. trifoliata (x 160) hy = hypodermal cells, s = stele bar= 100 m

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160 A B

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Figure 6-11 Electron rnicrographs of intercellular hyphae of Phytophthora spp and the effect on adjacent cortical cells of the host 168 hours after inoculation A Phytophthora palmivora on Citru s aurantium (x 5760) B. P. nicotianae on C. aurantium (x 5760) C. P. palmivora on Poncirus trifoliata (x 8210) D P nicotianae on P trifoliata (x 8210) h = hyphae bar = 2 m

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162

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Table 6-3 Effect of a susceptible variety sour orange (Citrus aurantium) and a tolerant variety, trifoliate orange (Poncirus trifoliata) on the combined colonization and percent intracellular hyphae of Phytophthora spp u in citrus roots Colonization ratingv Percent' intracellularw Variety 24 hours Y 48 hours 72 hours 24 hours 48 hours 72 hours Sour orange 2 3 a2 5 8 a 7 1 a 9%a 23%a 34%a Trifoliate 0 7 b 4.4 a 5.6 b 0b 12% b 18% b P-value 0 .01 0 3 0 .01 0 .01 0 .01 0 .01 uoata for Phytophthora nicotianae and P palmivora combined for each variety at each time v Average colonization rating of three sections from each of three different roots based upon the following scale : 0 = 0 hyphae per section (hps) ; 1 = 1-10 hps ; 2 = 11-25 hps ; 3 = 26-40 hps ; 4 = 41-50 hps ; 5 = 51-75 hps ; 6 = 76-90 hps ; 7 = 91-125 hps ; 8 = 126-175 hps ; and 9 = > 175 hps WR_oots were inoculated with zoospores of P pa/mivora and P nicotianae xvariety of host inoculated : sour orange (Citrus aurantium ; susceptible) and trifoliate (Poncirus trifoliata ; tolerant) Y Time sampled after inoculation with Phytophthora spp zoospores 2Means within the same column followed by the same letter are not significantly different (P::,0 05) according to least significant differences ..... 0\ w

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Table 6-4 Effect of Phytophthora spp on the colonization and percent intracellular hyphae in citrus roots Colonization ratingv Percent intracellularw -Phytophthora sp. x 24 hours Y 48 hours 72 hours 24 hours 48 hours 72 hours --P. palmivora 1.9 a2 6.4 a 7.4 a 7%a 18% a 31% a P. nicotianae 0 9 a 3 6 b 5 7 b 0.4%b 16%a 24%b P-value 0.1 0 .03 0 .01 0 02 0 52 0 .05 vcolonization of three sections from each of three different roots of sour orange and trifoliate varieties combined and rated on a scale based upon the following : 0 = 0 hyphae per section (hps) ; 1 = 1-10 hps ; 2 = 11-25 hps ; 3 = 26-40 hps ; 4 = 41-50 hps ; 5 = 51-75 hps ; 6 = 76-90 hps ; 7 = 91-125 hps; 8 = 126-175 hps ; and 9 = >175 hps ~oots were inoculated with zoospores of P. palmivora and P. nicotianae x variety of host inoculated : sour orange (Citrus aurantium ; susceptible) and trifoliate (Poncirus trifoliata ; tolerant) Y Time sampled after inoculation with Phytophthora spp zoospores 2Means within the same column followed by the same letter are not significantly different (P::S0 05) according to least significant differences _. 0\

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165 200 P palmivoralsusceptible 180 --oP nicotianae l s usceptible P pa/mivora l tolerant ~--6----6 a P nicotianae/to/erant a 160 I a 140 I 120 I /b ... I QI) 100 / t / ob = 80 / I /. 60 i / / 40 / 20 '/ / -~ .0 0 / 0 20 40 60 80 100 120 Time (hours) Figure 6-12 Electrical conductivity (S per 0.1 gram of fresh root weight) of suspensions with the susceptible sour orange (Citrus aurantium) or tolerant trifoliate 50-7 (Poncirus trifoliata) inoculated with Phytophthora nicotianae or P. palmivora zoospores Data are means of 10 replicates Points at a specific time followed by the same letter are not significantly different (P>0 05) according to Duncan's multiple range test.

