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Reduction of populations of Phytophthora spp. with soil solarization under field conditions and thermal inactivation of Phytophthora nicotianae

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Title:
Reduction of populations of Phytophthora spp. with soil solarization under field conditions and thermal inactivation of Phytophthora nicotianae
Creator:
Coelho, Lísias
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English
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ix, 136 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Cabbages ( jstor )
Chlamydospores ( jstor )
Pathogens ( jstor )
Phytophthora ( jstor )
Soil science ( jstor )
Soil solarization ( jstor )
Soil temperature regimes ( jstor )
Soil water ( jstor )
Soils ( jstor )
Solar temperature ( jstor )
Dissertations, Academic -- Plant Pathology -- UF ( lcsh )
Fungal diseases of plants ( lcsh )
Phytophthora nicotianae ( lcsh )
Plant Pathology thesis, Ph. D ( lcsh )
Soil microbiology ( lcsh )
Soil solarization ( lcsh )
Gadsden County ( local )
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1997.
Bibliography:
Includes bibliographical references (leaves 127-135).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Lísias Coelho.

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REDUCTION OF POPULATIONS OF PHYTOPHTHORA SPP.
WITH SOIL SOLARIZATION UNDER FIELD CONDITIONS
AND THERMAL INACTIVATION OF PHYTOPHTHORA NICOTIANAE









By


LISIAS COELHO















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 1997




























To Eliamar C. M. Coelho,

who has encouraged and supported me through the years.















ACKNOWLEDGMENTS


I would like to thank the Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) for the scholarship granted, and the Florida/Brazil Institute for the fee waiver, without which it would have been impossible for me to pursue a higher education.

I greatly appreciate the efforts of my major professor, Dr. David J. Mitchell, who with great patience taught the basics of research with soilborne plant pathogens, broadening my horizons, and, above all for his friendship; of Dr. Daniel O. Chellemi, who provided for all the field work, and made himself available whenever needed; and the other members of the supervisory committee, Dr. Robert J. McGovern, Dr. Harold C. Kistler, and Dr. David M. Sylvia, for their support and assistance.

Sincere appreciation is extended to Dr. Steven A. Sargent and Dr. Donald W.

Dickson, who provided space and equipment for parts of this study; to Gelman Sciences, and Arclad Adhesives Research, Inc. for providing materials for the field research; and to Rogers NK Seed Company for providing seeds for several tests.

In addition I would like to thank Patricia Rayside and Dr. Mary E. Mitchell for the support with lab procedures, "do's and don'ts," and helpful insight. Patricia E. Hill for the assistance in the lab, volunteered time beyond her duty, and mostly for her sincere friendship; G. Hank Dankers, Susan H. Lee, and Michelle L. Dankers, who helped with the tough part of the field work; Mircia Moura for the help with the controlled atmosphere experiment; and Mr. Jay Harrison for the support with the statistical analysis using SAS and making sense of a "bunch of numbers."





111














Finally, I appreciate the friendship of all of my colleagues in the department and the stimulus they provided to do a little more, a little better. Their companionship during our brief journey at the University of Florida made life a whole lot more enjoyable.








































iv















TABLE OF CONTENTS

page

ACKNOW LEDGEM ENTS .................................................................. ......... iii

TABLE OF CONTENTS ................. ...................................... v

A B S T R A C T ............................................................................. viii

CHAPTERS

1 INTRODUCTION AND LITERATURE REVIEW ..................................... .............. 1

2 THE EFFECT OF SOIL SOLARIZATION ON POPULATIONS OF
PHYTOPHTHORA NICOTIANAE AND P. CAPSICI UNDER FIELD
C O N D IT IO N S .................................................................................. . 13
Introduction ........................................... ............ 13
M aterials and M ethods .................................................. .......... ... 14
Characterization of the Solarization Sites ........................................ 14
Inoculum Production ......................................... 16
The Solarization Experiments ......................... ............... 17
Statistical Analysis ........................................ 20
R e su lts .................................................................................................................... 2 0
E nvironm ental C onditions ................................................................................. 20
Survival of Phytophthora nicotianae ........................................... ................ 27
Survival of Phytophthora capsici ........................................................ 29
Discussion .......................... .............................. 33
Temperature Changes and Soil Moisture Content ................... ....... .. ...... . 33
Survival of Phytophthora nicotianae ................................................. 34
Survival of Phytophthora capsici ......... ............................. .......... 3 5
Effect of Cabbage Amendment ................................... 36

3 THERMAL INACTIVATION OF PHYTOPHTHORA NICOTIANAE .................... 37
In tro d u ctio n ............................................................................................................ 3 7
Materials and Methods ...................................................... 39
Production of Chlamydospore Inoculum of Phytophthora nicotianae ................ 39
The Effect of Contant Temperature on the Inactivation of Chlamydospores
of Phytophthora nicotianae ........................................................ 39
The Effect of Cabbage Amendments on the Thermal Inactivation of
Chlamydospores of Phytophthora nicotianae ............ .. . ............... 41



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The Effect of Cycling Temperatures and Cabbage Amendments on the
Thermal Inactivation of Chlamydospores of Phytophthora nicotianae .......... 42
The Effect of Soil Water Matric Potential, Temperature Regimes, and
Cabbage Amendments on the Thermal Inactivation of Chlamydospores
of Phytophthora nicotianae ....................................................................... 44
The Effect of Three Nonpasteurized Soils, Temperature Regimes, and
Cabbage Amendments on the Thermal Inactivation of Chlamydospores
of Phytophthora nicotianae ................................ ................ ... ......... .. 45
Statistical A nalysis ........................................ 45
Results ............................................ ... ............ ............. 46
The Effect of Contant Temperature on the Inactivation of Chlamydospores
of Phytophthora nicotianae .................................. ............................. 46
The Effect of Cabbage Amendments on the Thermal Inactivation of
Chlamydospores of Phytophthora nicotianae ......................................... 51
The Effect of Cycling Temperatures and Cabbage Amendments on the
Thermal Inactivation of Chlamydospores of Phytophthora nicotianae .......... 57
The Effect of Soil Water Matric Potential, Temperature Regimes, and
Cabbage Amendments on the Thermal Inactivation of Chlamydospores
of Phytophthora nicotianae ........................................................................ 59
The Effect of Three Nonpasteurized Soils, Temperature Regimes, and
Cabbage Amendments on the Thermal Inactivation of Chlamydospores
of Phytophthora nicotianae ........................................................ 70
D isc u ssio n .............................................................................................................. 7 7

4 SUMMARY AND CONCLUSIONS ................................................. 87

APPENDICES

A PRODUCTION OF CHLAMYDOSPORES OF PHYTOPH7HORA
NICO TIANAE .................................................................................... 92
Intro du ctio n ............................ . .............................. ............................................ 9 2
Materials and Methods ................................. ......... ............ 93
Results and D iscussion .......................................................................... ...... ............... 95

B DETERMINATION OF SOIL WATER MATRIC POTENTIAL ........................... 98

C REGRESSION ANALYSES OF THE EFFECTS OF TEMPERATURE
REGIMES AND CABBAGE AMENDMENT ON SURVIVAL OF
PHYTOPHTHORA NICOTIANAE ................. .... ................................. 100

D SUMMARY TABLE OF THE STATISTICAL ANALYSIS OF THE
EFFECT OF SOIL WATER MATRIC POTENTIAL, TEMPERATURE
REGIMES, AND CABBAGE AMENDMENTS ON THE THERMAL
INACTIVATION OF CHLAMYDOSPORES OF PHYTOPHTHORA
NICOTIANAE ............... ............................................... ... ......... 121




VI









E SUMMARY TABLE OF THE STATISTICAL ANALYSIS OF THE
EFFECT OF THREE DIFFERENT SOILS, SOIL PASTEURIZATION,
TEMPERATURE REGIMES, AND CABBAGE AMENDMENTS ON THE
THERMAL INACTIVATION OF CHLAMYDOSPORES OF
PHYTOPHTHORA NICOTIANAE ........................................ 124

LIST OF REFERENCES ........... .................... .......... 127

BIOGRAPHICAL SKETCH ................... ........................................................... 136












































vii














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


REDUCTION OF POPULATIONS OF PHYTOPHTHORA SPP.
WITH SOIL SOLARIZATION UNDER FIELD CONDITIONS
AND THERMAL INACTIVATION OF PHYTOPHTHORA NICOTIANAE By

Lisias Coelho

May, 1997

Chairman: David J. Mitchell
Major Department: Plant Pathology


The effects of soil solarization in combination with cabbage amendments on the survival of populations of Phytophthora spp. were evaluated in North Florida. Soil under solarization for 45 to 55 days reached a maximum temperature of 470C on up to 10 days at 10 cm of depth. Soil solarization with a clear, gas impermeable, low density polyethylene film was as effective as methyl bromide in reducing populations of P. nicotianae at a depth of 10 cm; however, at 25 cm of depth the population of the pathogen was similar to that in the control treatments. Reduction of survival ofP. capsici in two sites in 1994 under solarization with either a clear, low density polyethylene or a clear, gas impermeable, low density polyethylene film was as effective as the methyl bromide treatment at the 10-cm depth, while at the 25-cm depth no control was observed. Incorporation of cabbage into the soil at a rate of 70 to 90 metric tons per hectare did not enhance the effectiveness of solarization.




viii









Studies on the thermal inactivation of P. nicotianae in the laboratory confirmed that the time required to inactivate chlamydospores of the pathogen is inversely proportional to the temperature of the treatment. The time required to reduce soil populations from 500 propagules per gram of soil to residual levels (0.2 propagule per gram of soil or less) was 10 minutes, 45 minutes, 4 hours, 12 hours, 4 days, and 16 days at 530, 500, 470, 440, 410 and 380C, respectively. The incorporation of cabbage into the soil reduced the time required to inactive the chlamydospores of P. nicotianae. Detection of P. nicotianae by the tomato-seedling baiting technique provided similar results to the soil plating procedure, except when only residual populations were present in the soil.

Temperature regimes that simulated solarization periods were effective in

eliminating P. nicotianae only when optimum regimes were used (470C for 3 hours daily and 44oC for 5 hours daily). Populations of P. nicotianae decreased at matric potentials of 0, -10, and -30 kPa with time of exposure to each temperature regime, but the lowest survival occurred in saturated soil.

Soils with different cropping history and pasteurization treatment may have

marked differences on the survival of P. nicotianae. Nonpasteurized soils from a fallow field, coupled with optimum temperature regimes, provided the greatest reduction in survival of the pathogen in relation to two other soils from commercial cropping systems.


















ix














CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW



Control of plant diseases caused by soilborne pathogens constitutes one of the most difficult aspects of disease management. In general, plant disease control is based on six principles: avoidance, exclusion, eradication, protection, disease resistance, and therapy (Maloy, 1993). Once a pathogen has been introduced in an area, efforts are generally directed to eradicating it or protecting the plant from disease. Tactics for control of diseases include disease resistance, crop rotation, and other cultural practices, or the use of chemicals to protect plants. For high value crops, one of the most effective methods for the control of soilbome plant pathogens is fumigation of soil with methyl bromide or other chemicals. The imminent ban on the production and use of methyl bromide has prompted a search for other alternatives for disease control. Among these strategies, removing a field from production for a considerable period would allow the reduction of populations of pathogens by attrition. However, this strategy may not be effective for pathogens that form resting structures that can survive for long periods in the absence of the host plant (Baker and Cook, 1974). The use of plastic films for solarization of soil in agriculture has opened new horizons for the control of soilborne plant pathogens. Solarization raises the soil temperature to levels that are lethal to most mesophyllic organisms; thus, soilborne plant pathogens of many crops may be controlled. The mechanisms by which disease control is achieved with this method are not fully understood and warrant further studies.

Soil solarization is a hydrothermal process in which moist soil is covered with transparent plastic and exposed to sunlight, allowing it to heat to temperatures under



1






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favorable conditions that are lethal to many plant pathogens, pests and weeds (DeVay, 1991b). The effectiveness of soil solarization depends on soil color and structure, soil moisture; air temperature, length of day, and intensity of sunlight (DeVay, 199 Ib). The process has been studied during the last 20 years as an alternative to chemical fumigation of soil for the management of soilborne plant pathogens (Katan, 1980, 1981; Katan et al., 1987). Solarization may reduce populations of several fungi, such as Fusarium spp. (Chellemi et al, 1994; Katan et al., 1976, Katan et al., 1983; Porter and Merriman, 1985; Ramirez-Villapudua and Munnecke, 1987, 1988), Phytophthora spp. (Barbercheck and Von Broembsen, 1986; Chellemi et al, 1994; Hartz et al., 1993; Juarez-Palacios et al., 1991; Kassaby, 1985; McGovern and Begeman, 1996; Moens and Aicha, 1990; Pinkas et al., 1984; Wicks, 1988), Pythium ultimum (Gamliel and Stapleton, 1993a, 1993b; Gamliel et al., 1989, 1993; Kulkami et al., 1992; Pullman et al., 1981a, 1981b; Stapleton and Garza-Lopez, 1988; Stapleton et al., 1995), Verticillium dahliae (Ghini et al., 1993; Hartz et al., 1993; Katan et al., 1976; Porter and Merriman, 1985; Pullman et al., 1981a, 1981b), and Rhizoctonia sp. (Gamliel et al., 1993; Grooshevoy et al., 1941; Keinath, 1995; Lewis and Papavizas, 1974; Pullman et al., 1981a, 1981b). However, disease management is not always achieved, as exemplified by the study of Stapleton and GarzaLopez (1988) in which a reduction in the population of Macrophomina phaseolina did not result in less disease in the indicator crop. Other soilborne pathogens that have not been controlled by soil solarization include Plasmodiophora brassicae, Sclerotium rolfsii, Pythium aphanidermatum and many others, including Fusarium oxysporum f. sp. radicislycopersici (Chellemi et al., 1994; Stapleton and DeVay, 1986).

Success of solarization for the control of plant diseases is closely associated with a combination of high ambient temperatures, maximum solar radiation, and optimum soil moisture (DeVay, 1991b; Souza, 1994). Early demonstration of the use of solarization came from work in arid climates by Katan (1980, 1981) and Katan et al. (1976, 1987) in Israel, by Stapleton and DeVay (1984, 1986) in California, and in semi-arid climates in





3


Mexico by Stapleton and Garza-Lopez (1988) . Regions where the summer coincides with the rainy season, such as in the southeastern U.S.A., may have less potential for successful solarization because of the cooling effect of frequent rain showers, as well as extended cloud cover, which reduce the solar radiation captured under the plastic tarp (Chellemi et al., 1994). Soil solarization has been used successfully in the Southeastern U.S.A. to manage Rhizoctonia solani, Didymella bryoniae, Fusarium spp. and P. nicotianae (Chellemi et al., 1994; Keinath, 1995, 1996). Populations of P. nicotianae were reduced to undetectable levels in soil depths of up to 15 cm in three sites in North Florida. However, reduction in populations below 25 cm of depth was noted in only one site. In contrast, methyl bromide nearly completely eradicated the pathogen in depths of up to 35 cm. Control of Fusarium spp. was limited to the top 5 cm of the soil, and no control was achieved at some sites (Chellemi et al., 1994). Keinath (1995) found that the number of organic fragments colonized by R. solani was lower in solarized than in nonsolarized soils; however, the control of belly rot on pickling cucumber was not as effective with solarization as was the chemical treatment with chlorothalonil.

The type of plastic used for solarization may also influence soil solarization

(DeVay, 1991a, 1991b; Malathrakis and Loulakis, 1989; Staplepton and DeVay, 1986). Stapleton and DeVay (1986) reported that thinner polyethylene sheets (25 jLm) were more effective than thicker sheets (50-100 jtm) for the transmission of solar radiation to the soil. DeVay (1991) noted that black polyethylene films lasted longer than transparent films that had not received a treatment for protection against UV rays; however, soil temperatures did not raise as much under the black films. Malathrakis and Loulakis (1989) compared the effectiveness of a clear, low density polyethylene film to a film of co-extruded polyethylene plus ethylene vinyl acetate (thermoplast). These authors found that the thermoplast was more effective than the clear film in raising soil temperatures and in trapping gases used for fumigation. Similarly, Chellemi et al. (1997) found that a gas impermeable film, consisting of a polyamide core sandwiched between two layers of





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polyethylene, was more effective than a co-extruded, white-on-black film for the reduction of populations of Paratrichodorus minor and Criconemella spp. and control of Fusarium wilt on tomatoes.

Indirect effects of soil solarization may include reduced fungistasis, or induction of shifts of microbial populations that affect the survival of spores of plant pathogens. Katan et al. (1976) studied the effect of heating soils to 450 to 50'C on the fungistasis of Fusarium oxysporum f. sp. lycopersici. These authors observed that autoclaving or preheating soil to 500C for 3 hours and cooling it to 250C before infestation induced an immediate increase in germination of conidia of F. oxysporum f. sp. lycopersici incubated during the first 24 hours. However, when conidia were incubated for longer periods of time (3 to 9 days) in pasteurized soils, the final population was reduced by 81%. The following three mechanisms were suggested to explain the increased control of pathogens in solarized or pasteurized soils: fungistasis may be partially nullified at 450 to 500C, allowing spores to germinate and then starve in the absence of the host or be killed by the existing microbiota; sublethal temperatures may weaken the resting structures, rendering them more vulnerable to antagonistic microbiota; a microbial population shift is induced which favors thermophylic saprophytes over pathogens.

Soil solarization alone may not be effective or consistent for the control of

soilborne pathogens, especially in regions where the rainy season occurs simultaneously with the warmer months of the year. In such cases, the use of soil amendments may enhance the performance of solarization (Gamliel and Stapleton, 1993a; Keinath, 1996; Ramirez-Villapudua and Munnecke, 1988). Cruciferous residues, due to their high content of isothiocyanates and aldehydes, have been suggested as amendments for use in combination with solarization (Mayton et al., 1996); cabbage is the primary amendment that has been studied in combination with soil solarization.

Lewis and Papavizas (1971) studied the effect of vapors from cabbage

decomposition on the control of Aphanomyces euteiches, and found that root rot of peas





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could be controlled with either cabbage amendment or with sulfur-containing volatile compounds. A later study on the survival of R. solani by these authors (Lewis and Papavizas, 1974) indicated that the major effect of the organic amendment resulted from either a change of the soil pH or from the low C:N ratio of the amendment, since fresh corn amendment produced similar results as the cabbage amendment.

Gamliel and Stapleton (1993b) characterized the antifungal volatiles from cabbage residue during solarization. The kinds of volatiles released from heated or nonheated soils differed, and the concentration of volatiles peaked during the first 2 weeks of heating. Spores of P. ultimum and sclerotia of S. rolfsii failed to germinate after exposure to cabbage volatiles for 20 days. The use of a crucifer amendment associated with sublethal heating (380C) reduced germination of P. ultimum and S. rolfsii in vitro (Stapleton et al., 1995). It is possible that the major benefits of the soil amendment with crucifer residues are associated with high but not lethal temperatures, when the heat alone is not sufficient to inactivate the pathogen; however, this aspect of the use of cabbage amendment has not been explored fully.

Solarization associated with cabbage amendments was evaluated by Keinath

(1996) and Ramirez-Villapudua and Munnecke (1987, 1988). The volatiles released by cabbage alone reduced the populations ofF. oxysporum f. sp. conglutinans; however, the association of solarization with the amendment was the most effective treatment for the control of this pathogen (Ramirez-Villapudua and Munnecke, 1987, 1988). High concentrations of amendment had no effect on the subsequent cabbage crop, but phytotoxicity was observed when tomato seedlings were transplanted into the treated soil.

Beneficial changes in soil microbiota have been suggested as one of the attributes of soil solarization, regardless of amendments; these changes may lead to higher yields or to longer periods in which the crop is not affected by the pathogen (Katan, 1981; Stapleton, 1991; Stapleton and DeVay, 1986). Solarization of soil amended with cabbage induced an increase in the populations of thermotolerant fungi and Bacillus spp., a





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corresponding decrease in populations of fluorescent Pseudomonas spp. and Fusarium spp., and did not affect the populations of actinomycetes. These changes in populations were correlated to a higher yield of watermelon (Keinath, 1996).

Although soil solarization is a hydrothermal process, depending on moisture for maximum heat transfer throughout the soil profile and to soilborne organisms (DeVay, 1991a; Mahrer et al., 1984), very little is known about the influence of moisture on the inactivation of soilborne pathogens. Moisture has been provided during soil solarization by different methods. Kulkarni et al. (1992) flood-irrigated solarized plots once a week, removing the plastic tarp. These authors found that recontamination of the solarized plots could occur with either contaminated water or soil movement with the irrigation water. Furrow irrigation of level fields has been suggested as an alternative to periodically replenish moisture to the soil under solarization ( Pullman et al., 1979; Stapleton and DeVay, 1986). However, a single irrigation before the plastic is laid down can provide the same control of soilborne plant pathogens as several irrigation events (Grinstein et al., 1979a, 1979b; Jacobsen et al., 1980; Katan et al., 1980; Pullman et al., 1979).

Studies of thermal inactivation of pathogens can yield important information on survival of specific types of propagules and, more importantly when simulating field conditions, can serve as the basis for estimation of the time needed for soil solarization to be an effective strategy for management of soilborne plant pathogens. Relationships between the effectiveness of soil solarization in the field and thermal inactivation in vitro have been studied with several pathogens (Benson, 1978; Myers et al., 1983; Pullman et al., 1981 a). However, there was a lack of quantification of the inoculum added or recovered after the heat treatment. Additionally, the propagules used in some studies for heat treatment were not the same as the propagules normally found in the soil, or the detection techniques did not allow the quantification of the surviving population (Ramirez-Villapudua and Munnecke, 1987,1988). Katan (1985) indicated the need for caution in the interpretation of the significance of heat mortality curves obtained under






7


laboratory conditions. Potential problems that need to be addressed include inoculum type, moisture level, medium containing the inoculum, and the procedure for heating the samples.

Early studies of thermal inactivation were more concerned with food processing, and very high temperatures were employed for short periods of time (Bigelow, 1921; Bigelow and Esty, 1920). While the emphasis at the time was on higher temperatures, Smith (1923) demonstrated that spores of Botrytis cinerea in aqueous solution could be inactivated at temperatures as low as 370C whenever exposure was long enough; spores, as an example, were inactivated over a range varying from 6 minutes at 50.30C to 23 hours at 37oC.

A logarithmic relationship between time and temperature in the inactivation of propagules has been observed (Bigelow, 1921; Bigelow and Esty, 1920; Pullman et al., 1981a; Smith, 1923). Pullman et al. (1981a) found that the exposure time in which 90% of the oospores of Pythium ultimum were killed (LD90) was, for each set of temperatures, 30 minutes at 500C, 110 minutes at 470C, 8 hours at 45oC, 42 hours at 420C, 13 days at 38.50C and 26 days at 370C.

Juarez-Palacios et al. (1991) found that the determination of the heat sensitivity of isolates ofPhytophthora spp. in the laboratory closely reflected their inactivation in solarized soil, and the results supported the possible use of soil solarization for management of these pathogens. These authors found that an isolate of P. megasperma tolerant to high temperatures survived exposure for 30 minutes at 450C in a heat sensitivity study, but that the number of leaf discs infected by the pathogen after solarization for 4 weeks declined. In contrast P. cinnamomi and a low-temperature isolate of P. megasperma did not survive either treatment.

Bollen (1985) noted that different types of propagules have different sensitivities to heat. Oospores of P. capsici were more thermotolerant than mycelium from either mating type of this pathogen. The difference in tolerance was more than 50C; oospores





8


survived at 500C for 30 minutes, but mycelium was eliminated at 42.50 to 450C for 30 minutes. Differences in the survival ofF. oxysporum were also observed; survival of this pathogen in soil cultures was greater at higher temperatures than survival of the pathogen in soil infested before the heat treatment.The difference may have been due to the presence of chlamydospores in the soil culture. However, the system used did not reflect field conditions, since soil was continuously flooded, which rarely occurs for extended duration in any field. Bollen (1985) did not analyze the relationship of time to temperature, and the temperature used, 500C, is reached most commonly under soil solarization only in arid or tropical climates.

One of the first attempts to control plant pathogens by exposure to intermittent heat was done by Grooshevoy et al. (1941). These authors demonstrated that exposing chlamydospores of Thielaviopsis basicola or sclerotia of a Rhizoctonia sp. or Sclerotinia sclerotiorum for 3 hours a day at 45'C for 5 days was sufficient to prevent the germination of the spores. However, no attempts were made to simulate the diurnal temperature fluctuations. Porter (1991) evaluated the effect of intermittent heat on the control of sclerotia of S. sclerotiorum in soil, and found that continuous heat was more effective than intermittent heat at reducing the number of viable sclerotia when the infested soil was heated for 6 hours daily over 14 days to temperatures ranging from 300 to 450C. In a similar study with several pathogens, Porter and Merriman (1983) observed that sclerotia of S. rolfsii tolerated exposure to 500C for 6 hours a day for 2 weeks, while Sclerotium cepivorum and Sclerotinia minor survived only under similar exposures at temperatures of 450C or less. Pythium irregulare and F. oxysporum survived at 500C, but Verticillium dahliae was killed at temperatures above 40oC; however, the type of fungal propagules used for this study was not determined (Porter and Merriman, 1983). Due to the labor-intensive nature of these studies and the demand for equipment, very little research has been done on the quantitative relationship of intermittent heat to the inactivation of spores of plant pathogens.