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166 greater from the tolerant roots infected with P. palmivora than from all other host pathogen combinations (Figure 6-13) There was no significant difference in the colonization frequency of the root tips after the completion of the study when plated on PARP-H selective medium (P < 0 05) Macerated noninoculated root tips in the tolerant and susceptible varieties had average electrical conductivities per 0 1 gram of roots of 182 and 148 respectively This was significantly different at P = 0 05 Discussion Cultivation and production of citrus probably originated in China before 2000 B C (Jackson, 1991) Although disease was present the pathosystems were in equilibrium and tolerable When Citrus spp were cultivated in areas outside of their native habitat or when new varieties were developed disease pressure often became more intense Methods for controlling or managing citrus diseases were developed and simple sanitation or cultural practices became important methods of managing diseases and keeping them from becoming threatening epidemics Over the past 50 years chemicals have been used extensively and often exclusively for the control of plant diseases However as information accumulates problems with fungicides may limit the i r use Applications of large quantities of pesticides have raised concern for human health and the environment. Effectiveness of some fungicides has been compromised by the development of resistance in some pathogens to chemicals (Fisher 1993) Thus the evaluation and selection of resistant varieties to normally destructive pathogens will

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220 / a 200 --P palmivoralsusceptible / -P nicotiana el susceptib/e / --P palmivoraltolerant / 180 -P nicotianaeltolerant / l::,. l::,. l::,. 160 / b / 0 l::,. / 140 / ... / 120 / 0 0 till .... / / C 100 / 0 / / ::s l::,./ / 0 80 / 0/ / / 0 / /-0 / C 60 / ,,,,,.. / / ~/ / / /0 ,,,,,.. 40 / __ ,,,,,.. / / o/o ,,,,,. .. /0 / 6 ,,,,,.20 / ,,,,,. / .b / g....0 ,,,,0 __ ,,,,,.0 0 20 40 60 80 100 120 Time (hours) Figure 6-13 Linear regression of electrical conductivity (S per 0 1 gram of fresh root weight) of suspensions with the susceptible sour orange ( Citrus aurantium) inoculated with Phytophthora palmivora ( o) or P. nicotianae (D) or tolerant trifoliate 507 (Poncirus trifoliata) inoculated with P nicotianae (0) or P. palmivora (t:.) zoospores Data are means of 10 replicates Lines followed by the same letter are not significantly different (P>0 05) according to orthogonal contrast analysis continue to be of great importance Overall this is probably the best way to control or manage diseases Several species of Citrus or related genera have been selected for tolerance to Phytophthora root rot (Graham 1995) 167

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168 In order to better understand resistance or tolerance in citrus the disease cycle must be studied Once this has been examined new approaches to screening for tolerance or development of new varieties can be implemented This study examined the disease cycle from initial penetration to colonization of tolerant and susceptible varieties In the early stages of infection there does not appear to be any difference in host response between the susceptible and tolerant varieties. Both species of Phytophthora encysted germinated and penetrated the host cells during the same time frame and in the same manner Broadbent (1969) also showed this behavior in P citrophthora This nonspecific action has been observed in other pathosystems involving Phytophthora spp (Beagle-Ristaino and Rissler 1983 ; Cahill et al., 1989 ; Tippett et al., 1977 ; Ward et al., 1989) Based on the electron micrographs observed it is difficult to conclude that the method of penetration is either enzymatic or mechanical Swollen germ tubes observed at the point of penetration may represent an appressorium-like structure such as that observed by Kraft et al. (1967) on bentgrass (Agrostis palustris L.) inoculated with Pythium aphanidermatum (Edson) Fitz zoospores These swollen germ tubes were also observed in P. cinnamomi (Cahill et al. 1989) and P. megasperma (Beagle-Ristaino and Rissler 1983) Lazarovits et al. (1981) and Broadbent (1969) called these structures appressoria However Beagle-Ristaino and Rissler (1983) were reluctant to refer to them as appressoria because adherance to the host was not investigated If these structures are appressoria penetration of the host by mechanical action may be indicated However, electron micrographs do not reveal an obvious disruption in the cell wall which might as