9



Phytophthora nicotianae Breda de Haan (syn. = P. parasitica Dastur) has been reported on more than 170 plant hosts in Florida (Alfieri et al., 1994). Soilborne diseases caused by P. nicotianae have limited production of several important crops, such as citrus, tobacco, ornamentals, and tomato.

Low levels of inoculum of P. nicotianae in the field can result in severe epidemics (Ferrin and Mitchell, 1986a; Kannwischer and Mitchell, 1981; Mitchell, 1978). Kannwischer and Mitchell (1981) found that 0.13 chlamydospore of P. nicotianae per gram of soil or 42 zoospores per plant were sufficient to cause 50% mortality on a susceptible cultivar of tobacco in a controlled environment. Residual population densities of 0.005 to 0.67 propagules per gram of soil were found in a tobacco nursery; mortality of a susceptible cultivar transplanted to this field reached 80% at the end of the growth season (Ferrin and Mitchell, 1986a).

Moisture plays an important role in the formation of sporangia, zoospore release, and subsequent epidemics. Sporangia of P. nicotianae are produced over a range of soil matric potentials (-4 to -1500 kPa), with the greatest number of sporangia being produced at the higher end of the range (-4 to -25 kPa) (Bernhardt and Grogan, 1982; Sidebottom and Shew, 1985a). Although flooding does not favor the formation of sporangia, it enhances zoospore release (Bernhardt and Grogan, 1982).

Lutz and Menge (1991) observed that populations ofP. nicotianae increased from 17 propagules per gram of soil before irrigation to 70 propagules per gram 2 days after a 24-hour furrow irrigation event in a citrus grove. The highest proportion of propagules at that time was comprised of sporangia and zoospores; chlamydospores increased 4 days after the irrigation event, and reached a maximum on the seventh day. When the citrus grove was irrigated with a drip system, the soil matric potential was maintained close to

-10 kPa, and the highest proportion of propagules consisted of sporangia and zoospores, with populations ranging from 71 to 93 propagules per gram of soil.






10



Ristaino et al (1988) analyzed the effect of irrigation frequency and duration on

the development of Phytophthora root rot of tomato and found that an irrigation regime in which plants were irrigated every 14 days, either for 4 to 8 hours or for 24 hours, increased root infection and decreased tomato yield. A less frequent irrigation schedule, such as 4 to 8 hours every 28 days, stressed the plants and also led to severe root infection. Zoospores of P. nicotianae can be carried in irrigation water and infect tomato plants more than 60 meters from inoculum sources (Neher and Duniway, 1992). In tomato, earlier infection led to higher disease intensity, and lower yield (Neher and Duniway, 1991; Neher et al., 1993). Infection of tobacco was favored by flooding of potted plants, or by saturating soil in Buchner funnel tension plates (Shew, 1983; Sidebottom and Shew, 1985b), and coincided with periods of high moisture levels in field trials (Ferrin and Mitchell, 1986b), indicating that zoospore movement is enhanced in saturated soils.

Temperature is another important factor in the development of epidemics caused by P. nicotianae. Lutz et al. (1991) found that heating naturally infested soils from citrus groves above 120C increased germination of chlamydospores. Stimulation was observed up to 340C, with maximum germination at 24'C and minimum at 360C. However, such a stimulus could be due to a temperature differential between the temperature in which the spores were formed and a temperature that would provide maximum germination, and not only an increase in temperature.

Even though extensive studies have been done on the relationships of inoculum density and disease, water status of the soil and disease, and temperature and increased recovery of chlamydospores in the soil (Ferrin and Mitchell, 1986b; Lutz and Menge, 1991; Ristaino et al., 1988; Shew, 1983; Sidebottom and Shew, 1985a, 1985b), there is a lack of information about the effects of interactions of moisture and temperature on the inactivation of P. nicotianae.






11


Phytophthora capsici Leonian was first described infecting chile pepper in New Mexico (Leonian, 1922). This pathogen has a broad host range, including perennial plants, such as cacao, rubber, and macadamia, and annuals, such as peppers, cucurbits and several solanaceous hosts (Alfieri et al., 1994). Important diseases caused by P. capsici in the United States include root rot, blight and fruit rot of pepper, eggplant, cantaloupe, squash and watermelon (McGovern et al., 1993). In 1994, severe outbreaks of P. capsici were reported on tomato in South Florida (Simone, personal information).

Phytophthora capsici survives in low numbers in soil (Papavizas et al., 1981). The effects of soil temperature and soil-water matric potential on survival of oospores, sporangia, and zoospores, as well as on oospores or mycelium in plant tissue, were studied by Bowers et al. (1990a). These authors found that zoospores survived no more than 3 weeks in field soil during the summer, when temperatures ranged from 200 to 300C; sporangia survived up to 8 weeks. Very little decrease in viability of the oospores was observed during the same period. In a winter test, viability of oospores free of organic residue in soil decreased from 67% to less than 10% after 27 weeks, while oospores in plant tissue did not survive more than 8 weeks.

Although long-term survival of P. capsici in the soil occurs in the form of

oospores (Bowers et al., 1990a), the primary infective structures generally are zoospores (Bowers and Mitchell, 1990; Hord and Ristaino, 1992; Luz and Mitchell, 1994; Ristaino et al., 1992). These authors found that flooding soils infested with oospores for as little as

2 hours resulted in increased disease, and several flooding events led to the eventual infection of all plants. Bernhardt and Grogan (1982) demonstrated that sporangia are formed within 24 hours in soils held at -30 kPa, and sporangia released zoospores 4 hours after the soil was flooded. Schlub (1983) observed that P. capsici spread in the field with rain splash. Bowers et al. (1990b) and Ristaino (1991) confirmed the influence of rainfall on disease, but noted that the movement of water over the soil surface or the plastic mulch had the most significant impact on disease development.





12


Knowledge of the primary survival and infection structures may be very important in the determination of strategies for the control of P. capsici. Still, the effects of moisture and temperature on the survival of this pathogen in the absence of a host plant are not known.

The objectives of this study were to determine the efficacy of soil solarization and organic amendment with cabbage for the control of P. nicotianae and P. capsici in the field; and to determine the relationships of time of exposure to temperature, cabbage amendment, soil water matric potential, and different soils on the thermal inactivation of propagules of P. nicotianae and its pathogenicity in tomato.














CHAPTER 2
THE EFFECT OF SOIL SOLARIZATION ON POPULATIONS OF PHYTOPHTHORA
NICOTIANAE AND P. CAPSICI UNDER FIELD CONDITIONS


Introduction


Phytophthora nicotianae Breda de Haan (syn.= P. parasitica Dastur) has been

reported on more than 170 plant hosts in Florida (Alfieri et al., 1994). Soilborne diseases caused by P. nicotianae have limited production of several important crops, such as citrus, tobacco, ornamentals and tomato (Erwin and Ribeiro, 1996).

Phytophthora capsici Leonian is an important pathogen on several solanaceous plants. Severe losses have resulted from root rot, blight, and fruit rot of pepper, eggplant, cantaloupe, squash, and watermelon (McGovern et al., 1993). Outbreaks of P. capsici have been observed in tomato, especially when soils are saturated with moisture.

Because these pathogens can survive in the soil for long periods of time in the absence of their hosts, or when weather conditions are not favorable for disease (Erwin and Ribeiro, 1996), the main strategy used by growers to reduce losses due to these pathogens, especially at the early stages of plant development, is the use of preplant fumigation with methyl bromide and application of fungicides.

The implication of methyl bromide as an ozone depleting substance has prompted a search for new alternatives for the control of soilborne plant pathogens. Soil solarization has been used in areas with arid climates for the management of soilborne diseases of high value crops (Hartz et al., 1993; Pullman et al., 1981b). Solarization has been evaluated in areas where the climate is considered marginally suitable for the procedure but results were not completely satisfactory (McSorley and Parrado, 1986). Soil amendments have been tested as components of soil solarization to enhance its


13





14


effectiveness (Chellemi et al., 1997; Gamliel and Stapleton, 1993a, 1993b). Cabbage residues have been shown to be a potential amendment for the control of soilborne plant pathogens due to the isothiocyanates and other volatiles that are released during the heating process (Gamliel and Stapleton, 1993a; Keinath, 1996; Ramirez-Villapudua, 1987, 1988; Stapleton et al., 1995) and could be useful in areas where the soil temperatures are not high enough to pasteurize the soil.

The objective of this study was to evaluate the effects of soil solarization and cabbage amendment on the survival of P. nicotianae and P. capsici in North Florida.




Materials and Methods



Characterization of the Solarization Sites


Two sites were selected for solarization in 1994 in commercial tomato production fields where control of soilborne diseases had been achieved previously through the use of a preplant application of methyl bromide plus chloropicrin. Site 1 was in the Naples Tomato Growers-Gargiulo farm number 4 in Decatur County, South Georgia. Site 2 was at the John Allen Smith farm located in Gadsden County, North Florida. In 1995, one experiment was conducted at the North Florida Research and Educational Center in Gadsden County (site 3, test 1) on soils that had been weed fallow for several years. In 1996, two experiments were conducted at the same center (site 3, tests 2 and 3) but in a different field. The soil at all locations consisted of Orangeburg or Tifton loamy fine sands (Typic Kandiudult: silicaceous, thermic), with pH values ranging from 4.7 to 6.6 (Table 2.1); these soils contained moderate amounts of kaolinitic clay and low amounts of organic matter.










Table 2.1. Characteristics of soils at the time of solarization.



Site Location pH Soil water Organic Percent sand- Soil class content" (-kPa) matter (%) silt-clay

1 NTG - Georgiaw 6.6 5 1.1 84.0-6.0-10.0 Kandiudultz 2 JAS - Floridax 5.1 15 0.5 88.5-4.5-7.0 Kandiudult

3, test 1 NFREC - Quincyy 4.7 25 1.5 88.5-4.0-7.5 Kandiudult 3, test 2 NFREC - Quincy 5.0 5 1.0 80.4-11.1-8.5 Kandiudult 3, test 3 NFREC - Quincy 5.0 50 1.2 82.8-10.0-7.2 Kandiudult ' Estimated by the gravimetric method with oven drying, prior to initiation of the experiment, and transformed to matric potential (Appendix B).
w NTG - Georgia = Naples Tomato Growers-Gargiulo farm number 4 in Decatur County, Georgia. x JAS - Florida = John Allen Smith farm located in Gadsden County, Florida. Y NFREC - Quincy = North Florida Research and Educational Center in Gadsden County. z Ultisol with profiles similar to paleudults but containing higher amounts of kaolinitic clay.





16


Inoculum Production


Inoculum ofP. nicotianae was produced by inoculating a 250-ml flask containing 20 g of wheat seeds and 30 ml of deionized water (autoclaved twice at a 24-hr interval) with four, 5-mm agar plugs of actively growing mycelium from a 4- to 7-day-old V8juice-agar plate containing isolate Pn21. The flask was incubated at 250C in the dark for 1 month and was shaken twice a week to ensure uniform growth of the isolate. Oospores of P. capsici were produced by inoculating a similarly prepared 250-ml flask of wheat seeds with three, 5-mm agar plugs of actively growing mycelium of each of two isolates of compatible mating types (Cp25 and Cp26). Isolate Pn21 was originally isolated from periwinkle, and isolates Cp25 and Cp26 were isolated from watermelon in South Florida; all isolates were maintained in the collection of Phytophthora spp. of the Plant Pathology Department of the University of Florida, Gainesville. The flask was incubated at 250C in the dark for 2 months and was shaken twice a week to ensure uniform growth of both isolates on the substratum and to allow for maximum contact between the two mating types.

Inoculum of P. nicotianae or P. capsici was incorporated into the soil by mixing 200 mg of shredded, infested wheat seeds with 3 grams of soil. Nonpasteurized soils from sites 1 and 2 were used in 1994, and the soils were infested 2 days before the samples were taken to the field. Pasteurized soil from site 3, test 1, was infested with the inoculum

2 days before the samples were taken to the field in 1995. To encourage augmented formation of resting structures in the two experiments in 1996, pasteurized soil from site

1 was infested with either P. nicotianae and incubated at 180C for 10 days, or with P. capsici and incubated at 250C for 10 days, before its placement in the field tests. In all tests, each 3-g sample was enclosed in a 25-cm2 nylon envelope (3-gtm-pore size; Versapor 3000, Gelman Sciences, Inc.), and the envelopes were buried in the soil at depths of 10 and 25 cm just before application of the solarization treatments.






17


The survival of the spores at the end of the solarization experiments was

determined by plating infested soil from the envelopes on a medium (PARPH) selective for pythiaceous fungi. The medium consisted of 17 g of cornmeal agar (Difco) in 1 liter of deionized water amended with 5 mg of pimaricin, 250 mg of ampicillin, 10 mg of rifampicin, 100 mg of pentachloronitrobenzene, and 50 mg of hymexazol (Mitchell and Kannwischer-Mitchell, 1992). In 1995 hymexazol was omitted from the selective medium.



The Solarization Experiments


Six solarization treatments with four replications each were selected for study at site 1, which consisted of an 18-treatment randomized complete block experiment on solarization and fumigation (Chellemi et al., 1997). The selected treatments included a 30-jm-thick, clear, gas impermeable plastic film (Bromotec film, Lawson Mardon Packaging, United Kingdom); a 30-gm-thick, coextruded white-on-black, low density polyethylene film (Edison Plastics, Lee Hall, VA); and each of the polyethylene films with or without cabbage residue (Brassica oleracea var. capitata L. cv. Constanza) incorporated into the soil at a rate of 80 tons/ha. Controls consisted of a nontarped treatment and a tarped treatment (white-on-black film) fumigated with a 67:33 formulation of methyl bromide plus chloropicrin at 39.2 g/m2.

At site 2, the six treatments selected for solarization from an 18-treatment

randomized complete block experiment with four replications (Chellemi et al., 1997) included a 30-tm-thick, clear, low density polyethylene film (Polydak film, Polyon Barkai Ltd., Kibutz Barkai, Israel); a 30-pjm-thick, clear, gas impermeable plastic film (Bromotec film) ; and each of the polyethylene films with or without cabbage residue incorporated into the soil at a rate of 66 tons/ha. Controls consisted of a nontarped





18


treatment and a tarped treatment (white-on-black film) fumigated with a 67:33 formulation of methyl bromide plus chloropicrin at 39.2 g/m2.

For test 1 at site 3, the eight treatments selected for solarization from an 15treatment randomized complete block experiment with four replications (Chellemi et al., 1997) included a 25-jim-thick, clear, low density polyethylene film (Polydak film); a 30jm-thick, clear, gas impermeable plastic film (Bromotec film); a 30-jim-thick, coextruded white-on-black, low density polyethylene film (Edison Plastics); and each of the polyethylene films with or without cabbage residue incorporated into the soil at a rate of 68 tons/ha. Controls consisted of a nontarped treatment and a tarped treatment (whiteon-black film) fumigated with a 67:33 formulation of methyl bromide plus chloropicrin at 39.2 g/m2

For tests 2 and 3 at site 3, six treatments were arranged in a randomized complete block design with four replications. Soil treatments included a 30-jim-thick, clear, gas impermeable plastic film (Bromotec film); a 30-jm-thick, coextruded white-on-black, low density polyethylene film (Edison Plastics); and each of the polyethylene films with or without cabbage residue incorporated into the soil at a rate of 89 tons/ha for test 2, and 81 tons/ha for test 3. Controls consisted of a nontarped treatment and a tarped treatment (white-on-black film) fumigated with a 67:33 formulation of methyl bromide plus chloropicrin at 39.2 g/m2.

At sites 1 and 2 and for test 1 at site 3, each replicate plot consisted of one raised,

0.20-m X 0.90-m X 20-m bed, prepared according to standard commercial production practices; row orientation was north/south. Preplant fertilizer was broadcast into the beds at 212 kg of N, 65 kg of P, and 212 kg of K/ha, and drip irrigation tubing was placed 5 cm beneath the soil on sites 1 and 2. At site 3 for test 1, fertilizer was applied at 196 kg of N, 26 kg of P, and 163 kg of K/ha. Drip irrigation tubing was laid on the surface of the bed prior to covering with plastic. At site 3 for tests 2 and 3, each replicate plot consisted of one raised, 0.20-m X 0.90-m X 4.0-m bed prepared according to standard commercial





19


production practices; row orientation was north/south. No fertilizer was applied and no irrigation tubing was placed on the beds.

Cabbage was grown and harvested in the plots at sites 1 and 2, and the residue was spread over the plots on 23 and 24 May 1994. Beds were prepared, the nylon envelopes containing the inoculum were placed in the soil, and fumigant and plastic were applied on 3 June 1994 at site 1. The solarization period was terminated on 22 July 1994, after 49 days, and the samples were removed for determination of survival. At site 2, beds were prepared, the nylon envelopes containing the inoculum were buried, and fumigant and plastic were applied on 15 June 1994; the solarization period was terminated on 2 August 1994, after 48 days, when the samples were removed. For the first test at site 3, cabbage was grown in plots near the experimental plots, harvested, and the head and wrapper leaves incorporated into the plots on 19 May 1995. Beds were prepared, the inoculum bags were buried, and fumigant and plastic applied on 26 May 1995. The solarization period was terminated on 20 July 1995, after 55 days, when the samples were removed. For tests 2 and 3 at site 3, cabbage was harvested from a commercial field in Hastings, FL; head and wrapper leaves were incorporated into the plots on 12 June for test 2, and on 19 June 1996 for test 3. Beds were prepared, two sets of nylon envelopes containing the inoculum were placed in each plot, and fumigant and plastic were applied on 21 June and 28 June 1996, respectively. The solarization period was terminated with the removal of the last samples on 6 and 13 August 1996, after 45 days, for tests 2 and 3, respectively.

Daily ambient temperature data were obtained from a weather station at the North Florida Research and Educational Center, in Quincy, located approximately 25 km from sites 1 and 2, while daily precipitation amounts were recorded at each site. In 1994, soil temperatures were monitored in site 2 at depths of 10 and 25 cm using thermocouple sensors connected to an electronic data logger (Omnidata International, Logan, UT). For all three tests at site 3, daily rainfall and ambient temperature data were recorded at the





20


site. For test 1 at site 3, hourly temperature changes were monitored for 33 days at an external test location approximately 500 m from test 1; however, temperatures were still recorded at 10 and 25 cm depths in the soil of the nontarped and the clear, gas impermeable treatments with thermocouple sensors connected to an electronic data logger (Campbell Scientific, Logan, UT). In 1996, temperature changes were monitored in test 2, at 10 and 25 cm depths in the soil.


Statistical Analysis

All data of survival of each pathogen were log-transformed (ln[ppg+1]) prior to analysis of variance. Analysis of variance was performed with PROC MIXED of SAS (SAS Institute, Cary, NC; release 6.11 for personal computers), considering that the environmental effects on the inoculum due to the depth factor could be correlated. Survival of P. nicotianae and P. capsici was compared using Tukey's Honestly Significant Difference procedure.



Results



Environmental Conditions

Environmental conditions varied from year to year, with 1994 being characterized by above average precipitation and below average temperatures (Figure 2.1). In 1995 both precipitation and temperatures were normal, and in 1996 precipitation was below average and temperatures were normal. The heating process of the soil was interrupted by precipitation during 61% of the days of the solarization period at site 1; 65% of the days at site 2; 30% of the days in test 1 at site 3; 42% of the days in test 2 at site 3; and 47% of the days in test 3 at site 3 (Table 2.2).






21


40 I 70
A 1994
35 - Temperature 60
Maximum 50
30 - Minimum ......
Precipitation 40
25 - Site I
Site 2 ..30
20- 20
10 - 20
15- I I I i . II - 10

40 I I I I - 70
B 1995 Temperature
B 1995Maximum 600 35- Minimum .... E S30- Preciptation 50 E
- 40 o
25- -CU
30
E .. 20 "D 15- - 10
10- 0 40 - I I I I I I I I 70 40 70
C 1996
35 - Temperature -60
Maximum -
30 - Mininimum --- 50
Precipitation 40
25- g
20 30
20
15
- 10
10, , Ol
o 0 o)(3) o) o ,
N CO CN -L 6 ) S "- 3 : -- 3 -3





Figure 2.1: Maximum and minimum air temperature and precipitation recorded at each site during the solarization period, from 1994 to 1996. A. Solid arrows (4) indicate beginning and end of solarization at Site 1; open arrows (0) indicate beginning and end of solarization at Site 2. B. Solid arrows (4) indicate beginning and end of solarization in test 1 at Site 3. C. Solid arrows (4) indicate beginning and end of solarization in test 2 at Site 3; open arrows (J) indicate beginning and end of solarization in test 3 at Site 3.










Table 2.2. Ambient weather conditions during solarization studies in 1994, 1995, and 1996.


Site Location Year Precipitation Number of days
(mm) Solarization Precipitation Ambient period occurred temperature >35
I Georgiax 1994 460 49 30 0 2 Floriday 1994 384 48 31 0

3, test 1 NFREC - Quincyz 1995 301 55 17 6 3, test 2 NFREC - Quincy 1996 232 45 19 6 3, test 3 NFREC - Quincy 1996 264 45 21 4 x Georgia = Naples Tomato Growers-Gargiulo farm number 4 in Decatur County, Georgia. Y Florida = John Allen Smith farm located in Gadsden County, Florida. z NFREC - Quincy = North Florida Research and Educational Center in Gadsden County.






23


Soil moisture conditions at the beginning of the solarization treatments varied from experiment to experiment and ranged from -5 to -50 kPa (Table 2.1). Even though there were several rain events during the solarization period in all experiments, it is possible that soil moisture was not replenished under the plastic films used due to the structure of the sandy soils studied; the high proportion of large size pores in these soils does not favor the lateral movement of water.

During the 48 days of solarization at site 2, there were 2 days in which

temperatures exceeded 410C at the 10-cm soil depth in the nontarped treatment; 410C was exceeded on 15 days in the clear plastic treatment and on 20 days in the gas impermeable treatment (Table 2.3). Temperatures exceeded 44oC at the 10-cm depth on 9 days under the clear plastic and on 13 days under the gas impermeable film. Temperatures were above 470C for 1 day and 3 days under the clear and the gas impermeable films, respectively. At the 25-cm depth, temperatures never reached the 41 C threshold under any of the treatments. Maximum temperatures attained in site 2 at the 10-cm depth under nontarped soil, clear low density polyethylene plastic, and clear, gas impermeable plastic were 41.50, 47.30, and 48.50C, respectively. At the 25-cm depth, maximum temperatures were 34.60, 37.90, and 380C under nontarped soil, clear low density polyethylene plastic, and clear, gas impermeable plastic, respectively. The average soil temperature change during the day is illustrated in Figure 2.2.A. Temperature was not monitored in site 1.

At site 3 for test 1, temperatures above 41 C were recorded at the 10-cm depth under the clear low density polyethylene plastic on 30 days, and under the nontarped treatment on 5 days; 44oC was reached on 20 days and 470C was reached on 6 days during the 33 days over which temperatures were monitored under the clear low density polyethylene film. At the 25-cm depth, temperatures never reached the 41oC threshold (Table 2.3). Maximum soil temperatures under nontarped soil and clear low density polyethylene plastic at the 10-cm depth were 42.40 and 48.70C, respectively; at the 25-cm







Table 2.3. Temperature profiles during soil solarization at site 2 and for tests 1, 2, and 3 at site 3.

Sitev/Test Depth Nontarpedw White-on-black Clear LDPE Gas Impermeable
(cm) 410C 440C 470C 410C 440C 470C 410C 440C 470C 410C 44"C 470C

Site 2 10 2 0 0 ntx nt nt 15y' 9 1 20 13 3
25 0 0 0 nt nt nt 0 0 0 0 0 0 Site3/1 10 5 0 0 nmz nm nm 30 20 6 nm nm nm 25 0 0 0 nm nm nm 0 0 0 nm nm nm Site 3/2 10 1 0 0 0 0 0 nt nt nt 32 24 10 25 0 0 0 0 0 0 nt nt nt 1 0 0 Site 3/3 10 0 0 0 0 0 0 nt nt nt 30 22 8 25 0 0 0 0 0 0 nt nt nt 1 0 0

Site 2 10 4 0 0 nt nt nt 63 25 2 82 42 7
25 0 0 0 nt nt nt 0 0 0 0 0 0 Site3/1 10 10 0 0 nm nm nm 161 84 14 nm nm nm 25 0 0 0 nm nm nm 0 0 0 nm nm nm Site 3/2 10 1 0 0 0 0 0 nt nt nt 225 125 24 25 0 0 0 0 0 0 nt nt nt 4 0 0 Site 3/3 10 0 0 0 0 0 0 nt nt nt 203 114 21 25 0 0 0 0 0 0 nt nt nt 4 0 0 SSite 2 = John Allen Smith farm located in Gadsden County, Florida (solarized for 48 days in 1994); Site 3 = North Florida Research and Educational Center in Gadsden County (solarized for 55 days in test 1, and for 45 days in tests 2 and 3). w Temperatures were measured in the untreated soil (nontarped); under a 30-pm-thick, coextruded white-on-black, low density polyethylene film (White-on-black); under a 30-jim-thick, clear, low density polyethylene film (Clear LDPE); and under a 30-jim-thick, clear, gas impermeable plastic film (Gas Impermeable).
x nt= not tested.
Y Temperatures measured during the first 42 days of solarization. nm = not measured.






25



45
- Nontarped 10 cm 1994 A
-- Nontarped 25 cm 1994
-A- White-on-black 10 cm
40 - - - White-on-black 25 cm
-- Clear 10cm
-- Clear 25 cm . - -[3.
--3- GI 10cm
35 --a- GI25cm









1995 B


_ 40
3" - . - . . o










25 I I
40





25






0 4 8996 C
..--El.