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169 other studies have concluded (Benhamou and Cote 1992 ; Hohl and Suter 1976) support penetration by enzymatic action rather than mechanical action Observations in the light microscopy of colonization by P nicotianae and P. palmivora on susceptible and tolerant hosts and in the electron microscopy of cell disruption support the data observed from the electrolyte leakage experiment. Significant amounts of electrolytes were detected after 24 hour only in roots of the susceptible variety infested with P palmivora This was the only treatment that showed disruption of the cells adjacent to the intercellular hyphae in micro graphs 24 hours after inoculation. After 48 hours all treatments showed cell disruption However in roots of the tolerant variety infected with P. nicotianae there were no electrolytes detected This might be explained by the lower colonization rating in this pathosystem and a lower percent of intracellular hyphae Most cortical cells in the vicinity of hyphae collapsed in both varieties 72 hours after inoculation with either P nicotianae or P palmivora After 72 hours the curves are significantly different than each other The roots of the tolerant variety infested with P. palmivora had the highest electrolyte leakage because it had a greater colonization rating than the roots of the tolerant variety infested with P. nicotianae and is greater than the roots of the susceptible variety infested with either P. palmivora or P nicotianae This is attributed to a higher concentration of electrolytes in the tolerant variety than that in the susceptible variety. The electrolyte leakage of the roots of the susceptible variety infested with P. palmivora was greater than the leakage of the roots of the susceptible or

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170 tolerant varieties infested with P nicotianae because the colonization was greater The electrolyte leakage of the roots of the susceptible variety infested with P. nicotianae was greater than the roots of the tolerant variety infested with P. nicotianae, because also the colonization is greater thus collapsing more cells and releasing more electrolytes Zilberstein and Pinkas (1987) used this test to screen for Phytophthora root rot-resistant varieties of avocado However such a test does not seem to be valid for varieties of Citrus spp due to the difference in cell electrolyte concentration between varieties The disruption of the adjacent cells to the intercellular hyphae may suggest the production of a toxin by the pathogens This collapse is similar to that observed by Hanchey and Wheeler (1969) in oat roots treated with the toxin victorin produced by Drechslera v ictoria e (Meehan & Mruphy) Suhr & Jain This reaction which has been termed cell necrosis has been observed in other pathosystems involving Phytophthora spp (Cahill et al., 1989 ; Tippett et al., 1977 ; Ward et al., 1989) However necros i s in advance of hyphae was not observed in soybean rootlets infected with P sojae (Slusher et al., 1974) Toxins have been isolated from Phytophthora spp in culture (BaUio et al., 1972 ; Breiman and Barash 1981; Graniti 1969 ; Keenan et al. 1985 ; Paxton 1972 ; Strange et al., 1982) Collapse of cells in advance of hyphae in both susceptible and tolerant varieties suggest that this is not a hypersensitive response This was also noted by Tippet et al. (I 977)

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CHAPTER 7 CONCLUSION Composted municipal waste was effective in suppressing the infection of citrus seedlings by Phytophthora nicotianae when amended into the planting soil. However, suppression was lost over time and the loss is accentuated by improper storage As demonstrated by the reduction in infection and colony growth a level of fresh CMW as low as 10% (v/v) incorporated into the planting soil was effective in reducing disease under greenhouse conditions The CMW significantly increased the growth of young citrus trees even in the presence of P. nicotianae However CMW did not reduce populations of P nicotianae in the field. The horticultural benefits demonstrated such as increasing tree growth and root density may be sufficient for the host to tolerate or outgrow the damage caused by the pathogen However if environemental conditions favor the pathogen disease pressure may become too intense and field tolerance will be overcome In the young tree plots there were no significant differences in the densities of fibrous roots recovered from cores among nonamended and CMW-amended treaments However in destructive sampling the CMW-amended plots had a significantly higher root density than the nonamended plots and there was a correlation between the total fibrous root weights and tree growth Thus core sampling of young trees to minimize damage to root systems was not adequate to detect differences in root growth. 171

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172 The most important factor to citrus growers is a yield response The CMW applied to the mature groves did not give an increase in yield although the fruit were significantly larger in the ValVB and GF-1 plots As in young tree plots populations of P nicotianae were not reduced significantly at any of the times sampled for any of the plots of mature trees Although white-tipped fibrous roots were observed growing in the CMW-layer half a year after application only in the Val-VB plot was there a significant increase in fibrous root density and only at one time period In fact in the very sandy well-drained soil in GF-1 and GF-11 plots the nontreated trees had significantly more roots than the treated trees except for one sampled date in the GF-11 plot. In the study of events during infection of roots by P. nicoti anae and P palmi v ora light and electron microscopy showed no differences between the susceptible and t olerant host response in the prepenetration and postpenetration phases Both Ph y tophthora spp on both citrus species were equal in timing of encystment and penetration and mode of penetration was similar However, differences in colonization and percentage of intracellular hyphae were observed 24 hours after inoculation ; the susceptible species was colonized more intensively than the tolerant variety and it had more intracellular hyphae The cellular response adjacent to intercellular hyphae in either the susceptible or tolerant species indicates that a toxin may be produced in situ by P. palmivora and P nicotianae. Based upon EM micrographs and electrolyte leakage the effectiveness of the proposed toxin to degrade cell content may depend upon the pathogen and the susceptibility of the host.