40 , s..



30


Z-.



25
0 4 8 12 16 20 24 Time of the day

Figure 2.2: Average temperature change during a 24 hour period at 10 and 25 cm of depth. Temperatures were measured in the untreated soil (nontarped); under the white-on-black, low density polyethylene film (White-on-black); under the clear, low density polyethylene film (Clear);and under the clear, gas impermeable plastic film (GI). A. Site 2 (John Allen Smith farm, Gadsden County); B. Site 3 (North Florida Research and Educational Center in Gadsden County) test 1; C. Site 3 test 2.






26


depth maximum temperatures were 35.10 and 40.30C. The average soil temperature change during the day is illustrated in Figure 2.2.B.

During the 45 days of solarization at site 3 for test 2, maximum temperatures attained at the 10-cm depth under nontarped soil, white-on-black plastic, and clear, gas impermeable plastic were 41.10, 39.00 and 49.10C, respectively. At the 25-cm depth, maximum temperatures were 37.40, 37.50, and 41.4oC under nontarped soil, white-onblack plastic, and clear, gas impermeable plastic, respectively. Under the clear, gas impermeable plastic, at the 10-cm depth, temperatures were above 410C for 32 days, above 440C for 24 days, and above 470C for 10 days; while at the 25-cm depth, 410C was reached on only 1 day. Temperatures in the nontarped treatment reached 41oC only once during the solarization period, and under the white-on-black plastic temperatures never reached that threshold. The average temperature change during the day is illustrated in Figure 2.2.C.

Soil temperature changes for test 3 at site 3 were monitored using thermocouples placed in test 2 at site 3, which was located a few meters away. Maximum temperatures attained at the 10-cm depth under nontarped soil, white-on-black plastic, and clear, gas impermeable plastic were 41.10, 44.20 and 49.1 0C, respectively. At the 25-cm depth, maximum temperatures were 37.4o, 37.50, and 41.40C under nontarped soil, white-onblack plastic, and clear, gas impermeable plastic, respectively. Under the clear, gas impermeable plastic, at the 10-cm depth, temperatures were above 410C for 30 days, above 44oC for 22 days, and above 470C for 8 days. Temperatures never reached these thresholds at the 25-cm depth. Temperatures in the nontarped treatment and in the whiteon-black plastic never reached the 410C threshold.

The highest accumulation of hours in which soil temperatures were greater than 410, 440 and 470C was observed at the 10-cm depth in test 2 at site 3, followed by test 3 at site 3. The lowest accumulation of hours was observed at site 2 (Table 2.3). At site 2 it was possible to compare the accumulation of hours above the given thresholds under the





27


solarization films. Under the gas impermeable film, temperatures were above 41', 440 and 47oC for 82, 42 and 7 hours, respectively; while under the clear low density polyethylene plastic the accumulation was of 63, 25, and 2 hours, respectively. In the nontarped treatment and at the 25-cm depth of all treatments, temperatures never reached the 440C threshold.


Survival of Phvtophthora nicotianae


Recovery of P. nicotianae varied from site to site and from year to year. At site 1 in 1994, no propagules were recovered from the nontarped or the methyl bromide treatments, or from the 10-cm depth in the gas impermeable treatment, with or without cabbage, after 49 days of solarization (Table 2.4). The highest survival was observed under the white-on-black polyethylene film at 10 cm of depth with or without cabbage amendment. At the 25-cm depth under the gas impermeable film, propagules were recovered at the same levels as in the white-on-black film treatment.

At site 2 in 1994, Burkholderia cepacia, a contaminant from the soil, covered the selective medium plates in such a way that very few propagules of P. nicotianae were recovered, and only from soil under the clear low density polyethylene at 10-cm of depth (data not shown).

The omission of hymexazol in the selective medium for the determination of

survival of P. nicotianae from site 3 for test 1, made it impossible to recover the pathogen from the soil because other microorganisms, especially Pythium spp., colonized the organic substratum (shredded wheat seeds) and may have inhibited P. nicotianae in the soil or on the dilution plates.

Propagules of P. nicotianae were recovered from all treatments in test 2 at site 3, except from soil treated with methyl bromide; however, there were no statistically significant differences among the treatments (Table 2.4).









Table 2.4. Survival ofPhytophthora nicotianae after soil solarization.
Treatment Depth Propagules/g of soil in 1994 Propagules/g of soil in 1996
(cm) Site 1V Site 3"- test 2 Site 3 - test 3 Gas impermeable 10 0.0x ay 0.4a 4.4a Gas impermeable 25 39.1 abc 2. la 202.3b Gas impermeable + Cabbage 10 0.0 a 0.9a 1.0a Gas impermeable + Cabbage 25 7.2 ab 2.3a 362.0b White-on-black 10 380.5 c 4.0a 733.9b White-on-black 25 12.9 abc 0.9a 697.8b White-on-black + Cabbage 10 439.1 c 14.8a 825.3b White-on-black + Cabbage 25 44.9 bc 12.3a 749.5b Methyl bromide 10 0.0 a 0.a 0.0a
ethyl bromide 25 0.0 a 0.Oa 0.Oa
on-tarped 10 0.0 a 14.9a 700.7b 00 on-tarped 25 0.0 a 4.5a 687.3b u Treatments consisted of untreated soil (Non-tarped); soil fumigated with methyl bromide (Methyl bromide); soil solarized under a 30-Gm-thick, coextruded white-on-black, low density polyethylene film (White-on-black); or soil solarized under a 30-mun-thick, clear, gas impermeable plastic film (Gas Impermeable). Soil was either nonamended or amended with cabbage (+ Cabbage) at 80, 89, and 81 tons/ha at site 1, and for tests 2 and
3 at site 3, respectively.
' Site I = Naples Tomato Growers-Gargiulo farm number 4 in Decatur County, Georgia (solarized for 49 days). w Site 3 = North Florida Research and Educational Center in Gadsden County (solarized for 45 days). x Weighted means ([Exp (mean}]-l).
Y Main effect means followed by the same letter do not differ according to Tukey's Honestly Significant Difference procedure (P< 0.05); data were transformed to In(ppg+l) for analysis.
Znt = not tested.





29


Trends similar to those observed in site 1 were observed in test 3 at site 3 (Table 2.4). Only methyl bromide eliminated the pathogen from the soil. The gas impermeable film was the next best treatment at the 10-cm depth, regardless of the cabbage amendment, for the reduction of the population of the pathogen; however, at the 25-cm depth the gas impermeable film was not effective, and more than 200 propagules survived in each gram of soil. These values were not statistically different from those observed under the white-on-black film, with or without cabbage amendment, or in the nontarped treatment. Survival in the nontarped treatments in tests 2 and 3, in contrast to no survival in Site 1 could have been due to the change in the procedure for soil infestation from that used for site 1; chlamydospores were already present at the beginning of the experiments in the soil prepared for tests 2 and 3, but not for test 1.

Samples removed after 17 days of solarization in tests 2 and 3 at site 3

demonstrated that the pathogen was present in the samples, except in the methyl bromide treatment, and that the populations declined with time of exposure to the solarization process (Table 2.5). Increases in population were observed in test 3, mostly at the 25-cm depth, in the nontarped, gas impermeable, and white-on-black treatments after the 45 days of solarization. Statistically significant differences were observed at the 10-cm depth between the two sampling dates under the gas impermeable film; the population of P. nicotianae dropped to levels comparable to those in the methyl bromide treatment.




Survival of Phvtophthora capsici


Oospores of Phytophthora capsici do not germinate readily in vitro, and this was clearly noted during the field experiments. Although the presence of oospores was verified in the inoculum before the soil was infested, there was not a corresponding high recovery





30


Table 2.5. The effect of time of exposure to solarization on the survival of Phytophthora nicotianae at Site 3.

Propagules per gram of soil

Treatmentx Depth Test 2 Test 3

(cm) 17 days 45 days 17 days 45 days Gas Impermeable 10 2.0' az 0.4a 544.6b 4.4a Gas Impermeable 25 4.1 a 2. la 168.0b 202.3b Gas Impermeable + Cabbage 10 2.7 a 0.9a 52.5b 1.0a Gas Impermeable + Cabbage 25 0.7 a 2.3a 491.3b 362.0b White-on-black 10 22.8 a 4.0a 196.6b 733.9b White-on-black 25 16.3 a 0.9a 186.4b 697.8b White-on-black + Cabbage 10 101.9 a 14.8a 830.5b 825.3b White-on-black + Cabbage 25 25.0 a 12.3a 608.3b 479.5b Methyl Bromide 10 0.0 a 0.Oa 0.a 0.0a Methyl Bromide 25 0.0 a O.Oa O.Oa 0.Oa Nontarped 10 21.6 a 14.9a 1138.7b 700.7b Nontarped 25 6.2 a 4.5a 311.8b 687.6b x Treatments consisted of untreated soil (Nontarped); soil fumigated with methyl bromide plus chloropicrin at 39.2 g/m2 (Methyl bromide); soil solarized under a 30-gm-thick, coextruded white-on-black, low density polyethylene film (White-on-black); or soil solarized under a 30-gm-thick, clear, gas impermeable plastic film (Gas Impermeable). Soil was either nonamended or amended with cabbage (+ Cabbage) at 89 and 81 tons/ha for tests 2 and 3, respectively.
Y Weighted means ([Exp {mean}]-1).
Main effect means at each date for each test followed by the same letter do not differ according to Tukey's Honestly Significant Difference procedure (P< 0.05); data transformed to (ln[ppg+l ]) prior to analysis.





31


of the pathogen, either before or after the solarization treatments. Recovery of the pathogen on the selective medium was erratic.

At site 1, no propagules were recovered at 10-cm depth after treatment with

methyl bromide, the gas impermeable plastic, or the gas impermeable plastic combined with cabbage (Table 2.6). Low levels of survival at the 25-cm depth under the methyl bromide treatment, the gas impermeable plastic, and the gas impermeable plastic combined with cabbage were not statistically different from the same treatments at the 10-cm depth. The highest survival was observed under the white-on-black plastic at the 10-cm depth, regardless of cabbage amendment, and in the nontarped treatment.

At site 2, no statistical differences were observed in the survival of P. capsici under the clear low density polyethylene film, clear low density polyethylene film plus cabbage, and the gas impermeable plastic at the 10-cm depth, or under the methyl bromide and the gas impermeable plastic combined with cabbage at both depths (Table

2.6). Survival was highest in the nontarped treatment at both depths, and populations were not significantly different from those at the 25-cm depth in all other treatments, except treatments with methyl bromide and gas impermeable plastic combined with cabbage.

The lack of hymexazol in the selective medium for the determination of survival of P. capsici from test 1 at site 3 made it impossible to recover the pathogen from the soil because other microorganisms colonized the organic substratum (shredded wheat seeds) and grew faster than the pathogen on the dilution plates. Not even the addition of 100 mg of lecithin per liter of medium to stimulate the germination of oospores of the pathogen could compensate for the lack of hymexazol.

Fewer oospores were observed in the inoculum used for tests 2 and 3 at site 3, than in previous experiments, and very few colonies were observed on the selective medium at the end of the experiments. No valid analysis could be performed and the data are not presented.





32


Table 2.6. Survival ofPhytophthora capsici after soil solarization in 1994.
Treatment Depth Site 1' Site 2'
(cm) (ppg) (ppg)
Gas Impermeable 10 0.0x ay 0.Oa as Impermeable 25 1.5 ab 7.0ab Gas Impermeable + Cabbage 10 0.0 a 0.4a Gas Impermeable + Cabbage 25 1.2 ab 0.5a Clear LDPE 10 ntz 0.Oa lear LDPE 25 nt 11.2ab Clear LDPE + Cabbage 10 nt 0.2a Clear LDPE + Cabbage 25 nt 7.9ab White-on-black 10 84.6 c nt White-on-black 25 47.4 c nt White-on-black + Cabbage 10 70.8 c nt White-on-black + Cabbage 25 16.3 bc nt Methyl Bromide 10 0.0 a 0.2a ethyl Bromide 25 1.1 ab 0.9a on-tarped 10 33.4 c 33.9b on-tarped 25 63.0 c 25.6b "Treatments consisted of untreated soil (Non-tarped); soil fumigated with methyl bromide plus chloropicrin at 39.2 g/m2 (Methyl bromide); soil solarized under a 30-pm-thick, coextruded white-on-black, low density polyethylene film (White-on-black); or soil solarized under a 30jm-thick, clear, low density polyethylene film (Clear LDPE); or a 30jim-thick, clear, gas impermeable plastic film (Gas Impermeable). Soil was either nonamended or amended with cabbage (+ Cabbage) at 80 and 68 tons/ha at sites 1 and 2, respectively. ' Site 1 = Naples Tomato Growers-Gargiulo farm number 4 in Decatur County, Georgia (49 days of solarization). w Site 2 = John Allen Smith farm located in Gadsden County, Florida (48 days of solarization).
x Weighted means ([Exp { mean]- 1). Y Main effect means followed by the same letter do not differ according to Tukey's Honestly Significant Difference procedure (P< 0.05); data were transformed to ln(ppg+ 1) for analysis. z nt = not tested.





33


Discussion



Temperature Changes and Soil Moisture Content


Soil solarization is a pasteurization process dependent on solar energy to heat the soil and kill pathogens. Effective pasteurization occurs when soil sustains temperatures that are lethal to the fungal propagules infesting the soil. In arid climates, soil temperatures between 480 and 540C have been reported as effective for the control of Rhizoctonia solani, Pythium spp., Thielaviopsis basicola (Pullman et al., 1979), Verticillium dahliae (Grinstein et al., 1979b; Pullman et al., 1979), Pyrenochaeta terrestris, Fusarium spp. (Katan et al., 1980) and Sclerotium rolfsii (Grinstein et al., 1979a). However, in climates where cloud cover and rainfall interrupt the heating process of the soil, maximum temperatures recorded were below 500C (Chellemi et al., 1994).

Temperature changes were monitored in site 2 for the experiments in sites 1 and 2, and in test 2 at site 3 for both tests 2 and 3. Inferences from one site in relation to the other have to be made with caution since soil color, proportion of sand, silt and clay content, and soil moisture were different. Each one of these factors can affect the temperature accumulation in soil, and when taken together could cause significant deviations in temperatures from the patterns observed at the site where temperatures were actually measured. These differences may hold for sites that are several kilometers apart (sites 1 and 2), as well as for sites a few meters apart, as was the case in site 3, between tests 2 and 3. Because tests 2 and 3 at site 3 were only a few meters apart, with the same type of soil, any differences in temperature could be explained best by the change in moisture content. As shown by Mahrer et al. (1984), the higher the water content of the soil, the higher the maximum temperature will be.





34


Survival of Phvtophthora nicotianae


Phytophthora nicotianae is believed to survive in root fragments and organic matter in the soil or as chlamydospores free in the soil (Erwin and Ribeiro, 1996). Infested wheat seeds were selected for inoculum to favor the formation of chlamydospores in soil and thus simulate natural spore formation for field survival. It is possible that lower temperatures during the first days of solarization allowed the formation of chlamydospores in the soil, and, therefore, a higher recovery was observed at the end of some experiments. Soil moisture also may have contributed to the variability in recovery of P. nicotianae from test to test and site to site. In test 2, the plastic treatments were laid down after a week of frequent rains, with soil moisture very uniform throughout the soil profile. In test 3, the week preceding the application of the plastic treatments was hot and dry, and irrigation was required to provide adequate moisture for the solarization. These factors were reflected in the results observed for the tests, especially in test 3, where higher numbers of surviving propagules were observed at lower depths, possibly indicating that temperatures did not rise as much as in test 2 due to the lack of moisture. According to Mahrer et al. (1984) temperature maxima of the soil increase with increasing soil moisture content. A 10-fold difference in the water status was observed between tests 2 and 3, which may have led to differences in survival of P. nicotianae at lower depths.

The absence of chlamydospores in the wheat seeds at the time of preparation of the inoculum and a subsequent lack of survival of P. nicotianae in the nontarped treatments in 1994 and 1995 prompted a search for methods that would augment the production of this type of inoculum. Ioannou and Grogan (1985) indicated that chlamydospores were formed abundantly when mycelial mats in liquid cultures were exposed to an environment containing 10% CO2 and 2% 02. This procedure was evaluated for the wheat seed inoculum, but increased chlamydospore production did not





35


occur (Appendix A). However, when soil was infested and incubated at 180 or 250C, large numbers of chlamydospores were formed (Appendix A). Since the highest number of chlamydospores was observed in the pasteurized soil from site 1 after incubation at 180C for 10 days, inoculum was prepared in 1996 by infesting microwaved soil from site

1 and incubating it at 180C for 10 days before burial in the field.

The clear, low density polyethylene and the gas impermeable films were effective in reducing populations of P. nicotianae at the 10-cm depth. At this depth control was similar to that achieved by fumigation with methyl bromide. However, at the 25-cm depth, no control was observed, and the populations recovered on the dilution plates were similar to those in the nontarped or the white-on-black plastic controls. Clearly, solarization alone cannot be relied upon to control P. nicotianae in soil under conditions encountered in these tests. Chellemi et al. (1994) found that soil solarization reduced populations of P. nicotianae to undetectable levels at a depth of 15 cm; however, at the 25-cm depth, no differences in survival were observed between the solarized and the control treatments in two sites.


Survival of Phvtophthora capsici


Although the same two isolates were used in all crosses to produce oospores for all tests, the numbers of oospores observed in wheat seeds were not consistent from year to year. Another problem associated with the evaluation of survival of P. capsici is the germination of the propagules in the selective medium. In preliminary studies it was found that approximately 1% of the oospores germinated in PARPH, which was confirmed by the work of Larkin et al. (1995). Phytophthora capsici survived better in the nontarped treatments and under the white-on-black plastic, regardless of the cabbage amendment. Control of the pathogen was most effective with methyl bromide, followed by the gas impermeable and clear low density polyethylene film at the 10-cm depth. In





36


1995 and 1996, fewer oospores were produced in the wheat seeds and the recovery of propagules after solarization was inconsistent, which precluded analysis of the results.



Effect of Cabbage Amendment

Cabbage did not affect the survival of either pathogen. This finding is contrary to that observed by other researchers (Keinath, 1996; Ramirez-Villapudua and Munnecke, 1987, 1988). It is possible that the differences observed are due to the preparation of the cabbage amendment and its incorporation into the soil. For all of the field plots, cabbage was either chopped with a machete (1994) or shredded in a shredder (1995 and 1996). In either case, the fragments were relatively large, and incorporation was done by disking the residue into the soil at the same time that the raised beds were being prepared, which may have led to an uneven distribution of the amendment in the soil profile. In contrast, Ramirez-Villapudua and Munnecke (1988) air dried the cabbage and ground it with a Wiley mill to a fine powder. This residue was incorporated into small amounts of soil, which were placed in plastic bags to be solarized. Further work by these authors (Ramirez-Villapudua and Munnecke, 1987) indicated that populations ofFusarium oxysporum f.sp. conglutinans were reduced by the incorporation of cabbage into the soil at rates that were higher than those used in the experiments reported here. Keinath (1996) found that gummy stem blight of watermelon was reduced in areas that received cabbage amendment and solarization; however, no information about the amount of residue incorporated into the soil was given. It may be that the effectiveness of cabbage as a soil amendment is influenced by the drying process, and, during the experiments reported here, rainfall during the drying time may have had a detrimental effect.















CHAPTER 3
THERMAL INACTIVATION OF PHYTOPHTHORA NICOTIANAE


Introduction


Studies of thermal inactivation of plant pathogens can yield important information on survival of specific types of propagules, and, using simulated conditions, can provide information for the estimation of time constraints for effective soil solarization. JuarezPalacios et al. (1991) found that determination of the heat sensitivity of isolates of Phytophthora spp. in the laboratory closely reflected their inactivation in solarized soil and indicated the possible use of soil solarization for management of those pathogens. These authors found that a high-temperature isolate of P. megasperma survived exposure for 30 minutes at 450C in a heat sensitivity study, and that leaf discs used as bait were infected by the pathogen after solarization for 4 weeks; however, P. cinnamomi and a low-temperature isolate of P. megasperma did not survive exposure to 450C for 20 minutes. In similar studies, Bollen (1985) observed that oospores of P. capsici were more thermotolerant than mycelium from either mating type used to produce the oospores. The temperature difference for tolerance was more than 50C, with oospores surviving at 500C for 30 minutes. However, the system used did not reflect field conditions, since the continuously flooded soil employed in the experiment would rarely be attained in any field. Bollen (1985) did not analyze the relationship of time to varying temperature, and the standard temperature of 50'C used in the laboratory tests is rarely obtained at soil depths below 10 cm. One interesting aspect of this study was the use of cucumber seedlings for a plant disease assay, which was more sensitive than soil dilution plating on


37






38


potato-dextrose agar (PDA, pH 5.6) for the detection of residual populations of the pathogen.

Pullman et al. (1981 a) observed a logarithmic relationship between time and temperature in the inactivation of propagules of four soilborne plant pathogens. These authors found that the exposure time required to reduce the population by 90% (LD,,0) for oospores of Pythium ultimum was 30 minutes at 500C, 110 minutes at 470C, 8 hours at 450C, 42 hours at 420C, 13 days at 38.50C and 26 days at 370C, respectively. This study was performed using soil moisture adjusted to field capacity, and it closely reflected common temperature profiles in soil solarization tests.

Studies of thermal inactivation can be complemented to simulate other conditions in the field, such as the addition of organic amendments to the soil prior to solarization (Gamliel and Stapleton, 1993a, 1993b; Ramirez-Villapudua and Munnecke, 1987, 1988). Cabbage residue associated with solarization was used successfully to control cabbage yellows, caused by Fusarium oxysporum f. sp. conglutinans, by Ramirez-Villapudua and Munnecke (1987, 1988). The addition of cabbage alone was only partially effective for the control of cabbage yellows, but, when cabbage residue was heated during solarization, there was significant control of the pathogen.

Due to the demands on time and equipment, thermal inactivation studies very seldom explore extensive combinations of both time and temperature ranges. Thus, the full benefits that can be derived from such experiments are often not realized. The objectives of this study were to evaluate the effects of both constant and pulsing temperatures, cabbage amendment, soil water matric potential, and three, pasteurized and nonpasteurized soils on the survival of Phytophthora nicotianae.





39


Materials and Methods



Production of Chlamydospore Inoculum of Phvtophthora nicotianae


Chlamydospores of Phytophthora nicotianae were produced in V8 broth, as

described by Mitchell and Kannwischer-Mitchell (1992). Four, 5-mm-diameter V8 juiceagar plugs of a culture with actively growing mycelium of isolate Pn21 were transferred to a 325-ml prescription bottle containing 25 ml of clarified V8 broth. After incubation at 250C in the dark for 24 hours, the bottle was shaken vigorously to fragment the mycelial mats. The hyphal fragments adhering to the walls were resuspended by slowly rotating the bottle. The bottle was incubated horizontally as a stationary culture at 250C for 6 days. One hundred milliliters of sterile deionized water were added to submerge the mycelial mat, and the culture was further incubated at 180C, vertically, for a minimum of

3 weeks.

The mycelial mats were rinsed in deionized water on a 400-mesh sieve,

transferred to a blender with enough water to make a slurry, and blended on high for 1 minute. The resulting slurry was ground about 30 times in a glass mortar and pestle and then subjected to two, 30-second cycles of sonication at 100 W (Model 450 Sonifier, Branson Ultrasonics Corporation, Danbury, CT 06810). The total number of chlamydospores in the suspension was estimated with a hemacytometer, and the suspension was immediately used to infest soil to a density of 500 chlamydospores per gram of soil.


Effect of Constant Temperature on the Inactivation of Chlamydospores of Phytophthora nicotianae


One-kilogram lots of moist soil from Site 1 (Naples Tomato Growers-Gargiulo farm number 4 in Decatur County, Georgia) were pasteurized in a microwave at 700 W






40


for 4 minutes in a plastic bag. After pasteurization soil moisture was adjusted to 6% (-15 kPa) with sterile water. Thirty grams of infested soil were dispensed into a test tube, which was loosely closed with a plastic cap to allow exchange of air.

A set of test tubes was placed in each of eight water baths held at each of the

following constant temperatures with circulation heaters: 250, 350, 380, 410, 440, 470, 500 and 530C. The time of exposure at each temperature varied from 5 to 120 minutes or from 2 to 480 hours. Three tubes were removed at each time interval and part of the soil (15 g) was diluted with soft agar (2.5 g of Difco agar per liter of deionized water) and plated on a medium selective for pythiaceous fungi within 24 hours. The selective medium (PARP) consisted of 17 g of cornmeal agar (Difco) in 1 liter of deionized water amended with 5 mg of pimaricin, 250 mg of ampicillin, 10 mg of rifampicin, and 100 mg of pentachloronitrobenzene (Mitchell and Kannwischer-Mitchell, 1992). The soil overlay was removed after 48 hours by gently washing the agar surface with tap water. The total number of colonies formed on PARP after 72 hours of incubation was recorded as an estimation of the number of chlamydospores surviving the heat treatment. The other part of the soil (15 g) was transferred to a Petri plate and covered with approximately 10 ml of sterile water to provide a thin layer of water over the soil. Three, 3-day-old tomato seedlings, cultivar solar set, were placed on the soil in each plate and incubated in a growth chamber at 270C for 3 days. The seedlings were rinsed twice in sterile water, blotted dry and plated on PARP to evaluate the colonization of tissues by P. nicotianae. The number of seedlings with fungal colonization was recorded. The experiment with times of exposure ranging from 5 to 120 minutes was repeated once, while the experiment with times of exposure ranging from 2 to 480 hours was repeated twice.






41


The Effect of Cabbage Amendments on the Thermal Inactivation of Chlamvdospores of Phvtophthora nicotianae


In order to determine the effect of cabbage amendments on the inactivation of chlamydospores of P. nicotianae, experiments similar to the ones described previously were conducted, but temperatures were restricted to 350, 380, and 41 C, and the time was limited from 2 to 480 hours. Cabbage leaves (Brassica oleracea var. capitata) were air dried in a greenhouse and ground in a Wiley mill with a 20-mesh screen. Pasteurized soil was amended with dry, ground cabbage leaves at 0, 0.125, 0.25 or 0.5% (w/w). Preliminary work indicated that concentrations above 0.5% were phytotoxic to tomato seedlings. Thirty grams of amended soil were dispensed into a test tube, a disk of filter paper was laid on top of the soil, and then thirty grams of amended and infested soil were dispensed on the top of the first layer. The tube was sealed with a gas impermeable plastic film (Bromotec film, Lawson Mardon Packaging, United Kingdom), and a plastic cap was placed over the tube to reduce the exchange of air and trap the volatiles released by heating the cabbage amended soil.

The test tubes were placed in water baths held at constant temperatures with

circulation heaters. Three tubes were removed at each time interval, and part of the soil (15 g) was diluted with soft agar and plated on PARP within 24 hours, as previously described. The soil overlay was removed after 48 hours by gently washing the agar surface with tap water, and the number of colonies was recorded. Fifteen grams of soil were transferred to Petri plates and covered with sterile water, and three, 3-day-old tomato seedlings were incubated on the soil slurry for 3 days. After incubation the seedlings were rinsed twice in sterile water, blotted dry, and plated on PARP. The number of colonized seedlings was recorded.

Another test was done to determine if the bottom layer was necessary to ensure adequate concentration of volatiles in the test tube. Soil infested with the pathogen was






42


dispensed into empty test tubes or into tubes containing a bottom layer of cabbageamended, noninfested soil. All test tubes were placed in the water baths at constant temperatures, as described above, and incubated for 1 week. The tubes were removed from the water baths, and the number of propagules were estimated using procedures previously described. No significant effects of the soil layering were observed when the data were analyzed. For this reason all other tests were done with only a single layer of infested, cabbage-amended soil.


The Effect of Cycling Temperatures and Cabbage Amendments on the Thermal Inactivation of Chlamvdospores of Phvtophthora nicotianae


A more critical analysis of the effects of daily temperature fluctuation, as

observed during soil solarization, on the survival of the pathogen was performed in this experiment. Two temperatures, representing average thresholds at 10 and 25 cm of depth, were selected, and the duration of the daily exposure at each temperature was determined by an analysis of published data from north Florida (Chellemi et al., 1994). The daily regimes selected were 5 hours at 410C and 8 hours at 350C, with a baseline temperature of 250C during the rest of the day. These temperatures were maintained with circulation heaters in water baths. After preliminary tests indicated a high percentage of the spores survived the heat treatment, a third temperature, 440C for 1.5 hours daily, was added. The duration of the daily exposure at 440C was based both on the results of previous tests with constant temperature and on the analysis of temperature profiles from field experiments in 1994 (Chellemi et al., 1994).

Microwaved and infested soil was adjusted to a final moisture of 6% with sterile water. Half of the infested soil was amended with dry, ground cabbage leaves at 0.125% (w/w). Thirty grams of soil were dispensed into a test tube, which was sealed with a piece






43


of the gas impermeable plastic film, and a plastic cap was placed over the tube to reduce the exchange of air and trap the volatiles released by heating the cabbage-amended soil.

The test tubes were placed in the water baths at 250C until the temperature of the soil and the water were equilibrated, and then the cycling was initiated. Temperatures were monitored using a CR10 datalogger (Campbell Scientific, Inc., Logan, Utah 84321). Three tubes were removed after 1, 2, and 3 days, and every 3 days thereafter during a period of 24 days. Part of the soil (15 g) was diluted with soft agar and plated on PARP within 24 hours, as previously described. The number of colonies was recorded. The other part of the soil was used in a plant disease assay.

At the end of the thermocycling experiment, all samples were transferred to 50ml, tripour plastic beakers and one, 1-month-old tomato seedling was transplanted into the soil in each beaker. A small amount of vermiculite was poured on the top of the soil to prevent the roots from desiccating. All plants were kept in an growth chamber at 27'C for 30 days. As plants died, the root systems were rinsed in water, surface disinfested for 30 seconds in 70% ethanol, rinsed twice in sterile water, and plated on PARP. At the end of the experiment, all plants were cut and the root systems plated on PARP for determination of infection.

The use of average temperatures achieved during soil solarization for the temperature cycling experiments was not sufficient to completely inactivate chlamydospores of P. nicotianae; therefore, an additional experiment was conducted using temperature regimes achieved during an optimum solarization day at the NFRECQuincy in 1995. Temperature regimes used were 470C for 3 hours, 440C for 5 hours, and350C for 8 hours daily. Throughout this experiment a baseline temperature of 250C was maintained during the rest of the day. The duration of each experiment was 15 days, and the experiment was repeated once.

The procedures for production of chlamydospores, treatment of soil, and

estimation of soil populations of P. nicotianae were the same as described previously. At






44



the end of the temperature cycling experiments, a plant disease assay was performed for 4 weeks with 1-month-old tomato seedlings, as described previously. All dead plants were plated on PARP to confirm the presence of mycelium typical of Phytophthora spp., and at the end of the experiment all surviving plants were plated to determine if any infection had occurred.


The Effect of Soil Water Matric Potential, Temperature Regimes, and Cabbage Amendments on the Thermal Inactivation of Chlamvdospores of Phytophthora nicotianae


The effect the soil water matric potential in association with temperature regimes that simulate soil solarization and cabbage amendments was examined. The soil water matric potentials used were 0 kPa, -10 kPa and -30 kPa. These potentials were selected due to their importance on the life cycle of the pathogen. The temperature regimes used were 1.5 hours at 440C, 5 hours at 410C and 8 hours at 35'C, or 3 hours at 470C, 5 hours at 440C and 8 hours at 350C, with a baseline temperature of 250C during the rest of the day. These temperatures were maintained with circulation heaters in water baths. The experiment was repeated once.

The procedures for production of chlamydospores, treatment of soil, and estimation of surviving populations ofP. nicotianae were the same as described previously. At the end of the temperature cycling experiments, a plant disease assay was performed for 4 weeks with 1-month-old tomato seedlings, as described previously. All dead plants were plated on PARP to confirm the presence of mycelium typical of Phytophthora spp., and at the end of the experiment all surviving plants were plated to determine if any infection had occurred.





45


The Effect of Three Nonpasteurized Soils. Temperature Regimes and Cabbage Amendments on the Thermal Inactivation of Chlamydospores of Phytophthora nicotianae


The effects of nonpasteurized soils and cabbage amendments were examined using soil from the three field sites where soil solarization was evaluated from 1994 to 1996 (see Chapter 2 for a description of the soil types). The soil water matric potential of all soils was adjusted to -10 kPa, and the following temperature regimes were evaluated in one test, with three replicates per combination of soil, pasteurization, temperature and exposure time: 470C for 3 hours, 44oC for 5 hours, 440C for 1.5 hours, 410C for 5 hours, and 350C for 8 hours. The experiment was repeated once.

The procedures for production of chlamydospores, treatment of soil, and estimation of surviving populations of P. nicotianae were the same as described previously. At the end of the temperature cycling experiments, a plant disease assay was performed for 4 weeks with 1-month-old tomato seedlings, as described before. This assay was done with the soil from temperature regimes of 41 oC and above, since all previous experiments indicated that all of the plants were infected at the lower temperature regimes after 30 days. All dead plants were plated on PARP, amended with 50 mg of hymexazol per liter of medium to confirm the presence of mycelium typical of Phytophthora spp., and at the end of the experiment all surviving plants were plated to determine if infection had occurred.


Statistical Analysis


The results of each experiment were analyzed individually. Whenever statistical analysis of the residues indicated that the results could be pooled due to the lack of variation, a final analysis was done with the pooled data.

The response surfaces were analyzed using the procedure PROC NLIN of SAS (SAS Institute, Cary, NC; release 6. 11 for personal computers). Survival data were






46


transformed using In (ppg+l) prior to analysis. Comparisons between the qualitative and quantitative methods used to determine the number of chlamydospores surviving the heat treatment were made.

The analyses of the effect of temperature cycling and cabbage amendment on the survival of chlamydospores of P. nicotianae were performed with Statgraphics Plus, version 2.1 (Manugistics, Inc., Rockville, MD), for the convenience of comparing several linearization models at once.

The experiments on the determination of the effects of soil water matric potential and the effects of different soils on survival of P. nicotianae were analyzed using PROC GLM of SAS for the analysis of variance. The determination of the effect of each individual factor was calculated as the average of that factor across all other factors; the analysis of each interaction was done by calculating the average for the secondary factor within the main factor across all other factors.




Results



Effect of Constant Temperature on the Inactivation of Chlamvdospores of Phytophthora nicotianae


Phytophthora nicotianae was consistently recovered throughout the 2-hour

experiment when temperatures were below 470C (Table 3.1). At 440C, a sharp decrease was observed after 30 minutes, but 10% of the initial population (500 ppg) survived to the end of the experiment. After 75 minutes of heat treatment at 470C, population levels dropped below 1 propagule per gram of soil, and after 2 hours populations had declined to levels undetectable by the procedure used to quantify the inoculum in the soil. At 500 and 530C, populations were very low after only 5 minutes of exposure.









Table 3.1. Effect of constant temperature and time on the survival of chlamydospores of Phytophthora nicotianae.


Temperature Number of propagules per gram of soil recovered over time minutesx
(0C) 5 10 15 30 45 60 75 90 105 120 25 110.5' 133.2 140.8 150.7 149.7 142.1 165.5 176.7 179.0 323.4 35 184.2 190.9 182.3 152.9 144.0 149.4 134.0 125.4 159.4 153.0 38 64.6 93.7 82.8 91.0 80.5 114.3 99.8 109.9 137.3 107.6 41 178.5 152.2 161.7 193.7 220.7 210.3 87.6 88.1 92.7 88.8 44 108.0 118.0 112.8 60.4 50.4 40.2 35.8 40.5 46.9 49.2 47 18.5 12.2 10.3 3.5 1.4 1.3 0.7 0.5 0.4 0.0
50 2.6 1.6 0.7 0.4 0.2 - - -
53 0.3 0.1 0.1 z
x Soil initially infested with 500 chlamydospores per gram; P. nicotianae recovered on a selective medium.
Y Each point consists of the average of two experiments with three repetitions each. zNot tested.






48


The decline in survival of the chlamydospores of P. nicotianae in soil exposed to constant heat above 380C for 5 to 120 minutes was best described by the equation In(ppg + 1) = (6.2 - 0.00108* time2 )*e-0.02208*temperature, based on the comparison of the plotting of Observed, Predicted Values, Residuals X Time, and Residuals X Temperature, and on the R2, = 0.8234, in which ppg is the number of propagules recovered per gram of soil.

The use of tomato seedlings as bait for the detection of survival of P. nicotianae indicated that survival was affected by the heat treatment only after 90% of the initial population of chlamydospores had been inactivated, which occurred generally only after exposure to 470C or higher (Tables 3.1 and 3.2). The detection of survival with the use of seedlings was not as sensitive as the detection with the soil dilution procedure used. For example, at 530C no seedlings were infected after the 3-day incubation period (Table

3.2); in contrast, the soil dilution procedure indicated that 0.1 propagule per gram of soil were still alive after 15 minutes of treatment (Table 3.1).

Exposure of chlamydospores of P. nicotianae to heat for 480 hours indicated that the pathogen can survive in the at 350C for a long period (Table 3.3). Populations declined to levels below 1 propagule per gram of soil when temperatures were maintained at 380C for more than 288 hours. After 96 hours of exposure at 410C, or 48 hours at 440C, no propagules were recovered from the soil. Exposure to 470C reduced the populations to residual levels after 4 hours.

The decline in survival of the chlamydospores of P. nicotianae in soil exposed to constant temperatures above 380C for 2 to 480 hours was best described by the equation In(ppg + 1) = 6.2* e(-0.00759*time-0.0138*temperature), based on the comparison of the plotting of Observed X Predicted Values, Residuals X Time, and Residuals X Temperature, and on the R2adj = 0.6773, in which ppg is the number of propagules recovered per gram of soil.










Table 3.2. Detection ofPhytophthora nicotianae by the tomato seedling assay after heat treatment for 5 to 120 minutes.


Temperatu Percentage of colonized tomato seedlings after soil treatment over time (minutes)
re
(oC) 5 10 15 30 45 60 75 90 105 120 25 100y 100 100 100 100 100 100 100 100 100 35 100 100 100 100 100 100 100 100 100 100 38 100 100 100 100 100 100 100 100 100 100 41 100 100 100 100 100 100 100 100 100 100 44 100 100 100 100 100 100 100 100 100 100 47 78 50 94 72 61 44 22 11 17 33 50 17 17 0 17 0 - - - -
53 0 0 0 -z
' Each point consists of the average of two experiments with three repetitions each. Z Not tested.











Table 3.3. Effect of constant temperature and extended periods of time on the survival of chlamydospores of Phytophthora nicotianae.


Temperature Number of propagules per gram of soil recovered over time (hours)x
(oC) 2 4 8 12 16 20 24 48 96 192 288 384 480 25 300.9' 214.6 270.1 271.8 274.9 290.7 329.4 524.2 326.5 294.1 275.7 297.2 241.8
35 150.9 231.5 344.8 430.0 362.6 398.5 402.8 385.1 461.2 447.4 311.5 88.1 68.9
38 125.8 162.8 268.3 300.1 354.0 397.2 417.1 89.8 34.9 9.5 0.9 0.2 0.0
41 146.0 169.6 150.6 91.6 99.1 78.3 50.4 11.7 0.0 0.0 0.0 0.0 0,0
44 35.2 10.2 1.6 0.1 0.0 0.1 0.1 0.0 0.0 - -
47 0.7 0.1 - - -
x Soil initially infested with 500 chlamydospores per gram; P). nicotianae recovered on a selective medium. Each point consists of the average of three experiments with three repetitions each. z Not tested.






51



Generally, the use of the tomato seedling assay provided similar results to the soil dilution plating procedure, except when only residual inoculum (0.1 propagule per gram of soil) was left (Tables 3.3, 3.4). When residual inoculum survived the heat treatment, baiting the soil with seedlings allowed detection of the fungus in two cases that were not detected by soil plating, but failed to detect the pathogen in three other instances that were detected by the plating procedure. All tomato seedlings were colonized by P. nicotianae whenever more than 50 propagules per gram of soil were recovered in the soil dilution plates with PARP; in one case 100% infection was observed when 10 propagules per gram of soil were recovered.




The Effect of Cabbage Amendments on the Thermal Inactivation of Chlamvdospores of Phytophthora nicotianae


The addition of dried, ground cabbage leaves changed the response of the

chlamydospores to heat (Table 3.5). As concentration of cabbage increased, inactivation also increased; thus, less time was required to inactivate spores in cabbage amended soil.

At 350C chlamydospores survived in the soil for 480 hours when no cabbage

amendment was present (Table 3.5). The addition of 0.125% cabbage to the soil reduced the population to residual levels after 192 hours. Ninety six hours were required with

0.25% cabbage to reduce population counts below one propagule per gram of soil; similar population reductions were attained after 48 hours when 0.5% cabbage was added to the soil. The addition of cabbage to soils incubated at 380C reduced pathogen populations in the following manner: the time required to reduce the inoculum to below 1 propagule per gram of soil dropped from 192 hours without cabbage amendment to 48 hours with

0.125% or 0.25% amendment, and to 24 hours with 0.5% amendment added to the soil. Survival of the chlamydospores in soil treated at 410C declined to undetectable levels










Table 3.4. Detection ofPhytophthora nicotianae by the tomato seedling assay after heat treatment for 2 to 480 hours.

Temperature Percentage of colonized tomato seedlings after soil treatment over time (hours)
(oC) 2 4 8 12 16 20 24 48 96 192 288 384 480 25 100Y 100 100 100 100 100 100 100 100 100 100 100 100 35 100 100 100 100 100 100 100 100 100 100 100 100 100
38 100 100 100 100 100 100 100 100 96 77 52 37 0 41 100 100 100 100 100 100 95 41 18 0 0 0 0 44 89 100 42 0 4 0 0 0 0 - - -
47 52 26 -z
' Each point consists of the average of three experiments with three repetitions each. z Not tested.









Table 3.5. Effect of constant temperature, cabbage amendment, and time on the inactivation of chlamydospores of Phytophthora nicolianae.


Temperature Cabbage Number of propagules per gram of soil recovered over time (hours)x
(oC) (%) 2 4 8 12 16 20 24 48 96 192 288 384 480 35 0 400.7y 415.1 327.8 434.6 472.6 471.4 379.9 149.2 226.0 159.2 68.2 0.0 26.5
35 0.125 102.8 77.0 79.6 79.8 60.9 58.3 36.8 35.2 1.5 0.1 0.0 0.0 0.0
35 0.25 116.5 57.7 102.2 38.0 42.6 44.7 24.7 2.8 0.6 0.0 0.0 0.0 0.1
35 0.5 101.0 96.1 53.8 39.2 18.1 17.1 13.1 0.5 0.0 0.0 0.0 0.0 0.0
38 0 202.0 185.9 146.5 280.7 182.1 201.7 218.6 41.9 3.6 0.1 0.0 0.0 0,5
38 0.125 88.5 91.2 82.8 672 80.6 15.2 4.9 00 0.0 0. 00 0.0 00 0.0
38 0.25 100.6 73.2 57.3 48.0 27.2 8.2 8.5 0.1 0.0 0.0 O.0 0.0 0.0
38 0.5 94.8 76.1 32.8 15.2 7.4 2.7 0.7 0.0 0.0 00 0..0 0.0 0.0
41 0 309.1 236.4 210.6 203.3 106.5 15.9 14.8 4.6 0.0 - -
41 0.125 99.8 79.5 30.6 16.6 10.4 2.9 0.6 0.0 0.0
41 0.25 72.7 40.4 9.9 4.0 2.0 1.9 0.0 0.0 0.0 - -
41 0.5 67.9 43.5 6.6 1.3 0.2 0.0 0.0 0.0 0.0 - -
Soil initially infested with 500 chlamydospores per gram.
Y Each point consists of the average of two experiments with three repetitions each. z Not tested.





54



after 96 hours in nonamended soil, 48 hours in soil amended with 0.125% cabbage, and 24 hours in soil amended with 0.25% cabbage (Table 3.5). Only 16 hours were required to reduce the population to 0.2 propagule per gram of soil in soil amended with 0.5% cabbage.

The decline in survival of the chlamydospores of P. nicotianae in soil amended with cabbage residue and exposed to constant temperature was best described by the linear-power model presented in Table 3.6. According to the model used for comparisons of survival of P. nicotianae, the equation describing the inactivation of chlamydospores in the absence of cabbage amendment is significantly different from the others (P<0.05); the equations generated for amendment with 0.125% and 0.25% cabbage are not different from each other, and the equation generated for amendment with 0.5% cabbage is different from all other equations.

Detection of survival of P. nicotianae with tomato seedlings as baits followed

similar trends as those observed with the soil dilution plating procedure (Tables 3.5, 3.7). As the number of propagules recovered in the soil plates decreased from 500 to 100 propagules per gram of soil, the percentage of colonized seedlings was relatively constant. Further decreases in the propagule counts generally reflected a decrease in the percentage of colonized seedlings. Of the 41 cases in which no propagules were detected by the soil dilution plating procedure, the survival of the pathogen was detected in 17 cases by the baiting technique. Generally, this discrepancy was observed right after the number of propagules recovered in the selective medium had dropped below 1 propagule per gram of soil.





55


Table 3.6. Equations of models that describe the thermal inactivation of chlamydospores ofPhytophthora nicotianae as a factor of constant temperature between 350C and 410C, time of exposure ranging from 2 to 480 hours, and cabbage amendment concentrations.

Cabbage (%) Equation R2adj.
0 In(ppg + 1) = (6.2 - 0.0141* Time)* e-0.0063*Temperature 0.5062
0.125 In(ppg + 1) = (6.2 - 0.0181* Time)* e-0.0191*Temperature 0.5474 0.25 In(ppg + 1) = (6.2 - 0.0183* Time)* e-0.0232*Temperature 0.6124 0.5 In(ppg + 1) = (6.2 - 0.0187* Time)* e-0.0282*Temperature 0.6928
z ppg = propagules per gram of soil.









Table 3.7. Detection of Phytophihora nicotianae by the tomato seedling assay after heat treatment of cabbage amended soil for 2 to 480 hours.


Temperature Cabbage Percentage of colonized tomato seedlings after soil treatment over time (hours)
(0C) (%) 2 4 8 12 16 20 24 48 96 192 288 384 480 35 0 100Y 100 100 100 100 100 100 100 100 100 83 11 33 35 0.125 100 100 100 100 100 100 100 100 83 17 0 0 22 35 0.25 100 100 100 100 100 100 100 100 17 6 6 0 22 35 0.5 100 100 100 100 89 100 100 89 33 0 0 0 0 38 0 100 100 100 100 100 100 100 100 6 7 17 0 0 6 38 0.125 83 56 100 100 56 10 100 11 33 0 0 0 0 38 0.25 100 78 100 100 72 94 83 6 28 0 0 0 0 38 0.5 100 100 100 100 50 72 56 11 28 11 0 0 0
41 0 100 100 100 100 100 100 100 100 44 -z
41 0.125 89 50 50 61 39 50 50 0 22 - - -
41 0.25 94 56 50 33 28 33 0 11 22 41 0.5 100 83 67 17 28 6 0 0 11
- Each point consists of the average of two experiments with three repetitions each. Not tested.





57


The Effect of Cycling Temperatures and Cabbage Amendments on the Thermal Inactivation of Chlamvdospores of Phvtophthora nicotianae


Generally, the use of pulsing temperatures increased the time required to inactivate the chlamydospores of P. nicotianae in comparison with the constant temperature treatment (Tables 3.5, 3.8). After 24 days at 350C for 8 hours daily, 40% of the initial infestation level of spores (500 chlamydospores per gram of soil) was still recovered from the nonamended soil in the selective medium. The addition of 0.125% cabbage to the soil reduced the population to levels below 10% after 18 days. The effect of cabbage was evident from the very first cycles. Cycling temperatures at 410C for 5 hours daily reduced the survival of the spores by more than 90% of the initial infestation level after 24 days; when cabbage was added, less than 5% of the propagules were recovered from the soil at the end of the experiment. Survival was below 5% of the initial infestation level after 21 days of exposure to 1.5 hours at 440C daily; with cabbage amendment, detection was lower than that observed in the nonamended soil.

Exposure to temperature regimes simulating optimum solarization periods

reduced the populations to very low levels (Table 3.8). Five hours daily at 440C for 15 days reduced the population to 0.4 propagule per grain of soil. The use of cabbage amendment reduced populations to the same levels after 6 days. Only 3 days of exposure at 470 for 3 hours daily were required to reduce populations to levels below 1 propagule per gram of soil; with cabbage amendment populations were reduced to levels below 1 propagule per gram of soil after 2 days. Populations were reduced to levels below detection only at the two highest temperature regimes (440C for 5 hours daily and 470C for 3 hours daily) after 9 days of treatment. Soil amendment with cabbage appeared to have a greater impact at lower temperatures, where the heat alone was not sufficient to inactivate the chlamydospores.






58


Table 3.8. The effect of temperature regime, cabbage amendment, and time on the survival
of chlamydospores of Phytophthora nicotianae.


Temperature Cabbage Number of propagules per gram of soil recovered over time (days)
Regime? (%) 1 2 3 6 9 12 15 18 21 24
35-8 0 321.9 336.4 304.1 334.6 342.2 314.1 324.4 256.7 234.0 199.3
35-8 0.125 114.5 121.2 129.4 130.5 108.8 84.0 80.8 41.7 32.9 30.9
41-5 0 25L4 329.1 359.0 353.0 250.2 247.2 155.9 80.7 51.9 41.3 41-5 0.125 47.8 58.2 42.6 41.3 37.2 22.2 24.5 18.6 215 18.7
44-1.5 0 98.0 93.8 91.5 75.5 41.0 36.8 33.2 25.3 22.0 19.8
44-1.5 0.125 31.4 33.1 35.0 24.0 20.0 16.8 15.6 16.3 12.6 14.4
44-5 0 42 34.::: 26. 5.2 2.5 1.7 f0.4 -2 44-5 0.125 34.4 14.6 2.7 0.4 0.0 0.0 0.0
47-3 0 3.2 1.2 0.3 0.1 0.2 0.0 0.1 -
47-3 0.125 2.2 0.1 0.0 0.1 0.0 0.0 0.0 x Soil initially infested with 500 chlamydospores per gram. Y Temperature regimes that simulated solarization consisted of temperatures increased daily to 350, 410, 440, 440, or 470C for 8, 5, 1.5, 5 or 3 hours, respectively; the remainder of each day was maintained at 250C.
z Not tested.