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APPENDIX A ANALYSES OF COMPOSTED MUNICIPAL WASTE Table A-1. Selected characteristics of composted municipal waste (CMW) used in ex perimental procedures Source w Batch v pW %C %NY C/N Ratio2 Reuter RI 7 66 34 7 0 .85 40 9 Reuter R3 7 50 24 0 1.07 22.4 Bedminster B2 7 .91 33.4 1.71 19. 9 Bedminster B4 7 52 w source of composted municipal waste : Bedminster = Bedminster Corp Sevierville TN; Reuter = Reuter Recycling Pembroke Pines FL. VOistinction of separate batches ofCMW: RI= received August 1996 ; R3 = received January 1994 ; B2 = received June 1994 ; B4 = received February 1996. w pH of the liquid extract from a mixture of 1 part CMW to 2 parts 0 .01 M CaCl2 "Percent carbon (C) of the total weight of the CMW Y_Percent nitrogen (N) of the total weight of the CMW 2Carbon : nitrogen (C/N) ratio of CMW 173

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174 Table A-2 Total phosphorous (P) calcium (Ca) magnesium (Mg) potassium (K), and sodium (Na) concentrations of composted municipal wastes (CMW) used in experimental procedures p Ca Mg K Na -------------------------------------Source x Batch Y mg Kgl z Reuter RI 1310 25400 1450 2500 3810 Reuter R3 2547 52510 2078 2267 3626 Bedminster B2 2625 33750 2310 3200 Bedminster B4 2870 24770 2430 3440 xsource of composted municipal waste : Bedminster= Bedminster Corp Sevierville TN; Reuter = Reuter Recycling Pembroke Pines FL YJ)istinction of separate batches ofCMW: Rl = received August 1996 ; R3 = received January, 1994 ; B2 = received June 1994 ; B4 = received February 1996 2Concentration expressed in mg of element per kilogram of CMW.

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175 Table A-3 Total zinc (Zn) copper (Cu) manganese (Mn), iron (Fe) cadmium (Cd) lead (Pb) and nickel (Ni) concentrations of composted municipal wastes (CMW) used in the experimental procedures Zn Cu Mn Fe Cd Pb Ni ---------------------------Source x Batch Y mg per Kg of CMW2 Reuter Rl 384 123 158 3240 5 272 18 Reuter R3 779 323 248 7610 5 406 42 Bedminster B2 563 175 260 13425 4 250 35 Bedminster B4 423 165 210 8805 2 212 34 xsource of composted municipal waste : Bedminster = Bedminster Corp., Sevierville TN; Reuter = Reuter Recycling Pembroke Pines FL. Yl)istinction of separate batches ofCMW: Rl = received August 1996 ; R3 = received January 1994 ; B2 = received June 1994 ; B4 = received February 1996 2Concentration e x pressed in mg of element per kilogram of CMW.

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Table A-4 Concentrations of nitrate nitrogen (N03-N) ammonium nitrogen (NH4-N), phosphorous (P) calcium (Ca) magnesium (Mg), potassium (K) and sodium (Na) in saturated water extracts of composted municipal wastes (CMW) used in experimental procedures Source x Reuter Reuter Bedminster Batch Y RI 0.2 R3 143.2 B2 0 0 NH4-N 82 6 79 1 45 6 p Ca Mg mg per liter of extract2 0 2 I.I 292 337 60 68 K 455 388 176 Na 695 991 xsource of composted municipal waste : Bedminster = Bedminster Corp. Sevierville TN; Reuter = Reuter Recycling Pembroke Pines FL. YDistinction of separate batches ofCMW: RI= received August 1996 ; R3 = received January 1994 ; B2 = received June 1994 2Concentration expressed in mg of element per liter of CMW extract.

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Table A-5 Concentrations of zinc (Zn) copper (Cu) manganese (Mn) iron (Fe) cadmium (Cd) lead (Pb) and nickel (Ni) in saturated water extracts of composted municipal wastes (CMW) used in the experimental procedures Zn Cu Mn Fe Cd Pb 177 Ni ----------------------------------------------... Source x Batch Y mg per liter of extract z Reuter Rl 2.4 0 8 0.9 3 3 0 0 0 2 0 3 Reuter R3 0 6 0 3 0 0 0.4 0 0 0 1 0 1 xsource of composted municipal waste : Reuter = Reuter Recycling Pembroke Pines FL. YDistinction of separate batches ofCMW: Rl = received August 1996 ; R3 = received January 1994 ZConcentration expressed in mg of element per liter of CMW extract.

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APPENDIXB EFFECT OF COMPOSTED MUNICIPAL WASTE ON SOIL TEMPERATURE AND MOISTURE 178

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a 35 ~----------------------------~ ---Soil only -oIncorporated CMW 30 -<>-Layered CMW 25 j 20 15 10 5 -'---+----+-----+-----+--~,----r-+-----------~ May June July Aug Sept Date Nov Dec Jan Figure B-1. Soil temperatures measured 7 5 cm below the surface of nonamended soil ( soil only) soil incorporated with composted municipal waste (CMW) at a level of20% (v / v) of the total volume (incorporated CMW) and soil with a 5-cm thick layer of CMW (layered CMW) Soil temperatures were measured every 30 minutes and averaged together for each treatment every 4 hours by a Campbell 21X micrologger (Campbell Scientific Inc., Logan UT) ....... -...J \0

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1.0 0 8 I 0 6 I i 1 0.4 0 2 0 0 --LayeredCMW Soilonly 0 Incorporated CMW May June July Date August September Figure B-2 Soil moisture measured 7 5 cm below the surface of nonamended soil (soil only) soil incorporated with composted municipal waste (CMW) at a level of 20% (v/v) of the total volume (incorporated CMW) and soil with a 5-cm-thick layer of CMW (layered CMW) The soil moisture was measured every 30 minutes and values were averaged together for each treatment every 4 hours by a Campbell 21X micrologger (Campbell Scientific Inc. Logan UT) 00 0

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181 Table B-1 The effect of composted municipal waste (CMW) on soil temperature 7 5 cm below the surface at two time periods Temperature (C) Treatment Y 10: 00 am2 4 : 00 pm Soil only 25.4 a 25 3 a Incorporated CMW 25. 2 a 25. 1 a Layered CMW 25. 6 a 25 0 a Y Soil temperature measured in soil only incorporated CMW and layered CMW. 2Time of day of temperature measurement.

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182 Table B-2 The effect of composted municipal waste (CMW) on soil moisture 7 5 cm below the surface at two time periods Soil moisture (bars) Treatment Y 4 : 00 pm2 10 : 00 pm Soil only 0 26 a 0 25 a Incorporated CMW 0 .21 b 0 20 b Layered CMW 0 .23 ab 0 22 ab Y Soil moisture measured in soil only incorporated CMW and layered CMW 2Time of day of temperature measurement.

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

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Table C-1 Incidence of root infection and percentage of root system of Citrumelo 507 seedlings infected in nonamended soil and soil amended with composted municipal waste (CMW) infested with Phytophthora palmivora chlamydospores (10 chlamydospores per cm3 ) Treatmentu Infestation v No. w -soil only -10 CMW -10 soil only + 10 CMW + 10 Incidence of infection x 0% 0% 50% 0% Percentage of root SignificanceY system infected2 ** 0 ** 0 ** 15 0 Significance ** ** NS uPasteurized Candler fine sand nonamended (soil only) or amended (CMW) with a 20% (v/v) of the total volume of CMW batch B4 from Bedminster Bioconversion (Sevierville TN) VJnfested or noninfested with P. palmivora chlamydospores w Total number of 5-week-old seedlings tested xpercent of the total seedlings infected with P. palmivora Y Statistical analysis of comparisons to infested nonamended treatments ; NS = not significant ** = significant at P:s0 .01. 2The mean percentage of the area of the total root system that was infected with P. palmivora ...... 00