59


The reduction in survival of P. nicotianae was modeled using linear regression. The model selected for the comparisons among temperature regimes and cabbage amendment was In(Y + 1) = a + b* X, in which Y is the number of propagules recovered per gram of soil and X is the number of days of exposure to the temperature regime. A lack of fit of the model to the data was observed at 350C, regardless of the cabbage amendment (Table 3.9). As temperature increased a better fit of the model was observed, but at the highest temperature regime tested a lower fit was obtained again.

The plant disease assay verifying the pathogenicity of the surviving populations of P. nicotianae indicated that temperature regimes below 440C for 5 hours daily did not reduce the infection of the tomato seedlings (Table 3.10). At 440C for 5 hours, infection was reduced in the soil amended with cabbage after 3 days. After 3 days of heat treatment at 470C for 3 hours daily, no infection of seedlings was observed. Mortality of the seedlings was observed in the lower temperature regimes (Table 3.11); however, when the soil was treated at 440C for 5 hours daily no seedlings died, except after 2 or 3 days in soil amended with cabbage. No seedlings died when the soil was heated to 470C for 3 hours daily.



The Effect of Soil Water Matric Potential, Temperature Regimes, and Cabbage Amendments on the Thermal Inactivation of Chlamydospores of Phytophthora nicotianae


The temperature regimes evaluated in these tests simulated either average daily

periods or optimum daily temperature periods observed during solarization. Generally, as temperature within the regimes increased, the number of chlamydospores recovered in the soil dilution plates decreased (Tables 3.12, 3.13, 3.14). Propagule survival in the two temperature regimes simulating optimum solarization periods (470C for 3 hours and 440C






60


Table 3.9. Regression coefficients of the linear regressions of temperature regimes and survival of chlamydospores of Phytophthora nicotianae; the model used for the analysis was In (ppg+l) = a + b*days.

Temperature Cabbage Parameters R2 P-Valuez Regimex % slope intercept 35-8 0 -0.01203 5.717 0.0267 0.062 35-8 0.125 -0.05508 4.808 0.2800 0.001 41-5 0 -0.09034 6.049 0.6679 0.001 41-5 0.125 -0.05091 3.812 0.2195 0.001 44-1.5 0 -0.07680 4.631 0.7264 0.001 44-1.5 0.125 -0.04426 3.454 0.2856 0.001 44-5 0 -026132 3.886 0.864 0.001 44-5 0125 -0.21618 2.531 0.5631 0.001 47-3 0 -0.05692 0.718 0.2422 0.001 47-3 0.125 -0.04026 0.455 0.2272 0.001 x Temperature regimes that simulated solarization consisted of temperatures increased daily to 350, 410, 440, 440, or 470C for 8, 5, 1.5, 5 or 3 hours, respectively; the remainder of each day was maintained at 250C.
z Significance associated to the coefficient of determination (R2).






61


Table 3.10. Infection of tomato seedlings after incubation in soil amended with cabbage, infested with Phytophthora nicotianae, and heat treated over time.


Temperature Cabbage Percent infection of tomato seedlings over time (days) Regimex (%) 1 2 3 6 9 12 15 18 21 24
35-8 0 100Y 100 100 100 100 100 100 100 100 100 35-8 0.125 100 100 100 100 100 100 100 100 100 100 41-5 0 100 10 100 100 100 100 100 100 100 100 41-5 i0.125 100 i 10 1 00 100 100 0 100 i 100 100 44-1.5 0 100 100 100 100 100 100 100 100 100 100 44-1.5 0.125 100 100 100 100 100 100 100 100 100 100
44-5 0 100 100 100 100 100 100 83
44-5 0.125 100 100 67 67 50 67 33 - -
47-3 0 100 100 0 0 0 0 0
47-3 0.125 100 83 0 0 0 0 0
x Temperature regimes that simulated solarization consisted of temperatures increased daily to 350, 410, 440, 440, or 470C for 8, 5, 1.5, 5 or 3 hours, respectively; the remainder of each day was maintained at 250C.
Y Each data point consists of the average of two experiments with three repetitions each. Z Not tested.






62


Table 3.11. Mortality of tomato seedlings after incubation in soil amended with cabbage, infested with Phytophthora nicotianae, and heat treated over time.


Temperature Cabbage Percent mortality of tomato seedlings over time (days)
Regimex (%) 1 2 3 6 9 12 15 18 21 24
35-8 0 33Y 27 27 27 33 33 33 33 44 44 35-8 0.125 33 27 20 40 27 40 33 44 44 33
41-5 0 57 33 33 33 33 44 33 33 44 56
41-5 0.125 44 33 44 33 44 33 33 44 44 22 44-1.5 0 44 44 33 44 33 22 33 11 22 22 44-1.5 0.125 33 33 33 33 33 45 0 22 11 11 44-5 0 0 0 0 0 0 0 0 -
44-5 0.125 0 16 33 0 0 0 0 47-3 0 0 0 0 0 0 0 0 47-3 0.125 0 0 0 0 0 0 0
xTemperature regimes that simulated solarization consisted of temperatures increased daily to 350, 410, 44", 440, or 470C for 8, 5, 1.5, 5 or 3 hours, respectively; the remainder of each day was maintained at 250C.
Y Each data point consists of the average of two experiments with three repetitions each. z Not tested.






63


Table 3.12. The effect of temperature regimes that simulate daily solarization periods and cabbage amendment, at a soil water matric potential of 0 kPa, on the survival of chlamydospores of Phytophthora nicotianae, and on the percentages of infection and mortality of tomato seedlings after 30 days of exposure in the previously treated soil.

Temperature Cabbage Test 1 Test 2
Regime (%) Days' PPGw Ix MY PPG I M
(%) (%) (%) (%)
35-8 0 1 223.08 ez 100 0 308.82 e 100 33 35-8 0 2 433.85 f 100 0 540.86 e 100 33 35-8 0 3 413.47 f 100 0 409.76 e 100 33 35-8 0 6 672.84 f 100 0 213.86 de 100 100 35-8 0.125 1 236.46 e 100 0 91.48 cde 100 33 35-8 0.125 2 226.47 ef 100 0 226.47 de 100 0 35-8 0.125 3 169.72 e 100 0 110.16 cde 100 17 35-8 0.125 6 153.47e 100 0 67.58 cde toO :100 41-5 0 1 150.41 e 100 0 214.51 de 100 33 41-5 0 3 248.39 ef 100 0 104.64 cde 100 33 41-5 0 6 126.74 e 100 0 62.37 bcd 100 67 41-5 0.125 1 90.84 de 100 0 37.78 bed 100 0
41-5 0.125 3 62.43 de 100 0 11.06 b 100 100 41-5 0.125 6 34.52 c 100 0 27.73 bc 100 33 44-1.5 0 1 34.16 cd 100 0 29.14 bc 100 33 44-1.5 0 3 19.29 bcd 100 0 15.61 b 100 67 44-1.5 0 6 14.80 bcd 100 0 10.32 b 100 67 44-4.5 0.125 1 49.91 de 100 0 42.90 bcd 100 33 44-1.5 0.125 3 54.15 de 100 0 12.46 b 100 67 .44-1.5 0125 6 20.98 ed 100 33 15.01 b 100 67 44-5 0 1 14.80 bcd 100 0 18.39 bc 100 0 44-5 0 2 7.76 bcd 100 0 11.26 b 100 0 44-5 0 3 6.54 bcd 100 0 9.32 b 100 0 44-5 0.125 1 6.17 be 100 0 10.94 b 100 0 44-5 0.125 2 5.42 bc 100 0 6.61 b 100 0 44-5 0.125 3 3.62 bc 100 0 6.24 b 100 0 47-3 0 1 0.62 a 100 0 0.78 a 100 0 47-3 0 2 0.00 a 67 0 0.00 a 100 0 47-3 0 3 0.00 a 67 0 0.00 a 100 0 47-3 0.125 1 0.52 a 100 0 0.92 a 100 0 47-3 0.125 2 0.00 a 33 0 0.00 a 100 0 47-3 0.125 3 0.00 a 50 0 0.00a 100 0
Temperature regimes that simulated daily solarization periods consisted of temperatures increased daily to 350, 410, 440C, 440C or 470C for 8, 5. 1.5, 5 or 3 hours, respectively; the temperature for the remainder of each day was maintained at 250C.
Days = duration of temperature regime.
w PPG = propagules per gram of soil: initial population was 500 chlamydospores per gram of soil. I = infection.
Y M = mortality.
SMain effect means followed by the same letter in each test do not differ according to Tukey's Honestly Significant Difference procedure (P< 0.05); data were transformed to ln(ppg+l) for analysis and presented as weighted means ([Exp {mean}l-1).






64


Table 3.13. The effect of temperature regimes that simulate daily solarization periods and cabbage amendment, at a soil water matric potential of -10 kPa, on the survival of chlamydospores of Phytophthora nicotianae, and on the percentages of infection and mortality of tomato seedlings after 30 days of exposure in the previously treated soils. Temperature Cabbage Test 1 Test 2
Regime" (%) Days' PPGw Ix MY PPG I M
(%) (%) (%) (%)
35-8 0 1 181.18 e 100 17 202.16 cd 100 0 35-8 0 2 247.14 e 100 0 287.59 d 100 0 35-8 0 3 168.02 e 100 0 147.41 cd 100 17 35-8 0 6 191.48 e 100 0 115.63 cd 100 67 35-8 :0.125 1 325.69 e 100 0 218.42 d 100 50 35-8 0.125 2 274.06 e 100 67 274.06 d 100 0 35-8 0.125 3 200.34 e 100 0 115.51 cd 100 17 35-8 0.125 6 107.85 de 100 0 65.02 cd 100 33 41-5 0 1 449.34 e 100 0 258.82 d 100 33 41-5 0 3 261.43 e 100 0 109.50 cd 100 100 41-5 0 6 209.61 e 100 0 73.22 cd 100 67 41-5 0.125 1 215.16 e 100 0 146.67cd 100 33 41-5 0.125 :3 127.77 e 100 0 77.89 cd 100 67 41-5 0.125 6 97.20 de 100 .0 5S5cd 100 67 44-1.5 0 1 44.15 cd 100 0 14.26 b 100 33 44-1.5 0 3 19.70 cd 100 0 6.24 b 100 33 44-1.5 0 6 14.03 c 100 0 8.73 b 100 33 44-1.5 0.125 I 70.95 de 100 0 74.19 cd 100 0 44-1.5 0.125 3 59.95 d 100 0 78.44 cd 100 33 44-1.5 0.125 6 45.34 cd 100 33 39.77 ecd 100 33 44-5 0 1 16.10 c 100 33 23.83 b 100 0 44-5 0 2 10.10 bc 100 0 16.99 b 100 0 44-5 0 3 4.64 b 100 0 6.92 b 100 0 44-5 0.125 1 8.38 bc 100 0 16.37 b 100 0
44-5 0.125 2 3.65 b 100 0 12.38 b 100 0 44-5 0125 3 1.80 ab 100 0 8.48 b 100 0 47-3 0 1 2.24 b 100 0 3.13 ab 100 0 47-3 0 2 0.30 a 100 0 0.25 a 100 0 47-3 0 3 0.09 a 100 0 0.00 a 100 0 47-3 0.125 1 1,70 ab 100 0 5.75b 100 0 47-3 0.125 2 0.52 a 100 33 0.45 a 100 0 47-3 0.125 3 0.45 a 100 0 0.28 a 100 0 " Temperature regimes that simulated daily solarization periods consisted of temperatures increased daily to 350, 410, 440C, 440C or 470C for 8, 5, 1.5, 5 or 3 hours, respectively; the temperature for the remainder of each day was maintained at 250C.
SDays = duration of temperature regime.
" PPG = propagules per gram of soil; initial population was 500 chlamydospores per gram of soil. x I = infection.
Y M = mortality.
z Main effect means followed by the same letter in each test do not differ according to Tukey's Honestly Significant Difference procedure (P<: 0.05); data were transfonned to ln(ppg 1) for analysis and presented as weighted means ([Exp {mean}l-I).






65


Table 3.14. The effect of temperature regimes that simulate daily solarization periods and cabbage amendment, at a soil water matric potential of -30 kPa, on the survival of chlamydospores ofPhytophthora nicotianae, and on the percentages of infection and mortality of tomato seedlings after 30 days of exposure in the previously treated soils. Temperature Cabbage Test 1 Test 2
Regime (%) Days" PPGw Ix MW PPG I M
(%) (%) (%) (%)
35-8 0 1 225.11 e 100 50 90.81 d 100 33 35-8 0 2 190.71 e 100 0 221.96 d 100 0 35-8 0 3 185.98 e 100 17 161.71 d 100 33 35-8 0 6 207.30 e 100 33 114.93 d 100 0
35-8 0125 1 i349.37 e 100 0 137.80 d 100 17 35-8 0.125 2 385.45 e 100 0 385.45 d 100 0 35-8 0.125 3 230.60 e 100 17 107.96 d 100 50 35-8 0.125 6 133.29 de 100 0 63.07 cd 100 100 41-5 0 1 183.93 e 100 0 288.17 d 100 33 41-5 0 3 253.68 e 100 0 153.93 d 100 33 41-5 0 6 240.29 e 100 0 82.18 cd 100 33 41-5 0.125 1 371.78e 100 0 116.20d 100 67 41-5 0.125 3 124.21 de 100 0 80.29 cd 100 67 41-5 0.125 6 85.57 de 100 0 56.34 cd 100 33 44-1.5 0 1 65.75 d 100 0 25.21 c 100 33 44-1.5 0 3 19.41 c 100 0 6.43 b 100 0 44-1.5 0 6 14.77 bc 100 0 6.55 b 100 33 44-1.5 0.125 1 86.01 de 100 0 95.06 d 100 0 44-1.5 0.125 3 53.27 cd 100 0 30.88 c 100 67 44-1.5 0.125 6 42.73 cd 100 33 28.11 c 100 100 44-5 0 1 10.67 bc 100 0 9.86 bc 100 0 44-5 0 2 9.52 bc 100 0 7.35 bc 100 0 44-5 0 3 5.83 b 100 33 4.80 b 100 0 44-5 0.125 1 9.31 bc 100 0 14.25 bc 100 0 44-5 0.125 2 601 b 100 0 8.55 bc 100 0 44-5 0.125 3 3.23 b 100 0 7.66bc 100 0 47-3 0 1 1.90 ab 100 0 1.44 ab 100 0 47-3 0 2 0.09 a 100 0 0.10 a 100 0 47-3 0 3 0.09 a 100 0 0.28 a 100 0 47-3 0.125 1 2.79 b 100 0 4.07 b 100 0 47-3 0.125 2 0.79 a 100 0 1,i15 ab 100 0 47-3 0.125 3 0.91 a 100 0 0.86 ab 100 0 Temperature regimes that simulated daily solarization periods consisted of temperatures increased daily to 350, 410, 440C, 440C or 470C for 8, 5, 1.5, 5 or 3 hours, respectively; the temperature for the remainder of each day was maintained at 250C.
Days = duration of temperature regime.
* PPG = propagules per gram of soil: initial population was 500 chlamydospores per gram of soil. SI = infection.
Y M = mortality.
z Main effect means followed by the same letter in each test do not differ according to Tukey's Honestly Significant Difference procedure (P<_ 0.05); data were transformed to ln(ppg+ 1) for analysis and presented as weighted means ([Exp {mean})-l).






66



for 5 hours daily) were significantly lower than those in the base temperature regime of 350C for 8 hours daily, and generally lower than those treated at 410C for 5 hours daily, regardless of the soil water matric potential (P<0.05).

The use of a plant disease assay to confirm the pathogenicity of the surviving population of P. nicotianae indicated that, when the soil was maintained at 0 kPa and treated at 470C for 3 hours daily, there was a reduction in infection of the seedlings in test

1 only (Table 3.12). In all other treatments under all three matric potentials, all of the seedlings were infected by P. nicotianae (Tables 3.12, 3.13, 3.14). A higher proportion of mortality of seedlings was observed in the second experiment than in the first experiment; however, little or no mortality occurred at any soil water matric potential when the soil was treated at 440C for 5 hours daily or at 470C for 3 hours daily.

Over all treatments, in both tests, soil water matric potential had a significant effect in reducing the populations of P. nicotianae in the soil (P<0.05) (Table 3.15; Appendix D). In both tests the lowest survival was observed in saturated soils (0 kPa) (Tables 3.12, 3.13, 3.14; Appendix D). No differences were observed in the survival of chlamydospores in soils maintained at either -10 kPa or -30 kPa in test 1; however, in test

2 survival at -10 kPa was higher than at -30 kPa.

As temperature increased, survival of P. nicotianae decreased (Table 3.15;

Appendix D), and survival at each temperature regime was significantly different from that in all others (P 0.05). Survival was lowest in soil maintained at 47'C for 3 hours daily (Appendix D).

The incorporation of cabbage amendment into soil significantly reduced the

populations of P. nicotianae in test 1, while no effect on the survival of the pathogen was observed in test 2 (P50.05) (Table 3.15, Appendix D). However, differences in populations between amended and nonamended soil exposed to specific temperature regimes were not evident from the analysis (Tables 3.12, 3.13, 3.14).






67


Table 3.15. Analysis of variance of the effect of temperature regimes that simulate solarization periods, cabbage amendment, and soil water matric potential on the survival of chlamydospores of Phytophthora nicotianae Source of Variation Test 1 Test 2 df MS F P MS F P Soil water matric potential (!V.)" 2 1.355 15.93 0.0001 1.967 16.68 0.0001 Temperatures 4 264.897 3114.24 0.0001 210.227 1782.67 0.0001 Cabbagey 1 0.924 10.87 0.0001 0.001 0.01 0.9149 Timez 3 6.740 79.24 0.0001 11.725 99.43 0.0001 xm xTemperature 8 1.096 12.89 0.0001 1.366 11.59 0.0001 Tkm x Cabbage 2 1.827 21.48 0.0001 6.789 57.57 0.0001 m x Time 6 0.375 4.41 0.0003 0.133 1.13 0.3442 Temperature x Cabbage 4 4.658 54.77 0.0001 8.255 70.01 0.0001 Temperature x Time 8 0.873 10.26 0.0001 2.024 17.16 0.0001 Cabbage x Time 3 0.643 7.57 0.0001 0.036 0.30 0.8230 Temperature x m x Cabbage x 54 0.225 2.65 0.0001 0.265 2.25 0.0001 Time
Residual 228 0.085 0.118 " Soil water matric potential adjusted to 0, -10, or -30 kPa. x Temperature regimes that simulated daily solarization periods consisted of temperatures increased daily to 350, 410, 44oC, 44oC or 47oC for 8, 5, 1.5, 5 or 3 hours, respectively; the temperature for the remainder of each day was maintained at 250C. Y Soil amended or not amended with dry, ground cabbage at a rate of 0.125% (w/w). Time = number of days of exposure to heat treatment at a given temperature regime.






68


Generally, the longer the soil infested with chlamydospores of P. nicotianae was exposed to the heat treatments, the lower the survival of the pathogen (Tables 3.12, 3.13,

3.14, 3.15). However, the effect of time has to be analyzed with care because of the different durations of exposure to the temperature regimes. Because the time required to kill chlamydospores is inversely proportional to temperature, shorter sampling times (1, 2, and 3 days) were used at the higher temperature regimes (470C for 3 hours daily and 440C for 5 hours daily) and longer sampling times (1, 3, and 6 days) were used at the average temperature regimes (440C for 1.5 hours daily and 410C for 5 hours daily); the base temperature (350C for 8 hours daily) was sampled at all time intervals (1, 2, 3, and 6 days). Therefore, the analysis of the effect of time across all other factors is biased in the following manner: the data for 2 days of exposure was obtained only from the temperature regimes that simulate optimum solarization periods and the base temperature regime; after 3 days of heat treatment, a final sample was collected from the optimum temperature regimes, and intermediate samples were collected from the average temperature regimes; after 6 days only the average temperature regimes and the base temperature regime were evaluated. Consequently, artificially higher survival rates were generated at 6 days as compared to 2 or 3 days because the lowest survival rates under the optimum temperature regimes had not been determined (Appendix D). The interactions of soil water matric potential or cabbage with time reflect the same bias, and the responses followed trends similar to those described here for time alone. Although these two interactions were significant (Table 3.15), the biased results did not allow insight into the roles of soil water matric potential or cabbage amendment in relation to time of exposure on the survival of the pathogen.

Within each of the soil water matric potentials evaluated, each of the temperature regimes significantly reduced the populations ofP. nicotianae in relation to the base temperature (35'C for 8 hours daily) (P<0.05) (Tables 3.12, 3.13, 3.14, 3.15; Appendix






69


D). Survival ofP. nicotianae at each temperature regime was significantly lower than that at all other preceding temperature regimes.

Although there was a significant interaction of soil water matric potential and cabbage amendment, the effect of the amendment on survival of P. nicotianae was not consistent throughout the range of soil water matric potentials evaluated (P<:0.05) (Tables

3.12, 3.13, 3.14, 3.15; Appendix D). Cabbage amendment reduced the survival of propagules in soil maintained at 0 kPa (Appendix D). However, when the soil was maintained at -30 kPa, higher survival of the pathogen was observed in soil amended with cabbage than in nonamended soil. At -10 kPa, the addition of cabbage to the soil had no impact on the survival of P. nicotianae in test 1, while in test 2 survival was greater in soil amended with cabbage than in nonamended soil.

The effect of cabbage amendment on survival of P. nicotianae within each

temperature regime was significant, but not consistent (P<0.05) (Table 3.15; Appendix D). At the two lower temperature regimes (35'C for 8 hours and 410C for 5 hours daily) the addition of cabbage to the soil reduced the number of propagules of P. nicotianae recovered on the selective medium (Tables 3.12, 3.13, 3.14). At 440C for 1.5 hours, and 470C for 3 hours daily, a higher proportion of the population of P. nicotianae survived in soil that was amended with cabbage than in nonamended soil. In test 1, at 440C for 5 hours daily the number of propagules of the pathogen recovered was lower in the soil amended with cabbage than in nonamended soil; in contrast, in test 2 there were no differences in the survival of P. nicotianae in soils amended or nonamended with cabbage.

Generally, longer exposure of the infested soil to each temperature resulted in lower recovery of propagules (Tables 3.12, 3.13, 3.14, 3.15; Appendix D). At the base temperature regime of 350C for 8 hours daily, the lowest survival was observed after 3 or

6 days. In test 1 no differences in survival were observed between 3 and 6 days or between 1 and 2 days of exposure to the heat treatment. In contrast, in test 2, the highest





70


survival was observed after 2 days of exposure, followed by 1 and 3 days of exposure to the heat treatment. When average temperature regimes were employed, the reduction in survival was not as consistent as with the optimum temperature regimes. At 41 0C for 5 hours daily, and 440C for 1.5 hours, survival decreased with increasing length of exposure in test 1; however, in test 2 no differences were observed between 3 and 6 days of exposure, in which survival was lower than after exposing the soil to 1 day of heat treatment. As time progressed, lower recovery was observed at 440C for 5 hours daily. Exposing the chlamydospore-infested soil to either 2 or 3 days at 470C for 3 hours daily resulted in lower survival as compared with a 1-day exposure.



The Effect of Three Nonpasteurized Soils, Temperature Regimes, and Cabbage Amendments on the Thermal Inactivation of Chlamydospores of Phytophthora nicotianae


Soils from the three sites where soil solarization was tested were used in this study. In all three soils the two higher temperature regimes (440C for 5 hours and 470C for 3 hours daily) were generally more effective in the inactivation of chlamydospores of P. nicotianae than the two lower regimes (350C for 8 hours and 410C for 5 hours daily) (Tables 3.16, 3.17, 3.18). Very few propagules were recovered from soil from Site 1 after

9 days at the temperature regime of 470C for 3 hours daily, and no propagules were recovered from the other soils after this treatment.