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APPENDIXD EFFECT OF ACETIC ACID ON CITRUS ROOT INFECTION 185

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Table D-1 Incidence of root infection and percentage of root system infected of 5-week-old sour orange seedlings in nonamended and acetic acid-amended soil, infested with Phytophthora nicotianae chlamydospores ( 10 chlamydospores per cm3); no infection occurred in plants grown in noninfested controls Incidence of Percentage of root Treatmentu Infestation v No_ w infection" Significanc&' system infectedz Significance Soil only 10 0% ** 0 ** Soil+150 10 0% ** 0 ** Soil only + 10 100% 25.4 Soil+50 + 10 70% NS 14 8 NS Soil+ 150 + 10 100% NS 15. 2 NS uPasteurized Candler fine sand nonamended (soil only) or amended to a final concentration of 50 ppm (soil+50) or 150 ppm (soil+ 150) glacial acetic acid 1:nfested or noninfested with P. nicotianae chlamydospores wTotal number of 5-week-old seedlings tested "Percent of the total seedlings infected with P. nicotianae Y Statistical analysis comparing to infested, nonamended treatments; NS = not significant ** = significant at P::;0 01. zThe mean percentage of the area of the total root system that was infected with P nicotianae ...... 00 0\

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APPENDIXE EFFECT OF ACREMONIUM SP. ON CITRUS ROOT INFECTION 187

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Table E-1 Effect of soil infestation with 10 chlamydospores of Phytophthora nicotianae per cm3 of soil on incidence of root infection and percentage of root system of sour orange seedlings infected in nonamended soil and soil amended with pasteurized composted municipal waste (CMW) (Reuter Recycling Pembroke Pines FL) supplemented with 1.4 X 107 colony forming units of Acremonium sp. per cm3 of soil. Incidence of Percentage of root Treatmentu Infestationv No. w infection x SignificanceY system infectedz Soil only Soil+antag Soil only SC+antag + + 10 10 10 10 0% 0% 90% 80% ** ** NS 0 0 56 0 25 5 Significance ** ** uPasteurized Candler fine sand nonamended (soil only) or amended with pasteurized CMW (20% v/v) and supplemented with Acremonium sp. (SC+antag) vrnfested or noninfested with P. nicotianae chlamydospores wTotal number of 5-week-old seedlings tested xpercent of the total seedlings infected with P. nicotianae Y Statistical analysis comparing to infested nonamended treatments ; NS = not significant, = significant at P ~ 0 .05, * = significant at P 0 01. z The mean percentage of the area of the total root system that was infected with P. nicotianae. ..... 00 00

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201 Lutz A.L. and Menge J.A. 1991. Population fluctuations and the numbers and types of propagules of Phytophthora parasitica that occur in irrigated citrus groves Plant Dis 75: 173-179 Lutz A., Menge J., and O' Connel N 1986 Hardpans claypans and other mechanical impedences Citrograph 71 : 57-61. Lyda S D 1982 Physical and chemical properties of suppressive soils Pages 9-27 in: Suppressive Soils and Plant Disease R.W Schneider ed APS Press St. Paul Minnesota MacDonald, J.D and Duniway J.M 1978 Influence of the matric and osmotic components of water potential on zoospore discharge in Phytophthora Phytopathology 68:751-757 MacDonald, J.D Stites J. and Kabashima J. 1990 Comparison serological and culture plate methods for detecting species of Phytophthora Pythium and Rhizoctonia in ornamental plants Plant Dis 74:655-659 Machado-Neto J.G and Vict6ria-Filho R. 1995 Dissipation of herbicide residues in the soil of a citrus orchard (Citrus sinensis L. Osbeck) after the ninth consecutive annual application Bull Environ Contam Toxicol. 55: 303-308 MacKenzie, D .R., Elliot V.J. Kidney B.A. King E.D Royer, M H. Theberge R.L. 1983 Application of modem approaches to the study of the epidemiology of diseases caused by Phytophthora Pages 303-313 in: Phytophthora : Its Biology Taxonomy Ecology and Pathology D C Erwin S Bartnicki-Garcia and P H. Tsao eds APS Press St. Paul Minnesota Malajczuk N 1983 Microbial antagonism to Phytophthora Pages 197-218 in: Phytophthora : Its Biology Taxonomy Ecology and Pathology D .C. Erwin S Bartnicki-Garcia and P .H. Tsao eds APS Press St. Paul Minnesota Malajczuk N., McComb A.J. and Parker C.A. 1975 An immunotluorescence technique for detecting Phytophthora cinnamomi Rands Aust. J. Bot. 23: 289-309 Malajczuk, N Nesbitt H.J., and Glenn A.R 1977 A light and electron microscope study of the interaction of soil bacteria with Phytophthora cinnamomi Rands Can J. Microbiol. 23: 1518-1525. Malathrakis, N E 1985 The fungusAcremonium alternatum Linc : Fr., a hyperparasite of the cucurbits powdery mildew pathogen Sphaerotheca fuliginea J. Plant Dis Protect. 92 : 509-515