Few tomato seedlings died after 30 days of incubation in the treated soils (Table

3.16, 3.17, 3.18). However, almost all seedlings were infected in all treatments, except at the highest temperature regime of 470C for 3 hours daily. At the highest temperature regime, infection was observed in the second test, even in treatments where no propagules were recovered. In contrast, in the first test, no infection of the root system










Table 3.16. The effect of pasteurization of soil from Site 1 (NTG farm, Decatur County, Georgia), temperature regimes, and cabbage amendment on the survival of chlamydospores of Phytophthora nicotianae, and on the percentages of infection and mortality of tomato seedlings after 30 days of exposure in the previously treated soils.
Test 1 Test 2
Temperature Pasteurized soil Nonpasteurized soil Pasteurized soil Nonpasteurized soil Regime' Cabbage Time PPGv 1w MX PPG I M PPG I M PPG I M
(%) (days) (%) (%) (%) (%) (%) (%) (%) (%) 35-8 0 3 15.6 bcdy ntz nt 34.6 cd nt nt 3.7 bc nt nt 109.8 d nt nt 35-8 0 9 29.4 cd nt nt 102.2 d nt nt 107.2 d nt nt 71.5 d nt nt 35-8 0.125 3 66.0 d nt nt 62.4 d nt nt 8.7 bc nt nt 139.6 d nt nt 35-8 0.125 9 27.0 cd nt nt 69.1 d nt nt 186.7 d nt nt 149.7 d nt nt 41-5 0 3 97.3 d 100 0 16.7 d 100 0 79.2 d 100 0 92 *d 100 0 41-5 0 9 412 cd. 100 0 81.4 d 100 0 54.7 d 100 0 77.1 d 100 0 41- 0.125 3 13.5 bcd :100 0 82.1 d 100 0 79.9 d 100 0 84.8 d 100 0 41-5 0.125 9 5.9 be. 100 0 23.8 cd 100 0 52.3 d 100 0 70.4 ed 100 0 44-1.5 0 3 11.2 bcd 100 0 27.8 cd 100 100 53.2 d 100 33 60.9 d 100 67 44-1.5 0 9 2.9 abc 100 33 13.5 bcd 100 0 49.8 cd 100 0 50.7 cd 100 0 44-1.5 0.125 3 3.1 abc 100 0 12.0 bcd 100 33 8.1 be 100 0 13.7 bcd 100 0 44-1.5 0.125 9 2.4 abc 100 33 5.7 bc 100 67 7.1 bc 100 0 6.8 b 100 0 44-5 0 3 1.3 ab 100 67 2.9 abc 67 0 128 bed 100 0 99 bc 67 33 44-5 0 .9 0.1 ab i100 0 0.5 ab 100 0 6.9 be 100 0 2.5 ab 100 0 44-5 0.125 3 1.1 ab 100 0 1.4 ab 100 0 1.2 ab 100 0 0.9 a 33 0 44-5 0.125 9 0.3 ab 100 33 0.5 ab 33 0 0.7 a 100 0 0.0 a 33 0 47-3 0 3 0.0 ab 0 0 0.1 ab 0 0 0.2 a 100 0 0.0 a 0 0 47-3 0 9 0.0 ab 0 0 0.0 ab 0 0 0.1 a 100 0 0.0 a 0 0 47-3 0.125 3 0.2 ab 0 0 0.1 ab 0 0 0.1 a 100 0 0.0 a 50 0 47-3 0.125 9 0.0 ab 0 0 0.0 ab 0 0 0.0 a 100 0 0.0 a 100 0 ' Temperature regimes that simulated solarization consisted of temperatures increased daily to 350, 4 1, 440, 440, or 470C for 8, 5, 1.5, 5 or 3 hours, respectively; the temperature for the remainder of each day was maintained at 250C. Days = duration of temperature regime.
PPG = propagules per gram of soil; the soil was initially infested with 500 chlamydospores per grain. w I = infection.
xM = mortality.
Y Main effect means followed by the same letter in each column do not differ according to Tukey's Honestly Significant Difference procedure (P< 0.05); data were transformed to ln(ppg+1) for analysis and presented as weighted means ([Exp {mean}l-1). Z nt = not tested.









Table 3.17. The effect of pasteurization of soil from Site 2 (John Allen Smith Farm, Gadsden County, Florida), temperature regimes, and cabbage amendment on the survival of chlamydospores of Phytophthora nicotianae, and on the percentages of infection and mortality of tomato seedlings after 30 days of exposure in the previously treated soils.
Test 1 Test 2
Temperature Pasteurized soil Nonpasteurized soil Pasteurized soil Nonpasteurized soil Regime' Cabbage Timeu PPGV I1 Mx PPG I M PPG I M PPG I M
(%) (days) (%) (%) (%) (%) (%) (%) (%) (%) 35-8 0 3 16.6 bcy ntz nt 36.2 bc nt nt 14.2 cd nt nt 107.3 d nt nt 35-8 0 9 44.2 c nt nt 45.8 c nt nt 47.3 d nt nt 50.1 d nt nt 35-8 0.125 3 54.3 c nt nt 8.2 abc nt nt 1.7 ab nt nt 23.4 bcd nt nt 35-8 0.125 9 63.8 c ut nt 22.4 bc nt nt 115.2 d nt nt 69.7 d nt nt, 41-5 0 3 84.2 c 100 0 95,3 c 100 0 39.l d 100 379 ed 100 0 41-5 0 9 46.3 c 100 33 43.7 c 100 0 31.9 bcd 100 33 357 bcd 100 0 :1-5 0.125 3 1.4 ab 100 0 10.6 abe 100o 3.0 bc: 100 10.5 bad 100 0 41-5 0.125 9 3.2 ab 100 0 9.3 abc 100 0 2.9 bc 100 0 10.0 bed 100 0 44-1.5 0 3 23.1 c 100 0 35.1 bc 100 67 10.8 bcd 100 0 12.1 bcd 100 0 44-1.5 0 9 6.9 abc 100 0 9.0 abc 100 0 8.9 bcd 100 0 10.5 bcd 100 0 44-1.5 0.125 3 3.4 ab 100 0 3.7 ab 100 0 2.4 abc 100 33 5.3 bc 100 33 44-1.5 0.125 9 4.0 abc 100 0 1.9 ab 100 33 1.0 a 100 0 6.8 bc 100 0 44-5 0 3 . b 0 0 0.5 a 100 33 1.0 a 100 0 0.5 a 100 o 44-5 0 9 0.Oa 100 0.0 a 67 0 0.0 a 100 0 0.2a 100 0 44-5 0.125 3 ,0 a 100 0 0.6 ab 100 0 00 a 100 0 0.4 a 100 44-5 0.125 9 0.0 a 100 0 0.0 a 100:.: 0 0.0 a 100 0 2 a 100 0 47-3 0 3 0.0 a 0 0 0.0 a 0 0 0.0 a 100 33 0.0 a 33 0 47-3 0 9 0.0 a 0 0 0.0 a 0 0 0.0 a 100 0 0.0 a 0 0 47-3 0.125 3 0.0 a 0 0 0.0 a 0 0 0.0 a 100 33 0.0 a 0 0 47-3 0.125 9 0.0 a 0 0 0.0 a 0 0 0.0 a 100 33 0.0 a 0 0 ' Temperature regimes that simulated solarization consisted of temperatures increased daily to 350, 41 , 440, 440, or 470C for 8, 5, 1.5, 5 or 3 hours, respectively; the temperature for the remainder of each day was maintained at 250C. " Days = duration of temperature regime.
' PPG = propagules per gram of soil; the soil was initially infested with 500 chlamydospores per gram.
* I = infection.
M = mortality.
Y Main effect means followed by the same letter in each column do not differ according to Tukey's Honestly Significant Difference procedure (P<_ 0.05); data were transformed to ln(ppg+1) for analysis and presented as weighted means ((Exp {mean}]-l). Z nt = not tested.









Table 3.18. The effect of pasteurization of soil from Site 3 (NFREC-Quincy, Gadsden County, Florida), temperature regimes, and cabbage amendment on the survival of chlamydospores of Phytophthora nicotianae, and on the percentages of infection and mortality of tomato seedlings after 30 days of exposure in the previously treated soils.
Test 1 Test 2
Temperature Pasteurized soil Nonpasteurized soil Pasteurized soil Nonpasteurized soil Regime' Cabbage Time PPGv' 1 Mx PPG I M PPG I M PPG I M
(%) (days) (%) ) (%) (%) (%) (%) (%) (%) (%) 35-8 0 3 46.80 cdy ntz nt 37.47 cd nt nt 1000.24 e nt nt 123.84 cd nt nt 35-8 0 9 60.19 d nt nt 39.81 cd nt nt 237.88 d nt nt 137.10 cd nt nt 35-8 0.125 3 62.50 d nt nt 13.61 bcd nt nt 306.97 de nt nt 192.06 cd nt nt 35-8 0.125 9 21.46 cd nlt nt 0.00 ab it nt 567.50 e nt nt 138.49 cd nt nt 4105 0 3 93.07 d 100 0 7009 d 100 0 120.02 cd :100 0 9958 cd 100 33 41-5 0 9 34.98 cd 100 0 45.99 cd 100 0 91.20 cd 100 33 73.51 cd 100 0 41-5 0.125 3 26,55 cd 100 0 8.40 cd 100 0 110.83 ed 100 0 32,72 bcd 100 0 .41-5 0.125 9 17.89 cd 100 0 36.15 cd 100 0 78.52 cd 100 0 3.08 b 100 33 44-1.5 0 3 25.08 cd 100 33 38.21 cd 100 67 54.26 cd 100 33 41.65 bcd 100 33 44-1.5 0 9 9.25 bcd 100 0 23.80 cd 100 67 52.09 cd 100 33 45.34 bcd 100 33 44-1.5 0.125 3 22.08 cd 100 67 26.77 cd 100 33 29.72 bcd 100 0 1.80 a 100 0 44-1.5 0.125 9 7.55 bcd 100 33 18.43 cd 100 67 21.04 bc 100 33 1.80 a 67 33 44-5 0 3 2.28 abc 100 0 6.46 bcd 100 0 11.58 b 100 13 2,98 b 100 0 44-5 0 9 0.23 ab 100 0 1.39 abc 33 0 2.05 a 100 67 0.15 a 100 0 44-5 0.125 3 0.00 ab 100 0 0.25 ab 33 0 1.33 a 100 33 0.96 a 100 0 44-5 0.125 9 0.00 ab 100 0 0.13 ab 33 0 0.62 a 100 33 0.15 a 100 0 47-3 0 3 0.00 ab 0 0 0.00 ab 0 0 0.28 a 0 0 0.00 a 0 0 47-3 0 9 0.00 ab 0 0 0.00 ab 0 0 0.00 a 0 0 0.00 a 0 0 47-3 0 125 3 0.00 ab 0 0 0.14 ab 0 0 0.08 a 0 0 0.00 a 67 0 47-3 0.125 9 0.00 ab 0 0 0.00 ab 0 0 0.00 a 0 0 0.00 a 33 0 t Temperature regimes that simulated solarization consisted of temperatures increased daily to 350, 410, 440, 440, or 470C for 8, 5, 1.5, 5 or 3 hours, respectively: the temperature for the remainder of each day was maintained at 250C. Days = duration of temperature regime.
PPG = propagules per gram of soil; the soil was initially infested with 500 chlamydospores per gram. " I = infection
xM = mortalty.
Y Main effect means followed by the same letter in each column do not differ according to Tukey's Honestly Significant Difference procedure (P< 0.05): data were transformed to ln(ppg+l) for analysis and presented as weighted means (IExp {mean}l-1). z nt = not tested.






74


was observed in the highest temperature regime, even in treatments from which a few propagules had been recovered.

The numbers of propagules recovered from the soil from site 2 were generally lower than the numbers recovered from the other two soils in both tests (Tables 3.16,

3.17, 3.18, 3.19; Appendix E). No differences in survival were observed between sites 1 and 3 in test 1, while survival was higher in soils from site 3 than in soils from site 1 in test 2 (P<0.05).

The effect of pasteurization of the soil on the survival of P. nicotianae was

significant in test 1; however, no differences in survival were observed in test 2 (Table

3.19). Populations were lower in pasteurized soil than in nonpasteurized soil in test 1 (P<0.05) (Appendix E).

Lower numbers of propagules were recovered from the soils as temperature

regimes increased (Tables 3.16, 3.17, 3.18, 3.19; Appendix E). Temperature regimes of 440C for 1.5 hours or higher were more effective than 350C for 8 hours or 410C for 5 hours at reducing the populations of P. nicotianae in test 1 (P50.05) (Appendix E). In test 2, survival under each temperature regime was significantly lower than that at each preceding regime.

The incorporation of cabbage amendment into soil significantly reduced the

populations of P. nicotianae in the soil (Table 3.19; Appendix E). However, differences in populations between amended and nonamended soil exposed to specific temperature regimes were not evident from the analysis (Tables 3.16, 3.17, 3.18).

The duration of the heat treatment significantly affected survival of P. nicotianae in test 1, but not in test 2 (Table 3.19). Significantly fewer propagules were recovered in the selective medium after 9 days of heat treatment than after 3 days in test 1 (P<0.05) (Appendix E); in contrast, no differences in survival between the two sampling dates were observed in test 2.






75


Table 3.19. Analysis of variance of the effects of three soils, pasteurization, temperature regimes that simulate daily solarization periods, cabbage amendment, and time on the survival of chlamydospores ofPhytophthora nicotianae. Source of variation Test 1 Test 2 df MS F P MS F P Soil' 2 3.88 19.64 0.0001 36.18 267.87 0.0001 Pasteurization" 1 3.33 16.81 0.0001 0.35 2.59 0.1086 Temperatures 4 190.82 964.25 0.0001 249.99 1850.83 0.0001 Cabbagey 1 36.19 182.88 0.0001 38.69 286.47 0.0001 Time 1 10.74 54.27 0.0001 0.44 3.25 0.0725 Soil x Pasteurization 2 4.09 20.66 0.0001 14.05 104.04 0.0001 Soil x Temperature 8 2.50 12.65 0.0001 5.75 42.58 0.0001 Soilx Cabbage 2 1.71 8.64 0.0001 0.16 1.18 0.3105 Soil x Time 2 0.75 3.77 0.0245 2.73 20.20 0.0001 Temperature x Cabbage 4 6.91 34.94 0.0001 7.58 56.09 0.0001 Temperature x Time 4 1.61 8.16 0.0001 5.36 39.69 0.0001 Cabbage x Time 1 0.34 1.74 0.1889 1.29 9.58 0.0022 Soil x Pasteurization x 87 1.01 5.12 0.0001 1.19 8.78 0.0001 Temperature x Cabbage x Time
Residual 240 0.20 0.13 " Soils used were from Site 1 (Naples Tomato Growers-Gargiulo farm number 4 in Decatur County, Georgia), Site 2 (John Allen Smith farm located in Gadsden County, Florida), and Site 3 (North Florida Research and Educational Center in Gadsden County, Florida).
* Soils (1-kg lots) were either nonpasteurized or pasteurized in a microwave oven at 700 W for 4 minutes, after moisture had been adjusted to 5% (w/w).
x Temperature regimes that simulated solarization consisted of temperatures increased daily to 350, 410, 440, 440, or 470C for 8, 5, 1.5, 5 or 3 hours, respectively; the temperature for the remainder of each day was maintained at 250C.
Y Soil amended or not amended with dry, ground cabbage at a rate of 0.125% (w/w). z Time = number of days of exposure to heat treatment at a given temperature regime.





76


A significant interaction between soils and pasteurization was observed in both tests (Table 3.19). In test 1, no differences in survival of P. nicotianae were observed between pasteurized and nonpasteurized soils from sites 2 and 3, and survival was higher in nonpasteurized than pasteurized soil from site I (P_0.05) (Appendix E). In test 2, survival was higher in nonpasteurized than pasteurized soil from sites 1 and 2, and lower in pasteurized soil from site 3.

The interaction of soils with temperature regimes was significant in both tests

(Table 3.19). Generally, survival of P. nicotianae decreased with increasing temperatures in regimes in each of the three soils tested (Tables 3.16, 3.17, 3.18; Appendix E).

The effect of cabbage amendment on the survival of P. nicotianae within each soil was significant in test 1, but not in test 2 (P<0.05) (Table 3.19). Within each soil the addition of cabbage amendment lowered the number of propagules recovered from the soils (Appendix E).

The effects of interactions of soils with duration of heat treatment on the survival of populations of P. nicotianae were significant (Table 3.19). In test 1, lower survival was found after 9 days of heat treatment than after 3 days in soils from sites 1 and 3; no differences in survival between the two sampling dates were observed in soils from site 2 (P<0.05) (Appendix E). Lower population levels were detected after 9 days of heat treatment than after 3 days in soils from site 3 in test 2; no differences in survival were observed between the two sampling dates in soils from either site 1 or from site 2 in test

2.

The effect of cabbage amendment on survival of P. nicotianae within each

temperature regime was significant (Table 3.19). In test 1, cabbage amendment reduced the survival of P. nicotianae, except at 47'C for 3 hours (P<0.05) (Appendix E). In test 2, no differences in survival were found at 35'C for 8 hours or 470C for 3 hours daily; at all other temperature regimes the cabbage amendment reduced the populations of P. nicotianae.





77



The influence of the interaction of temperature regimes and time on survival was significant for both tests (Table 3.19). No propagules were recovered after either sampling time at 470C for 3 hours daily in both tests (P<0.05) (Appendix E). Lower recovery of P. nicotianae was found after 9 days than after 3 days of heat treatment at 440C for 1.5 hours and at 440C for 5 hours in test 1; no differences in survival between the sampling times were observed at 350C for 8 hours, 410C for 5 hours or 470C for 3 hours. In test 2, significantly fewer spores germinated in the selective medium after 9 days of heat treatment than after 3 days in all temperature regimes, except at 440C for 1.5 hours.

The interaction of cabbage amendment and time in reducing populations of P.

nicotianae was significant only in test 2 (Table 3.19). Within each cabbage concentration fewer spores were recovered in test 1 after 9 days than after 3 days (P<0.05) (Appendix E). In contrast, no differences were observed when cabbage was added to the soils in test

2.


Discussion


The findings of this study are in general agreement with related work on the

thermal inactivation of spores of Phytophthora spp. (Barbercheck and Von Broembsen, 1986; Benson, 1978; Bollen, 1985; Juarez-Palacios et al., 1991). Bollen (1985) reported that a soil culture of P. cryptogea required 30 minutes at 450C to be completely inactivated, and a soil culture of P. capsici had to be heated to 500C for 30 minutes before oospores was killed. Benson (1978) found that culture disks of P. cinnamomi containing chlamydospores were killed after 90 minutes at 390C or 4.5 minutes at 440C; in contrast, Barbercheck and Von Broembsen (1986) noted that a suspension of chlamydospores of P. cinnamomi in water was inactivated after 10 minutes at 440C. The differential heat sensitivity of isolates of the same species was demonstrated by Juarez-Palacios et al.





78



(1991). These authors found that chlamydospores of P. cinnamomi or oospores of a lowtemperature isolate of P. megasperma added to soil did not survive 20 minutes at 450C; in contrast, a high-temperature isolate of P. megasperma survived more than 30 minutes at the same temperature. The discrepancies among the values reported in the literature could be related to the different types of substratum used to produce the spores and to the different media used for the heat treatment in each study. It is expected that spore suspensions in water would be inactivated faster than soil cultures saturated with water, which in turn would die faster than spores added to soil at a lower soil water matric potential. These assumptions are based on the differential transmission of heat throughout the substrata, the formation of air pockets, or simply due to the nature of the spores formed in each substratum, as noted by Myers et al. (1983) and Katan (1981).

The use of heat to inactivate spores of microorganisms in diverse media began as early as 1920 (Bigelow and Esty, 1920). Soon after that the advantages of the logarithmic transformation for the analysis of the data also were realized (Bigelow, 1921; Smith, 1923). The logarithmic transformation indicated that a constant proportion of the spore population was killed per unit of time, and also served the purpose of linearizing the data. Simple linear regressions, then, could be used for the analysis of the effect of time on the survival of spores at each temperature employed; or, coefficients for lethal dosages (LDs or LD,,) could be interpolated from the data. Pullman et al. (1981a) demonstrated that the logarithmic relationship between time and temperature on the survival of four plant pathogens was maintained at temperatures below 500C. However, Anderson et al. (1996) and Cole et al. (1993) pointed out that the use of the 'log-linear' model assumes that all spores in a population have equal heat sensitivity and that death of an individual is dependent on it receiving sufficient heat. Deviations from this basic assumption have been observed and a vitalistic model has been proposed by these authors. The concept in this theory is that individuals in a population do not have identical heat resistances and that these differences are permanent. It is possible that a better understanding of the





79


survival of P. nicotianae would be achieved by using the vitalistic model described by Cole et al. (1993).

In the present study the relationship of time and temperature on the survival of P. nicotianae was evaluated over a series of temperatures and times (350 to 530C for 5 to 120 minutes, and 350 to 470 for 2 to 480 hours), for which a surface response approach allowed the simultaneous analysis of the effects of both time and temperature on the survival of the pathogen. The models that best described survival of P. nicotianae were linear-exponential or exponential.

The analysis of the effect of temperature regimes on the survival of P. nicotianae indicated that a single model is not adequate to describe the changes in survival over time as temperature increased. At the base temperature regime (350C for 8 hours daily), none of the models accurately described survival, which was little affected by the temperature regime. As temperature increased, the change in survival was best explained by other models, such as an exponential model, then a model of square root of either time or survival, then a logarithmic model of time, and finally a model of the reciprocal of time. The changes in spore survival that resulted in these models may have occurred as constant numbers of spores died per unit time at lower temperatures, a constant proportion of the population was killed per unit time at intermediate temperatures, and larger proportions of populations were inactivated early during the exposure to the highest temperature.

The use of tomato seedlings as baits to detect survival of P. nicotianae in the

treated soil demonstrated that this method can be as sensitive as the soil dilution plating procedure, except when residual populations are present (less than 1 propagule per gram of soil). The reasoning for using the baiting test was that heat treatment at temperatures that did not eliminate the pathogen from the soil could possibly have affected the ability of the surviving population to infect and colonize a susceptible host, or to germinate in the presence of an external stimulus, such as root extracts. However, it is clear from these





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tests that if P. nicotianae is present in the soil, even in residual levels, infection of a tomato seedling can occur. The baiting technique has the following disadvantages: population of the pathogen are not quantified, extensive amounts of time and space may be required to grow the seedlings and incubate the baited soil, and the soil samples may be cross contaminated due to handling procedures.

Amending infested soils with dried, ground cabbage leaves and exposing it to heat treatment generally reduced survival of P. nicotianae in the present study. The effectiveness of cabbage amendment could be observed in two ways: first, at a given temperature, less time was required to achieve the same reduction in population as cabbage concentration increased; second, with increasing concentrations of cabbage amendment, lower temperatures were required to achieve the same reduction in populations of the pathogen. Populations of P. nicotianae were eliminated after 24 hours at 410C or after 12 days at 38' and 350C in soil amended with 0.5% cabbage; after 4 days at 410C or after 20 days at 350C only residual populations of the pathogen were detected. After 12 days at 380C no propagules were detected in soils amended with 0.125% or

0.25% cabbage. These findings contrast with the results obtained in the field work, where no additional reductions in populations of P. nicotianae were achieved with cabbage amendments. The amount of cabbage incorporated into the soil in the field experiments ranged from 0.125% to 0.25%; however, leaf pieces were much larger and during the drying period may have been rained on, which could have started a decay process with subsequent loss of volatiles. Furthermore, incorporation of the amendment into the field soil was done by disking, and it could not be determined if uniform distribution throughout the soil profile was attained.

The use of cabbage amendment without any additional soil heating has been

shown to be effective for the control of Aphanomyces euteiches (Lewis and Papavizas, 1971), or inconsistent for control of Fusarium oxysporum f. sp. conglutinans (RamirezVillapudua and Munnecke, 1987, 1988). Heating cabbage amended soils induces the





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volatilization of certain compounds that are fungicidal, as demonstrated by Gamliel and Stapleton (1993b). These authors determined that heating soil amended with cabbage released volatile compounds, such as methanol, isothiocyanates and aldehydes, which could be directly correlated with the inactivation of the spores of Pythium ultimum and sclerotia of Sclerotium rolfisii. The effect of cabbage amendment appears to be directly proportional to its concentration in the soil, as observed by Ramirez-Villapudua and Munnecke (1988). These authors found that increasing the concentration of cabbage from

0.25% to 2% reduced the population ofF. oxysporum f. sp. conglutinans to undetectable levels in 12 days instead of the 30 days required at the lower concentration. Other crucifers, such as kale (Brassica oleracea var. viridis) and mustard (B. nigra) were also effective for the control F. oxysporum f. sp. conglutinans. Another factor that may determine the effectiveness of the crucifer amendment is the concentration of glucosinolates present in the crop (Mayton et al., 1996). These authors found that some cultivars of Brassica spp. were more effective than others in the control ofF. sambucinum. Even though Ramirez-Villapudua and Munnecke (1988) demonstrated that dried cabbage residue is more effective than fresh residue for the control ofF. oxysporum f. sp. conglutinans, other Brassica spp. may be more effective as fresh amendment, as demonstrated by Subbarao and Hubbard (1996) with broccoli (B. oleracea var. botrytis) and Verticillium dahliae.

One major benefit of the use of intermittent heat in thermal inactivation studies is the provision of estimations of the effectiveness of soil solarization to control plant pathogens. However, cycling temperatures have not been employed routinely, possibly due to the compounded difficulties of establishing appropriate temperature regimes, the daily requirement of adjusting each temperature, and the longer time required to reach desirable control of the organism under study. Examples of the use of pulsing temperatures are provided by Gamliel and Stapleton (1993b), Porter and Merriman (1983), Tjamos and Fravel (1995) and Wicks (1988).





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Wicks (1988) analyzed the effect of intermittent heat on the survival of mycelium of Phytophthora cambivora. None of the isolates tested survived after 1 day at a regime of 450C for 6 hours and 200C for 18 hours daily. The response of the isolates was variable and inconclusive at either 400 or 35'C for 6 hours daily during 4 days. However, mycelium is not the most likely survival structure of P. cambivora in the soil.

Tjamos and Fravel (1995) evaluated intermittent heat on the survival of a

suspension of microsclerotia of Verticillium dahliae over 4 days. The following daily temperature regimes were used: base temperature of 31 C for 10 hours and high temperature at 350C for 14 hours, base temperature of 330C for 10 hours and high temperature at 36'C for 14 hours, and base temperature of 350C for 10 hours and high temperature at 380C for 14 hours. After 4 days at the highest temperature regime, less than 1% of the sclerotia germinated. In their study the use of sclerotium suspensions in water negated the insulating effect of air pockets in the soil, and the duration of the high temperature in each regime was longer than that which would normally occur under field conditions.

A more comprehensive study was done by Porter and Merriman (1983) with

several soilborne plant pathogens. These authors used infested soil held at field capacity, and over 15 days evaluated cycles of low temperature at 250C for 18 hours, followed by 6 hours daily at a supplemental temperature of 250, 30', 350, 400, 450 or 50'C. Survival of each pathogen depended on the heat sensitivity of the type of propagule being evaluated. For example, V. dahliae did not survive for 15 days when the high temperature was above 400C; in contrast, Pythium irregulare was recovered at 5x104 propagules per gram of soil at 500C, even at the end of the experiment.

Gamliel and Stapleton (1993b) selected two temperature regimes similar to those found in the San Joaquin Valley in California to evaluate the effectiveness of the regimes and cabbage amendment on the control of P. ultimum and S. rolfsii. Both pathogens were eliminated after 4 days in the cabbage amended soil at either temperature regime of 380C





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or 450C for 4 hours daily plus 20 hours at 300C; however, propagules of these pathogens could be recovered from the nonamended soil after 4 days at the same temperature regimes.

Comparisons between studies are further complicated due to the differences in

length of the experiments, amplitude between low and high temperatures in each regime, and the duration of the high temperature. If a given in vitro experiment is to be compared with soil solarization in the field, then the duration of the in vitro study should be similar to the solarization trial, and the temperature regimes should simulate those observed during solarization.

In the present study populations ofP. nicotianae decreased to residual levels after 15 days only when temperature regimes simulating optimum solarization conditions (470C for 3 hours daily) were used; under these circumstances no infection of tomato seedlings was observed after as little as 3 days of heat treatment. The use of 44oC for 5 hours daily also reduced populations to levels below 1 propagule per gram of soil; however, infection of tomato seedlings exposed to this regime was observed throughout the experiment. The use of temperature regimes simulating average field temperature regimes reduced the populations to at least 40% of the initial infestation level, but all seedlings were colonized at these regimes.

Athough the effect of soil moisture on reproduction and dispersal of Phytophthora spp. has been researched extensively (Browne and Mircetich, 1988; Ferrin and Mitchell, 1986; Lutz and Menge, 1991; McIntosh, 1972; Neher and Duniway, 1992; Ristaino et al., 1992; Sidebottom and Shew, 1985a, 1985b), the effect of soil moisture and heat on the inactivation of spores of this genus has not been addressed before. Survival of chlamydospores of P. nicotianae was lower in saturated soil (0 kPa) than at the two other soil water matric potentials evaluated (-10 and -30 kPa). The two lower soil water matric potentials (-10 and -30 kPa) used for this study are close to what is normally considered field capacity of a soil and they represent an optimum for thermal inactivation studies.





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However, in field studies, these conditions are very difficult to maintain for the time required for soil solarization without supplemental irrigation.

Porter et al. (1991) evaluated the effect of two moisture contents (field capacity and 10% of field capacity) and constant temperature on survival of Plasmodiophora brassicae in two soils. Only 15 days were required to kill all spores of P. brassicae in either soil at 400C or above when the soil moisture was at field capacity. In dry soil, however, inactivation was observed only at 500 or 550C.

The effect of soil water matric potential on the thermal inactivation process is twofold. First, temperature maxima of the soil increase with increasing soil moisture content (Mahrer et al., 1984). This principle is important in field experiments, but is not operative in an in vitro system as used in the present study. The second effect of moisture is the increase in heat transfer or conduction in the soil, with a subsequent reduction of air pockets that could provide an insulation for the spores (DeVay, 1991 a; Stapleton and DeVay, 1986).

One of the key biological principles of soil solarization is that, due to the

relatively low temperatures of solar heating, stimulation of microbiological activities in solarized soils may lead to biological control of plant pathogens (Katan, 1985). This indirect effect of soil solarization may be related to a differential thermal sensitivity of other soil microbiota, such as actinomycetes and certain bacteria, or to a greater saprophytic competitive ability of these microorganisms in relation to pathogens once the soil has cooled down (Stapleton and DeVay, 1986). Several researchers have enumerated some of the groups of microorganisms that may be involved in the eventual biological control of plant pathogens in solarized soils (DeVay, 1991; DeVay and Katan, 1991; Gamliel and Katan, 1993; Gamliel and Stapleton, 1993a; Keinath, 1996; RamirezVillapudua and Munnecke, 1988; Stapleton, 1991; Stapleton and DeVay, 1984). Gamliel and Stapleton (1993a) determined the microbial activity in the soil using an indirect measure of respiration with fluorescein diacetate. Initially, heated soils had less microbial





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activity than nonheated control soil, but after 2 weeks the activity was similar in both soils. Keinath (1996) determined populations of thermotolerant fungi, fluorescent Pseudomonas spp., Bacillus spp., and actinomycetes by soil dilution plating. Populations of thermotolerant fungi increased during solarization and declined in the months following solarization, but remained higher than in nonsolarized soils. Fluorescent Pseudomonas spp. were not found in the soil immediately after solarization; populations of actinomycetes were not affected by solarization; and populations of Bacillus spp. increased both in solarized and nonsolarized soils.

All previous work on thermal inactivation of various plant pathogens has been done with spores in water suspension (Benson, 1978; Smith, 1923; Tjamos and Fravel, 1995), with pasteurized soil (Myers et al., 1983), or with naturally infested soil (Bollen, 1985; Juarez-Palacios et al., 1991; Kulkarni et al., 1992; Pullman et al., 1981a). One objective of the present research was to determine if lower survival of P. nicotianae occurred in nonpasteurized soils than in pasteurized soils. Two of the soils evaluated (Site

1 and Site 2) were collected from fields where tomato was commercially grown and soil fumigation with methyl bromide was routinely used. In these soils, survival of P. nicotianae was either higher in nonpasteurized soils or similar in nonpasteurized and pasteurized soil. In soil from Site 3, which had been weed fallow for several years, survival of P. nicotianae was either lower in nonpasteurized soil or similar under both soil treatments. Soils that have been fallow for some time may have a greater diversity of microorganisms that could inhibit, compete with, or lyse the structures of plant pathogens, such as P. nicotianae, thus decreasing their survival. In contrast, soils that have been routinely used for commercial production of a crop and have been fumigated with methyl bromide regularly, may provide a biological vacuum after each fumigation, allowing the pathogen to reestablish in the soil and colonize it (Maloy, 1993). Possibly the duration of the tests was too short (9 days) to observe any definite trends in relation to the effects of natural soils and temperature regimes on the inactivation of chlamydospores





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of P. nicotianae. Although there were limitations in time of exposure in this study, this is the first attempt to determine the biological effects of three nonpasteurized soils on survival of P. nicotianae in an in vitro system.














CHAPTER 4
SUMMARY AND CONCLUSIONS



The effects of soil solarization and cabbage amendment on the survival of

Phytophthora spp. were evaluated in North Florida. Soil solarization was applied as a potential soil disinfestation technique in mid-summer of 1994, 1995, and 1996, in five field trials at three sites for 49, 48, 55, 45, and 45 days. During the field trials precipitation occurred on 30, 31, 17, 19, and 21 days, respectively. Ambient air temperature exceeded 350C only in 1995 and 1996. Soil temperatures exceeded temperature thresholds of 44o and 470C only under clear, low density polyethylene film and under clear, gas impermeable plastic.

Soil solarization using clear, low density polyethylene film, or with clear, gas impermeable plastic film was an effective strategy for the reduction of populations of Phytophthora nicotianae and P. capsici in the top 10-cm layer of soil from Site 1 or Site

3. However, at the 25-cm depth, survival of P. nicotianae was similar to that in the whiteon-black low density polyethylene, and survival of P. capsici was similar to that in the nontarped treatment. The incorporation of 70 to 90 metric tons of cabbage into the soil did not affect survival of either pathogen. Phytophthora nicotianae could not be quantified in the selective medium for any of the treatments in Site 2 (1994) due to bacterial contamination by Burkholderia cepacia, or from the first test in Site 3 (1995). Phytophthora capsici was not recovered from any of the trials in Site 3 in 1995 or 1996. In 1996 no oospores of P. capsici were observed in the inoculum at the time of soil infestation, and this might have contributed to the lack of survival of this pathogen. Overall conditions for solarization were excellent in 1995; the soil was at field capacity (87






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5 kPa), air temperature exceeded 350C six times, precipitation occurred only on 17 days, and solarization was carried on for 55 days. During the solarization period soil temperatures under the clear, low density polyethylene film exceeded 440 and 470C for 20 and 6 days respectively. All these factors coupled with the potentially diverse soil microbiota present in a field that had been fallow for several years might account for the lack of survival of the Phytophthora spp. in any of the treatments, even the controls.

Some of the possible changes to allow better evaluation of the effectiveness of soil solarization in combination with amendment for the control of Phytophthora spp. include: disking the cabbage residue into the soil before making the beds for solarization, and thus promoting a better distribution of the amendment throughout the soil profile; irrigating the field for a few days to ensure that soil moisture is optimum down to 30 cm of depth; and making sure that the appropriate survival structures are present in the inoculum before it is taken to the field.

The temporal analysis of survival of P. nicotianae in soil from Site 3 indicated

that populations of the pathogen declined with time at the 10-cm depth in plots that were solarized from 17 to 45 days. At the 25-cm depth and in the nontarped treatment, populations of P. nicotianae were not affected. No propagules of the pathogen were recovered at either depth or sampling date from soil treated with methyl bromide. Further studies following the course of inactivation of survival structures, such as chlamydospores or oospores, during soil solarization would help to determine the minimum time required for soil solarization to effectively control Phytophthora spp. in the soil.

The time required to eliminate P. nicotianae from the soil in laboratory thermal

inactivation studies decreased with increasing temperatures. Populations of the pathogens were reduced from the infestation level of 500 chlamydospores per gram of soil at the beginning of the tests to 0.2 propagule per gram of soil after 10 minutes at 530C, 45 minutes at 500C, 4 hours at 470C, 12 hours at 440C, 4 days at 410C, and 16 days at 380C.





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More than 10% of the initial population survived for 20 days at 35C . The incorporation of cabbage amendment into the soil reduced the time required to inactivate the chlamydospores of P. nicotianae at all temperatures tested. Populations of the pathogen dropped below a detectable level after 4 days in soil amended with 0.5% cabbage and heated to 350C. Populations also declined below a detectable level in soils amended with 0.125, 0.25, and 0.5% cabbage and heated at 380C. Similar reductions in populations of the pathogen were not observed in the field experiment, possibly due to the different procedures in the preparation and incorporation of cabbage into the soil. For the laboratory experiments, cabbage wrapper leaves were air dried in a greenhouse, ground in a Wiley mill with a 20-mesh screen; in contrast, for the field test, cabbage heads and wrapper leaves were chopped with a machete, or shredded in a mechanical shredder, air dried for 1 week on the soil in the plots and disked into the soil as the solarization beds were prepared. These differences may have resulted in a loss of potential volatiles during the drying period and uneven incorporation of the amendment throughout the soil profile, which may have prevented the release of volatiles at the lower depths of the soil (25 cm).

Although no propagules were detected by the soil dilution plating in soils

maintained at 410C, the presence of the pathogen was detected by the tomato seedling baiting technique. Detection of P. nicotianae by the baiting technique provided similar results to the soil dilution plating procedure, except when residual populations were present in the soil (less than 0.2 propagule per gram of soil). At this low level the pathogen was more frequently detected by one or the other method due to the restricted volume of soil used in the assays.

The use of temperature regimes that simulated solarization periods increased the time required to inactivate the chlamydospores of P. nicotianae in relation to the use of constant temperature. Of all temperature regimes evaluated, only those simulating optimum solarization periods (440C for 5 hours daily and 470C for 3 hours daily) eliminated the pathogen from the infested, amended soil and prevented mortality of





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tomato seedlings; infection was prevented only in soil treated at the highest temperature regime. All other temperature regimes (350C for 8 hours daily, 410C for 5 hours daily and 44oC for 1.5 hours daily) did not prevent infection or mortality of the seedlings.

One of the few factors that can be controlled during soil solarization is the soil water matric potential through the use of irrigation. The analysis of the effect of soil water matric potential and temperature regimes on the inactivation of chlamydospores of P. nicotianae in cabbage amended soils indicated that survival was lowest in saturated soil; and as temperature increased, survival of the pathogen decreased at all soil water matric potentials evaluated (0, -10 and -30 kPa). The soil water matric potentials evaluated represent optimum levels for the study of thermal inactivation; however, under field conditions lower potentials may be found, as in the third trial in Site 3. Extending the range of soil water matric potentials to -50 kPa, or -100 kPa and the treatment time to 15 to 20 days would allow better comparisons with the field data.

The study with different soils and soil pasteurization, coupled with cabbage

amendment and heat simulating solarization periods indicated that soils with potentially greater microbiological activity can be more suppressive to populations of P. nicotianae. The duration of the tests (9 days) did not allow the full expression of any of the biological factors that may have been present.

Further studies should be conducted on the effects of temperature regimes, such as 440C for 1.5 or 5 hours daily, on the inactivation of chlamydospores of P. nicotianae over time periods similar to those observed during solarization. Another promising area of study is the possibility of inducing suppressiveness of a soil after heat treatment. Such a study could be carried by heating nonpasteurized soils for 15 days, incubating it for another 15 days (average time between the end of a solarization period and transplanting the field with seedlings), and then adding the inoculum and further incubating it to follow the dynamics of the population of the pathogen.





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Another area that needs clarification is the relationship of inoculum density of P. nicotianae to disease incidence in tomato. The studies in this dissertation indicated that very low levels of the pathogen could cause infection of 1-month-old tomato seedlings, even after the stress of the heat treatments. It is not known how closely the disease responses described here would relate to root infection of tomato with P. nicotianae or P. capsici and subsequent disease under field conditions.




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REDUCTION OF POPULATIONS OF PHYTOPHTHORA SPP. WITH SOIL SOLARIZATION UNDER FIELD CONDITIONS AND THERMAL INACTIVATION OF PHYTOPHTHORA NICOTIANAE By LISIAS COELHO 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 1997

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To Eliamar C, M. Coelho, who has encouraged and supported me through the years.

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ACKNOWLEDGMENTS I would like to thank the Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) for the scholarship granted, and the Florida/Brazil Institute for the fee waiver, without which it would have been impossible for me to pursue a higher education. I greatly appreciate the efforts of my major professor. Dr. David J. Mitchell, who with great patience taught the basics of research with soilborne plant pathogens, broadening my horizons, and, above all for his friendship; of Dr. Daniel O. Chellemi, who provided for all the field work, and made himself available whenever needed; and the other members of the supervisory committee, Dr. Robert J. McGovern, Dr. Harold C. Kistler, and Dr. David M. Sylvia, for their support and assistance. Sincere appreciation is extended to Dr. Steven A. Sargent and Dr. Donald W. Dickson, who provided space and equipment for parts of this study; to Gelman Sciences, and Arclad Adhesives Research, Inc. for providing materials for the field research; and to Rogers NK Seed Company for providing seeds for several tests. In addition I would like to thank Patricia Rayside and Dr. Mary E. Mitchell for the support with lab procedures, "do's and don'ts," and helpful insight. Patricia E. Hill for the assistance in the lab, volunteered time beyond her duty, and mostly for her sincere friendship; G. Hank Dankers, Susan H. Lee, and Michelle L. Dankers, who helped with the tough part of the field work; Marcia Moura for the help with the controlled atmosphere experiment; and Mr. Jay Harrison for the support with the statistical analysis using SAS and making sense of a "bunch of numbers."

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Finally, I appreciate the friendship of all of my colleagues in the department and the stimulus they provided to do a little more, a little better. Their companionship during our brief journey at the University of Florida made life a whole lot more enjoyable. IV

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TABLE OF CONTENTS page ACKNOWLEDGEMENTS iii TABLE OF CONTENTS v ABSTRACT viii CHAPTERS 1 INTRODUCTION AND LITERATURE REVIEW 1 2 THE EFFECT OF SOIL SOLARIZATION ON POPULATIONS OF PHYTOPHTHORA NICOTIANAE AND P. CAPSICI UNDER FIELD CONDITIONS 13 Introduction 13 Materials and Methods 14 Characterization of the Solarization Sites 14 Inoculum Production 16 The Solarization Experiments 17 Statistical Analysis 20 Results 20 Environmental Conditions 20 Survival of Phytophthora nicotianae 27 Survival of Phytophthora capsici 29 Discussion 33 Temperature Changes and Soil Moisture Content 33 Survival of Phytophthora nicotianae 34 Survival of Phytophthora capsici 35 Effect of Cabbage Amendment 36 3 THERMAL INACTIVATION OF PHYTOPHTHORA NICOTIANAE 37 Introduction 37 Materials and Methods 39 Production of Chlamydospore Inoculum of Phytophthora nicotianae 39 The Effect of Contant Temperature on the Inactivation of Chlamydospores of Phytophthora nicotianae 39 The Effect of Cabbage Amendments on the Thermal Inactivation of Chlamydospores of Phytophthora nicotianae 41 V

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The Effect of Cycling Temperatures and Cabbage Amendments on the Thermal Inactivation of Chlamydospores of Phytophthora nicotianae 42 The Effect of Soil Water Matric Potential, Temperature Regimes, and Cabbage Amendments on the Thermal Inactivation of Chlamydospores of Phytophthora nicotianae 44 The Effect of Three Nonpasteurized Soils, Temperature Regimes, and Cabbage Amendments on the Thermal Inactivation of Chlamydospores of Phytophthora nicotianae 45 Statistical Analysis 45 Resuhs 46 The Effect of Contant Temperature on the Inactivation of Chlamydospores of Phytophthora nicotianae 46 The Effect of Cabbage Amendments on the Thermal Inactivation of Chlamydospores of Phytophthora nicotianae 51 The Effect of Cycling Temperatures and Cabbage Amendments on the Thermal Inactivation of Chlamydospores o^ Phytophthora nicotianae 57 The Effect of Soil Water Matric Potential, Temperature Regimes, and Cabbage Amendments on the Thermal Inactivation of Chlamydospores of Phytophthora nicotianae 59 The Effect of Three Nonpasteurized Soils, Temperature Regimes, and Cabbage Amendments on the Thermal Inactivation of Chlamydospores of Phytophthora nicotianae 70 Discussion 77 4 SUMMARY AND CONCLUSIONS 87 APPENDICES A PRODUCTION OF CHLAMYDOSPORES OF PHYTOPHTHORA NICOTIANAE 92 Introduction 92 Materials and Methods 93 Resuhs and Discussion 95 B DETERMINATION OF SOIL WATER MATRIC POTENTIAL 98 C REGRESSION ANALYSES OF THE EFFECTS OF TEMPERATURE REGIMES AIvID CABBAGE AMENDMENT ON SURVIVAL OF PHYTOPHTHORA NICOTIANAE 100 D SUMMARY TABLE OF THE STATISTICAL ANALYSIS OF THE EFFECT OF SOIL WATER MATRIC POTENTIAL, TEMPERATURE REGIMES, AND CABBAGE AMENDMENTS ON THE THERMAL INACTIVATION OF CHLAMYDOSPORES OF PHYTOPHTHORA NICOTIANAE 121 VI

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E SUMMARY TABLE OF THE STATISTICAL ANALYSIS OF THE EFFECT OF THREE DIFFERENT SOILS, SOIL PASTEURIZATION, TEMPERATURE REGIMES, AND CABBAGE AMENDMENTS ON THE THERMAL INACTIVATION OF CHLAMYDO SPORES OF PHYTOPHTHORA NICOTIANAE 124 LIST OF REFERENCES 127 BIOGRAPHICAL SKETCH 136 vii

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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 Doctor of Philosophy REDUCTION OF POPULATIONS OF PHYTOPHTHORA SPP. WITH SOIL SOLARIZATION UNDER FIELD CONDITIONS AND THERMAL INACTIVATION OF PHYTOPHTHORA NICOTIANAE By Lisias Coelho May, 1997 Chairman: David J. Mitchell Major Department: Plant Pathology The effects of soil solarization in combination with cabbage amendments on the survival of populations of Phytophthora spp. were evaluated in North Florida, Soil under solarization for 45 to 55 days reached a maximum temperature of 47°C on up to 10 days at 10 cm of depth. Soil solarization with a clear, gas impermeable, low density polyethylene film was as effective as methyl bromide in reducing populations of P. nicotianae at a depth of 10 cm; however, at 25 cm of depth the population of the pathogen was similar to that in the control treatments. Reduction of survival of P. capsici in two sites in 1994 under solarization with either a clear, low density polyethylene or a clear, gas impermeable, low density polyethylene film was as effective as the methyl bromide treatment at the 10-cm depth, while at the 25-cm depth no control was observed. Incorporation of cabbage into the soil at a rate of 70 to 90 metric tons per hectare did not enhance the effectiveness of solarization. viii

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Studies on the thermal inactivation of P. nicotianae in the laboratory confirmed that the time required to inactivate chlamydospores of the pathogen is inversely proportional to the temperature of the treatment. The time required to reduce soil populations from 500 propagules per gram of soil to residual levels (0.2 propagule per gram of soil or less) was 10 minutes, 45 minutes, 4 hours, 12 hours, 4 days, and 16 days at 53°, 50°, 47°, 44°, 41° and 38°C, respectively. The incorporation of cabbage into the soil reduced the time required to inactive the chlamydospores of P. nicotianae. Detection of P. nicotianae by the tomato-seedling baiting technique provided similar results to the soil plating procedure, except when only residual populations were present in the soil. Temperature regimes that simulated solarization periods were effective in eliminating P. nicotianae only when optimum regimes were used (47°C for 3 hours daily and 44°C for 5 hours daily). Populations of P. nicotianae decreased at matric potentials of 0, -10, and -30 kPa with time of exposure to each temperature regime, but the lowest survival occurred in saturated soil. Soils with different cropping history and pasteurization treatment may have marked differences on the survival of P. nicotianae. Nonpasteurized soils from a fallow field, coupled with optimum temperature regimes, provided the greatest reduction in survival of the pathogen in relation to two other soils from commercial cropping systems. ix

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CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Control of plant diseases caused by soilbome pathogens constitutes one of the most difficult aspects of disease management. In general, plant disease control is based on six principles: avoidance, exclusion, eradication, protection, disease resistance, and therapy (Maloy, 1993). Once a pathogen has been introduced in an area, efforts are generally directed to eradicating it or protecting the plant from disease. Tactics for control of diseases include disease resistance, crop rotation, and other cultural practices, or the use of chemicals to protect plants. For high value crops, one of the most effective methods for the control of soilbome plant pathogens is fumigation of soil with methyl bromide or other chemicals. The imminent ban on the production and use of methyl bromide has prompted a search for other alternatives for disease control. Among these strategies, removing a field from production for a considerable period would allow the reduction of populations of pathogens by attrition. However, this strategy may not be effective for pathogens that form resting structures that can survive for long periods in the absence of the host plant (Baker and Cook, 1974). The use of plastic films for solarization of soil in agriculture has opened new horizons for the control of soilbome plant pathogens. Solarization raises the soil temperature to levels that are lethal to most mesophyllic organisms; thus, soilbome plant pathogens of many crops may be controlled. The mechanisms by which disease control is achieved with this method are not fully understood and warrant further studies. Soil solarization is a hydrothermal process in which moist soil is covered with transparent plastic and exposed to sunlight, allowing it to heat to temperatures under 1

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2 favorable conditions that are lethal to many plant pathogens, pests and weeds (DeVay, 1991b). The effectiveness of soil solarization depends on soil color and structure, soil moisture; air temperature, length of day, and intensity of sunlight (DeVay, 1991b). The process has been studied during the last 20 years as an alternative to chemical fumigation of soil for the management of soilbome plant pathogens (Katan, 1980, 1981; Katan et al., 1987). Solarization may reduce populations of several fungi, such as Fusarium spp. (Chellemi et al, 1994; Katan et al, 1976, Katan et al, 1983; Porter and Merriman, 1985; RamirezVillapudua and Munnecke, 1987, 1988), Phytophthora spp. (Barbercheck and Von Broembsen, 1986; Chellemi et al, 1994; Hartz et al, 1993; Juarez-Palacios et al, 1991; Kassaby, 1985; McGovem and Begeman, 1996; Moens and Aicha, 1990; Pinkas et al, 1984; Wicks, 19S8), Pythium ultimum (Gamliel and Stapleton, 1993a, 1993b; Gamliel et al, 1989, 1993; Kulkami et al, 1992; Pullman et al, 1981a, 1981b; Stapleton and Garza-Lopez, 1988; Stapleton et al, 1995), Verticillium dahliae (Ghini et al, 1993; Hartz et al, 1993; Katan et al, 1976; Porter and Merriman, 1985; Pullman et al, 1981a, 1981b), and Rhizoctonia sp. (Gamliel et al, 1993; Grooshevoy et al, 1941; Keinath, 1995; Lewis and Papavizas, 1974; Pullman et al, 1981a, 1981b). However, disease management is not always achieved, as exemplified by the study of Stapleton and GarzaLopez (1988) in which a reduction in the population of Macrophomina phaseolina did not result in less disease in the indicator crop. Other soilbome pathogens that have not been controlled by soil solarization include Plasmodiophora brassicae, Sclerotium rolfsii, Pythium aphanidermatum and many others, including Fusarium oxysporum f sp. radicislycopersici (Chellemi et al, 1994; Stapleton and DeVay, 1986). Success of solarization for the control of plant diseases is closely associated with a combination of high ambient temperatures, maximum solar radiation, and optimum soil moisture (DeVay, 1991b; Souza, 1994). Early demonstration of the use of solarization came from work in arid climates by Katan (1980, 1981) and Katan et al. (1976, 1987) in Israel, by Stapleton and DeVay (1984, 1986) in Cahfomia, and in semi-arid climates in