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202 Malek RB. and Gartner J.B. 1975 Hardwood bark as a soil amendment for suppression of plant parasitic nematodes on container-grown plants HortScience 10: 33-35 Mandelbaum R Habar Y., and Chen Y. 1988 Composting of agricultural wastes for their use as container media : Effect of heat treatments on suppression of P y th i um aphanid e rmatum and microbial activity in substrates containing compost. Biological Wastes 26 : 261-274 Mauch F., Mauch-Mani B. and Boller T. 1988 Antifungal hydrolases in pea tissue II Inhibition of fungal growth by combinations of chitinase and P-1, 3-glucanase Plant Physiol. 88 : 936-942 Maurhofer M., Hase C., Meuwly P., Metraux J.P. and Defago G 1994 Induction of systemic resistance of tobacco to tobacco necrosis virus by the root colonizing P s eudomonasfluoresc ens strain CHAO: Influence of the gacA gene and of pyoverdine production Phytopathology 84 : 139-146 Maynard A.A 1989 Agricultural compost as amendments reduce nitrate leaching from the soil. Frontiers of Plant Sci Fall : 2-4 Maynard D G and Kalra Y.P 1993 Nitrate and exchangeable ammonium nitrogen Pages 25-38 in: Soil Sampling and Methods of Analysis M R Carter ed Lewis Publishers Boca Raton FL Mays D .A., Terman G .L., and Duggan J.C 1972 Municipal compost: Effects on crop yields and soil properties J. Environ Qual. 2 : 89-92 McIntyre J.L. and Hankin L. 1978 An examination of enzyme production by Phytophthora spp on solid and liqu i d media Can J. Microbiol. 24 : 75-78 Menge J., Morse J., Hare D., Coggins C Pehrson J., Meyer J., Embleton T., VanGundy, S Dodds A., Arpa i a M .L., Takele E., Adams C., Strawn A., Pond E., and Atkin D 1990 Integrated crop management increases citrus growth and yields Calif Agric 44 : 10-12 Michelmore R 1995 Molecular approaches to manipulation of disease resistance genes Annu Rev Phytopathol. 33 : 393-427 Mircetich S M and Zentmeyer G .A. 1970 Germination of chlamydospores of Phytophthora Pages 112-115 in: Root Diseases and Soilbome Pathogens Tousson T.A. Bega RV., and Nelson P E., eds University of California Press Los Angeles

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Sneh B., Humble S.J and Lockwood J.L. 1977 Parasitism of oospores of Phytophthora megasperma var. sojae, P cactorum Pythium sp., and Aphanomyces euteiches in soil by oomycetes chytridiomycetes hyphomycetes actinomycetes and bacteria Phytopathology 67 : 622-628. Sommers L.E Harris RF., Dalton F N., and Gardner W .R. 1970. Water potential relations of three root-infection Phytophthora species Phytopathology 60 : 932-934. Spencer, S and Benson D M 1981. Root rot of Aucubajaponica caused by Phytophthora cinnamomi and P. citricola and suppressed with bark media Plant Dis. 65: 918-921 Spencer, S and Benson D M 1982 Pine bark hardwood bark compost and peat amendment effects on development of Phytophthora spp and lupine root rot. Phytopathology 72 : 346-351 207 Stamps D.J. Waterhouse G M., Newhook, F.J. and Hall G.S 1990 Revised tabular key to the species of Phytophthora CAB Int. Mycol. Inst. Mycol. Papers No. 162 28 pp Steffen A. and Pawliszyn J. 1996 Analysis of flavor volatiles using headspace solid phase microextraction J. Agric. Food Chem. 44 : 2187-2193 Stephens C.T. Herr L.J Hoitink H.A.J. and Schmitthenner AF. 1981. Suppression of Rhizoctonia damping-offby composted hardwood bark medium. Plant Dis. 65 : 796-797 Stephenson RE. and Schuster C.E. 1945 Effect of mulches on soil properties Soil Sci 59 : 219-230 Stossel P., Lazarovits, G., and Ward E.W .B. 1981. Differences in the mode of penetration of soybean hypocotyls by two races of Phytophthora megasperma var. sojae Can J. Bot. 59 : 1117-1119 Strange RN., Pippard D .J. and Strobel G .A. 1982 A protoplast assay for phytotoxic metabolites produced by Phytophthora dreschleri in culture Physiol. Plant Pathol. 20 : 359-364 Sun S and Huang J. 1985 Formulated soil amendment for controlling Fusarium wilt and other soilborne diseases Plant Dis 69 : 917-920 Tarjan A.C 1977 Use of municipal refuse compost on nematode-infected citrus Citrus Veg Mag 40 : 44-49 Thurston HD. 1992 Sustainable Practices for Plant Disease Management in Traditional Farming Systems Westview Press Boulder, Colorado

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211 Wolf, D C and Skipper H.D 1994 Soil sterilization. Pages 41-51 in : Methods of Soil Analysis Part 2 Microbiological and Biochemical Properties R.W Weaver et al., eds Soil Science of Society of America Inc Madison Wisconsin Woloshuk C P., Meulenhoff, J.S Sela-Buurlage M., van den Elzen P J.M., and Cornelissen B.J C 1991. Pathogen-induced proteins with inhibitory activity toward Phytophthora infestans. Plant Cell 3 : 619-628 Yang C., Menge J.A. and Cooksey, D .A. 1991. Mutations affecting hyphal colonizat i on and pyoverdine production in pseudomonads antagonistic toward Phytophthora para s itica Appl. Environ Microbiol. 60 : 473-481. Yu L.M 1995 Elicitins from Phy t oph t hora and basic resistance i n tobacco Proc Natl. Acad Sci USA 92 : 4088-4094 Zentmeyer G .A. 1983 The world of Phytophthora Pages 1-7 in: Phytophthora : Its Biology Taxonomy Ecology and Pathology D C Erwin S Bartnicki-Garcia and P H Tsao eds APS Press St. Paul Minnesota Zhang Z and Pawliszyn J. 1993 Head space solid-phase microextraction Anal. Chem 65: 1843-1852 Zilberstein M and Pinkas Y. 1987 Detached root inoculation a new method to evaluate resistance to Phytophthora root rot in avocado trees Phytopathology 77 : 841844 Zitko S E and Timmer L.W 1994 Competitive abilities of Phytophthora para s itica and P. palmivora on fibrous roots of citrus Phytopathology 84 : 1000-1004 Zitko S E Timmer L.W and Castle W S 1987 Survey of Florida citrus nurseries for Phytophthora spp Proc Fla State Hortic Soc 100 : 82-85 Zitko, S E., Timmer L.W and Sandler H.A. 1991. Isolation of Phytophthora palmivora pathogenic to citrus in Florida. Plant Dis 75: 532-535 Zucconi F., Pera A., Forte M and de Bertoldi M 1981. Evaluating toxicity of immature compost. Biocycle 22 : 54-57

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BIOGRAPHICAL SKETCH Timothy L. Widmer was born January 21, 1964 in Albany Oregon He received his high school diploma in June, 1982 from South Albany High School. He graduated in August, 1987 from Goshen College, Goshen, Indiana receiving his Bachelor of Arts degree in chemistry Enrolling in the graduate program of the Plant Pathology Department at the University of Florida he completed his research at the Citrus Research and Education Center in Lake Alfred FL. He expects to earn his Doctor of Philosophy degree in December 1996 212

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy David J. MiYchell, Chair Professor of Plant Pathology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality as a dissertation for the degree of Doctor of Philosophy H. Graham Jr. Cochair Professor of Soil and Water Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy Lavern W Timmer Professor of Plant Pathology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality I as a dissertation for the degree of Doctor of Philosophy L ~,~ J.&dn mes W Kimbrough Professor of Plant Pathology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy ~A-~ Donald A. Graetz Professor of Soil and Water Science

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This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. )~ ;t', J{, December 1996 Dean College of AgricllUfe Dean Graduate School

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