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3 Mexico by Stapleton and Garza-Lopez (1988) . Regions where the summer coincides with the rainy season, such as in the southeastern U.S.A., may have less potential for successful solarization because of the cooling effect of frequent rain showers, as well as extended cloud cover, which reduce the solar radiation captured under the plastic tarp (Chellemi et ai, 1994). Soil solarization has been used successfully in the Southeastern U.S.A. to manage Rhizoctonia solani, Didymella bryoniae, Fusarium spp. and P. nicotianae (Chellemi etal, 1994; Keinath, 1995, 1996). Populations of P. nicotianae were reduced to undetectable levels in soil depths of up to 15 cm in three sites in North Florida. However, reduction in populations below 25 cm of depth was noted in only one site. In contrast, methyl bromide nearly completely eradicated the pathogen in depths of up to 35 cm. Control of Fusarium spp. was limited to the top 5 cm of the soil, and no control was achieved at some sites (Chellemi et al, 1994). Keinath (1995) found that the number of organic fragments colonized by R. solani was lower in solarized than in nonsolarized soils; however, the control of belly rot on pickling cucumber was not as effective with solarization as was the chemical treatment with chlorothalonil. The type of plastic used for solarization may also influence soil solarization (DeVay, 1991a, 1991b; Malathrakis and Loulakis, 1989; Staplepton and DeVay, 1986). Stapleton and DeVay (1986) reported that thinner polyethylene sheets (25 |xm) were more effective than thicker sheets (50-100 ^m) for the transmission of solar radiation to the soil. DeVay (1991) noted that black polyethylene films lasted longer than transparent films that had not received a treatment for protection against UV rays; however, soil temperatures did not raise as much under the black films. Malathrakis and Loulakis (1989) compared the effectiveness of a clear, low density polyethylene film to a film of co-extruded polyethylene plus ethylene vinyl acetate (thermoplast). These authors found that the thermoplast was more effective than the clear film in raising soil temperatures and in trapping gases used for fumigation. Similarly, Chellemi et al. (1997) found that a gas impermeable film, consisting of a polyamide core sandwiched between two layers of

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4 polyethylene, was more effective than a co-extruded, white-on-black film for the reduction of populations oi Paratrichodorus minor and Criconemella spp. and control of Fusarium wilt on tomatoes. Indirect effects of soil solarization may include reduced fungistasis, or induction of shifts of microbial populations that affect the survival of spores of plant pathogens. Katan et al. (1976) studied the effect of heating soils to 45° to 50°C on the fungistasis of Fusarium oxysporum f sp. lycopersici. These authors observed that autoclaving or preheating soil to 50°C for 3 hours and cooling it to 25°C before infestation induced an immediate increase in germination of conidia of F. oxysporum f. sp. lycopersici incubated during the first 24 hours. However, when conidia were incubated for longer periods of time (3 to 9 days) in pasteurized soils, the final population was reduced by 81%. The following three mechanisms were suggested to explain the increased control of pathogens in solarized or pasteurized soils: fungistasis may be partially nullified at 45° to 50°C, allowing spores to germinate and then starve in the absence of the host or be killed by the existing microbiota; sublethal temperatures may weaken the resting structures, rendering them more vulnerable to antagonistic microbiota; a microbial population shift is induced which favors thermophylic saprophytes over pathogens. Soil solarization alone may not be effective or consistent for the control of soilbome pathogens, especially in regions where the rainy season occurs simultaneously with the warmer months of the year. In such cases, the use of soil amendments may enhance the performance of solarization (Gamliel and Stapleton, 1993a; Keinath, 1996; RamirezVillapudua and Munnecke, 1988). Cruciferous residues, due to their high content of isothiocyanates and aldehydes, have been suggested as amendments for use in combination with solarization (Mayton et al., 1996); cabbage is the primary amendment that has been studied in combination with soil solarization. Lewis and Papavizas (1971) studied the effect of vapors from cabbage decomposition on the control of Aphanomyces euteiches, and found that root rot of peas

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5 could be controlled with either cabbage amendment or with sulfur-containing volatile compounds. A later study on the survival of R. solani by these authors (Lewis and Papavizas, 1974) indicated that the major effect of the organic amendment resulted from either a change of the soil pH or from the low C:N ratio of the amendment, since fresh com amendment produced similar results as the cabbage amendment. Gamliel and Stapleton (1993b) characterized the antifimgal volatiles from cabbage residue during solarization. The kinds of volatiles released from heated or nonheated soils differed, and the concentration of volatiles peaked during the first 2 weeks of heating. Spores of P. ultimum and sclerotia of S. rolfsii failed to germinate after exposure to cabbage volatiles for 20 days. The use of a crucifer amendment associated with sublethal heating (38°C) reduced germination of P. ultimum and S. rolfsii in vitro (Stapleton et al, 1995). It is possible that the major benefits of the soil amendment with crucifer residues are associated with high but not lethal temperatures, when the heat alone is not sufficient to inactivate the pathogen; however, this aspect of the use of cabbage amendment has not been explored fiilly. Solarization associated with cabbage amendments was evaluated by Keinath (1996) and RamirezVillapudua and Munnecke (1987, 1988). The volatiles released by cabbage alone reduced the populations of F. oxysporum f sp. conglutinans; however, the association of solarization with the amendment was the most effective treatment for the control of this pathogen (RamirezVillapudua and Munnecke, 1987, 1988). High concentrations of amendment had no effect on the subsequent cabbage crop, but phytotoxicity was observed when tomato seedlings were transplanted into the treated soil. Beneficial changes in soil microbiota have been suggested as one of the attributes of soil solarization, regardless of amendments; these changes may lead to higher yields or to longer periods in which the crop is not affected by the pathogen (Katan, 1981; Stapleton, 1991; Stapleton and DeVay, 1986). Solarization of soil amended with cabbage induced an increase in the populations of thermotolerant fimgi and Bacillus spp., a

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6 corresponding decrease in populations of fluorescent Pseudomonas spp. and Fusarium spp., and did not affect the populations of actinomycetes. These changes in populations were correlated to a higher yield of watermelon (Keinath, 1996). Although soil solarization is a hydrothermal process, depending on moisture for maximum heat transfer throughout the soil profile and to soilbome organisms (DeVay, 1991a; Mahrer et al, 1984), very little is known about the influence of moisture on the inactivation of soilbome pathogens. Moisture has been provided during soil solarization by different methods. Kulkami et al. (1992) flood-irrigated solarized plots once a week, removing the plastic tarp. These authors found that recontamination of the solarized plots could occur with either contaminated water or soil movement with the irrigation water. Furrow irrigation of level fields has been suggested as an alternative to periodically replenish moisture to the soil under solarization ( Pullman et al., 1979; Stapleton and DeVay, 1986). However, a single irrigation before the plastic is laid down can provide the same control of soilbome plant pathogens as several irrigation events (Grinstein et al., 1979a, 1979b; Jacobsen et al, 1980; Katan et al, 1980; Pullman et al, 1979). Studies of thermal inactivation of pathogens can yield important information on survival of specific types of propagules and, more importantly when simulating field conditions, can serve as the basis for estimation of the time needed for soil solarization to be an effective strategy for management of soilbome plant pathogens. Relationships between the effectiveness of soil solarization in the field and thermal inactivation in vitro have been studied with several pathogens (Benson, 1978; Myers et al, 1983; Pulhnan et al, 1981a). However, there was a lack of quantification of the inoculum added or recovered after the heat treatment. Additionally, the propagules used in some studies for heat treatment were not the same as the propagules normally found in the soil, or the detection techniques did not allow the quantification of the surviving population (RamirezVillapudua and Munnecke, 1987,1988). Katan (1985) indicated the need for caution in the interpretation of the significance of heat mortality curves obtained under

PAGE 16

7 laboratory conditions. Potential problems that need to be addressed include inoculum type, moisture level, medium containing the inoculum, and the procedure for heating the samples. Early studies of thermal inactivation were more concerned with food processing, and very high temperatures were employed for short periods of time (Bigelow, 1921; Bigelow and Esty, 1920). While the emphasis at the time was on higher temperatures. Smith (1923) demonstrated that spores of Bottytis cinerea in aqueous solution could be inactivated at temperatures as low as 37°C whenever exposure was long enough; spores, as an example, were inactivated over a range varying from 6 minutes at 50.3°C to 23 hours at 37°C. A logarithmic relationship between time and temperature in the inactivation of propagules has been observed (Bigelow, 1921; Bigelow and Esty, 1920; Pullman et al, 1981a; Smith, 1923). Pullman et al. (1981a) found that the exposure time in which 90% of the oospores of Pythium ultimum were killed (LD50) was, for each set of temperatures, 30 minutes at 50°C, 110 minutes at 47°C, 8 hours at 45°C, 42 hours at 42°C, 13 days at 38.5°C and 26 days at 37°C. Juarez-Palacios et a/. (1991) found that the determination of the heat sensitivity of isolates of Phytophthora spp. in the laboratory closely reflected their inactivation in solarized soil, and the results supported the possible use of soil solarization for management of these pathogens. These authors found that an isolate of P. megasperma tolerant to high temperatures survived exposure for 30 minutes at 45°C in a heat sensitivity study, but that the number of leaf discs infected by the pathogen after solarization for 4 weeks declined. In contrast P. cinnamomi and a low-temperature isolate of P. megasperma did not survive either treatment. Bollen (1985) noted that different types of propagules have different sensitivities to heat. Oospores of P. capsici were more thermotolerant than mycelium from either mating type of this pathogen. The difference in tolerance was more than 5°C; oospores

PAGE 17

8 survived at 50°C for 30 minutes, but mycelium was eliminated at 42.5° to 45°C for 30 minutes. Differences in the survival of F. oxysporum were also observed; survival of this pathogen in soil cultures was greater at higher temperatures than survival of the pathogen in soil infested before the heat treatment.The difference may have been due to the presence of chlamydospores in the soil culture. However, the system used did not reflect field conditions, since soil was continuously flooded, which rarely occurs for extended duration in any field. Bollen (1985) did not analyze the relationship of time to temperature, and the temperature used, 50°C, is reached most commonly under soil solarization only in arid or tropical climates. One of the first attempts to control plant pathogens by exposure to intermittent heat was done by Grooshevoy et al. (1941). These authors demonstrated that exposing chlamydospores of Thielaviopsis basicola or sclerotia of a Rhizoctonia sp. or Sclerotinia sclerotiorum for 3 hours a day at 45 °C for 5 days was sufficient to prevent the germination of the spores. However, no attempts were made to simulate the diurnal temperature fluctuations. Porter (1991) evaluated the effect of intermittent heat on the control of sclerotia of S. sclerotiorum in soil, and found that continuous heat was more effective than intermittent heat at reducing the number of viable sclerotia when the infested soil was heated for 6 hours daily over 14 days to temperatures ranging from 30° to 45°C. In a similar study with several pathogens. Porter and Merriman (1983) observed that sclerotia of S. rolfsii tolerated exposure to 50°C for 6 hours a day for 2 weeks, while Sclerotium cepivorum and Sclerotinia minor survived only under similar exposures at temperatures of 45°C or less. Pythium irregulare and F. oxysporum survived at 50°C, but Verticillium dahliae was killed at temperatures above 40°C; however, the type of fungal propagules used for this study was not determined (Porter and Merriman, 1983). Due to the labor-intensive nature of these studies and the demand for equipment, very little research has been done on the quantitative relationship of intermittent heat to the inactivation of spores of plant pathogens.

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9 Phytophthora nicotianae Breda de Haan (syn. = P. parasitica Dastur) has been reported on more than 170 plant hosts in Florida (Alfieri et al, 1994). Soilbome diseases caused by P. nicotianae have limited production of several important crops, such as citrus, tobacco, ornamentals, and tomato. Low levels of inoculum of P. nicotianae in the field can result in severe epidemics (Ferrin and Mitchell, 1986a; Kannwischer and Mitchell, 1981; Mitchell, 1978). Karmwischer and Mitchell (1981) found that 0.13 chlamydospore of P. nicotianae per gram of soil or 42 zoospores per plant were sufficient to cause 50% mortality on a susceptible cultivar of tobacco in a controlled environment. Residual population densities of 0.005 to 0.67 propagules per gram of soil were found in a tobacco nursery; mortality of a susceptible cuhivar transplanted to this field reached 80% at the end of the growth season (Ferrin and Mitchell, 1986a). Moisture plays an important role in the formation of sporangia, zoospore release, and subsequent epidemics. Sporangia of P. nicotianae are produced over a range of soil matric potentials (-4 to -1500 kPa), with the greatest number of sporangia being produced at the higher end of the range (-4 to -25 kPa) (Bernhardt and Grogan, 1982; Sidebottom and Shew, 1985a). Although flooding does not favor the formation of sporangia, it enhances zoospore release (Bernhardt and Grogan, 1982). Lutz and Menge (1991) observed that populations of P. nicotianae increased from 17 propagules per gram of soil before irrigation to 70 propagules per gram 2 days after a 24-hour furrow irrigation event in a citrus grove. The highest proportion of propagules at that time was comprised of sporangia and zoospores; chlamydospores increased 4 days after the irrigation event, and reached a maximum on the seventh day. When the citrus grove was irrigated with a drip system, the soil matric potential was maintained close to -10 kPa, and the highest proportion of propagules consisted of sporangia and zoospores, with populations ranging from 71 to 93 propagules per gram of soil.

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10 Ristaino et al (1988) analyzed the effect of irrigation frequency and duration on the development of Phytophthora root rot of tomato and found that an irrigation regime in which plants were irrigated every 14 days, either for 4 to 8 hours or for 24 hours, increased root infection and decreased tomato yield. A less frequent irrigation schedule, such as 4 to 8 hours every 28 days, stressed the plants and also led to severe root infection. Zoospores of P. nicotianae can be carried in irrigation water and infect tomato plants more than 60 meters from inoculum sources (Neher and Duniway, 1992). In tomato, earlier infection led to higher disease intensity, and lower yield (Neher and Duniway, 1991; Neher et al, 1993). Infection of tobacco was favored by flooding of potted plants, or by saturating soil in Buchner fiinnel tension plates (Shew, 1983; Sidebottom and Shew, 1985b), and coincided with periods of high moisture levels in field trials (Ferrin and Mitchell, 1986b), indicating that zoospore movement is enhanced in saturated soils. Temperature is another important factor in the development of epidemics caused by P. nicotianae. Lutz et a/. (1991) found that heating naturally infested soils fi-om citrus groves above 12°C increased germination of chlamydospores. Stimulation was observed up to 34°C, with maximum germination at 24°C and minimum at 36°C. However, such a stimulus could be due to a temperature differential between the temperature in which the spores were formed and a temperature that would provide maximum germination, and not only an increase in temperature. Even though extensive studies have been done on the relationships of inoculum density and disease, water status of the soil and disease, and temperature and increased recovery of chlamydospores in the soil (Ferrin and Mitchell, 1986b; Lutz and Menge, 1991; Ristaino et al, 1988; Shew, 1983; Sidebottom and Shew, 1985a, 1985b), there is a lack of information about the effects of interactions of moisture and temperature on the inactivation of P. nicotianae.

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11 Phytophthora capsici Leonian was first described infecting chile pepper in New Mexico (Leonian, 1922). This pathogen has a broad host range, including perennial plants, such as cacao, rubber, and macadamia, and annuals, such as peppers, cucurbits and several solanaceous hosts (Alfieri et al, 1994). Important diseases caused by P. capsici in the United States include root rot, blight and fruit rot of pepper, eggplant, cantaloupe, squash and watermelon (McGovem et al, 1993). In 1994, severe outbreaks of P. capsici were reported on tomato in South Florida (Simone, personal information). Phytophthora capsici survives in low numbers in soil (Papavizas et al., 1981). The effects of soil temperature and soil-water matric potential on survival of oospores, sporangia, and zoospores, as well as on oospores or mycelium in plant tissue, were studied by Bowers et al. (1990a). These authors found that zoospores survived no more than 3 weeks in field soil during the summer, when temperatures ranged fi-om 20° to 30°C; sporangia survived up to 8 weeks. Very little decrease in viability of the oospores was observed during the same period. In a winter test, viability of oospores free of organic residue in soil decreased from 67% to less than 10% after 27 weeks, while oospores in plant tissue did not survive more than 8 weeks. Although long-term survival of P. capsici in the soil occurs in the form of oospores (Bowers et al., 1990a), the primary infective structures generally are zoospores (Bowers and Mitchell, 1990; Hord and Ristaino, 1992; Luz and Mitchell, 1994; Ristaino et al., 1992). These authors found that flooding soils infested with oospores for as little as 2 hours resulted in increased disease, and several flooding events led to the eventual infection of all plants. Bernhardt and Grogan (1982) demonstrated that sporangia are formed within 24 hours in soils held at -30 kPa, and sporangia released zoospores 4 hours after the soil was flooded. Schlub (1983) observed that P. capsici spread in the field with rain splash. Bowers et al. (1990b) and Ristaino (1991) confirmed the influence of rainfall on disease, but noted that the movement of water over the soil surface or the plastic mulch had the most significant impact on disease development.

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12 Knowledge of the primary survival and infection structures may be very important in the determination of strategies for the control of P. capsici. Still, the effects of moisture and temperature on the survival of this pathogen in the absence of a host plant are not known. The objectives of this study were to determine the efficacy of soil solarization and organic amendment with cabbage for the control of P. nicotianae and P. capsici in the field; and to determine the relationships of time of exposure to temperature, cabbage amendment, soil water matric potential, and different soils on the thermal inactivation of propagules of P. nicotianae and its pathogenicity in tomato.

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CHAPTER 2 THE EFFECT OF SOIL SOLARIZATION ON POPULATIONS OF PHYTOPHTHORA NICOTIANAE AND P. UNDER FIELD CONDITIONS Introduction Phytophthora nicotianae Breda de Haan (syn .= P. parasitica Dastur) has been reported on more than 170 plant hosts in Florida (Alfieri et al, 1994). Soilbome diseases caused by P. nicotianae have limited production of several important crops, such as citrus, tobacco, ornamentals and tomato (Erwin and Ribeiro, 1996). Phytophthora capsici Leonian is an important pathogen on several solanaceous plants. Severe losses have resulted from root rot, blight, and fruit rot of pepper, eggplant, cantaloupe, squash, and watermelon (McGovem et al, 1993). Outbreaks of P. capsici have been observed in tomato, especially when soils are saturated with moisture. Because these pathogens can survive in the soil for long periods of time in the absence of their hosts, or when weather conditions are not favorable for disease (Erwin and Ribeiro, 1996), the main strategy used by growers to reduce losses due to these pathogens, especially at the early stages of plant development, is the use of preplant fumigation with methyl bromide and application of fiingicides. The implication of methyl bromide as an ozone depleting substance has prompted a search for new alternatives for the control of soilbome plant pathogens. Soil solarization has been used in areas with arid climates for the management of soilbome diseases of high value crops (Hartz et al, 1993; Pullman et al, 1981b). Solarization has been evaluated in areas where the climate is considered marginally suitable for the procedure but results were not completely satisfactory (McSorley and Parrado, 1986). Soil amendments have been tested as components of soil solarization to enhance its 13

PAGE 23

14 effectiveness (Chellemi et ai, 1997; Gamliel and Stapleton, 1993a, 1993b). Cabbage residues have been shown to be a potential amendment for the control of soilbome plant pathogens due to the isothiocyanates and other volatiles that are released during the heating process (Gamliel and Stapleton, 1993a; Keinath, 1996; RamirezVillapudua, 1987, 1988; Stapleton et ai, 1995) and could be useful in areas where the soil temperatures are not high enough to pasteurize the soil. The objective of this study was to evaluate the effects of soil solarization and cabbage amendment on the survival of P. nicotianae and P. capsici in North Florida. Materials and Methods Characterization of the Solarization Sites Two sites were selected for solarization in 1994 in commercial tomato production fields where control of soilbome diseases had been achieved previously through the use of a preplant application of methyl bromide plus chloropicrin. Site 1 was in the Naples Tomato Growers-Gargiulo farm number 4 in Decatur County, South Georgia. Site 2 was at the John Allen Smith farm located in Gadsden County, North Florida. In 1995, one experiment was conducted at the North Florida Research and Educational Center in Gadsden County (site 3, test 1) on soils that had been weed fallow for several years. In 1996, two experiments were conducted at the same center (site 3, tests 2 and 3) but in a different field. The soil at all locations consisted of Orangeburg or Tifton loamy fine sands (Typic Kandiudult: silicaceous, thermic), with pH values ranging from 4.7 to 6.6 (Table 2.1); these soils contained moderate amounts of kaolinitic clay and low amounts of organic matter.

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15 O 00 T3 C 1/5 o PL, '5 on C .2 '+-• o o <1> -4-» v5 1) k3 » o c o o o 'C a. 06 c c > o ITS 'ob o O >^ c 3 O U o c o s go X) CO O PQ H .
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16 Inoculum Production Inoculum of P. nicotianae was produced by inoculating a 250-ml flask containing 20 g of wheat seeds and 30 ml of deionized water (autoclaved twice at a 24-hr interval) with four, 5-mm agar plugs of actively growing mycelium from a 4to 7-day-old V8juice-agar plate containing isolate Pn21. The flask was incubated at 25°C in the dark for 1 month and was shaken twice a week to ensure uniform growth of the isolate. Oospores of P. capsici were produced by inoculating a similarly prepared 250-ml flask of wheat seeds with three, 5-mm agar plugs of actively growing mycelium of each of two isolates of compatible mating types (Cp25 and Cp26). Isolate Pn21 was originally isolated from periwinkle, and isolates Cp25 and Cp26 were isolated from watermelon in South Florida; all isolates were maintained in the collection of Phytophthora spp. of the Plant Pathology Department of the University of Florida, Gainesville. The flask was incubated at 25°C in the dark for 2 months and was shaken twice a week to ensure uniform grovv1:h of both isolates on the substratum and to allow for maximum contact between the two mating types. Inoculum of P. nicotianae or P. capsici was incorporated into the soil by mixing 200 mg of shredded, infested wheat seeds with 3 grams of soil. Nonpasteurized soils from sites 1 and 2 were used in 1994, and the soils were infested 2 days before the samples were taken to the field. Pasteurized soil from site 3, test 1, was infested with the inoculum 2 days before the samples were taken to the field in 1995. To encourage augmented formation of resting structures in the two experiments in 1996, pasteurized soil from site 1 was infested with either P. nicotianae and incubated at 18°C for 10 days, or with P. capsici and incubated at 25°C for 10 days, before its placement in the field tests. In all tests, each 3-g sample was enclosed in a 25-cm^ nylon envelope (3-^m-pore size; Versapor 3000, Gelman Sciences, Inc.), and the envelopes were buried in the soil at depths of 10 and 25 cm just before application of the solarization treatments.

PAGE 26

17 The survival of the spores at the end of the solarization experiments was determined by plating infested soil from the envelopes on a medium (PARPH) selective for pythiaceous fimgi. The medium consisted of 17 g of commeal agar (Difco) in 1 liter of deionized water amended with 5 mg of pimaricin, 250 mg of ampicillin, 10 mg of rifampicin, 100 mg of pentachloronitrobenzene, and 50 mg of hymexazol (Mitchell and Kannwischer-Mitchell, 1992). In 1995 hymexazol was omitted from the selective medium. The Solarization Experiments Six solarization treatments with four replications each were selected for study at site 1, which consisted of an 18-treatment randomized complete block experiment on solarization and fumigation (Chellemi et al, 1997). The selected treatments included a 30-|im-thick, clear, gas impermeable plastic film (Bromotec film, Lawson Mardon Packaging, United Kingdom); a 30-^m-thick, coextruded white-on-black, low density polyethylene film (Edison Plastics, Lee Hall, VA); and each of the polyethylene films with or without cabbage residue {Brassica oleracea var. capitata L. cv. Constanza) incorporated into the soil at a rate of 80 tons/ha. Controls consisted of a nontarped treatment and a tarped treatment (white-on-black film) fiimigated with a 67:33 formulation of methyl bromide plus chloropicrin at 39.2 g/m^ At site 2, the six treatments selected for solarization from an 1 8-treatment randomized complete block experiment with four replications (Chellemi et al., 1997) included a 30-^m-thick, clear, low density polyethylene film (Polydak film, Polyon Barkai Ltd., Kibutz Barkai, Israel); a 30-nm-thick, clear, gas impermeable plastic film (Bromotec film) ; and each of the polyethylene films with or without cabbage residue incorporated into the soil at a rate of 66 tons/ha. Controls consisted of a nontarped

PAGE 27

18 treatment and a tarped treatment (white-on-black film) fumigated with a 67:33 formulation of methyl bromide plus chloropicrin at 39.2 g/m^. For test 1 at site 3, the eight treatments selected for solarization from an 15treatment randomized complete block experiment with four replications (Chellemi et al., 1997) included a 25-^m-thick, clear, low density polyethylene film (Polydak film); a 30Hm-thick, clear, gas impermeable plastic film (Bromotec film); a 30-nm-thick, coextruded white-on-black, low density polyethylene film (Edison Plastics); and each of the polyethylene films with or without cabbage residue incorporated into the soil at a rate of 68 tons/ha. Controls consisted of a nontarped treatment and a tarped treatment (whiteon-black film) fumigated with a 67:33 formulation of methyl bromide plus chloropicrin at 39.2 g/m' For tests 2 and 3 at site 3, six treatments were arranged in a randomized complete block design with four replications. Soil treatments included a 30-^m-thick, clear, gas impermeable plastic film (Bromotec film); a 30-|im-thick, coextruded white-on-black, low density polyethylene film (Edison Plastics); and each of the polyethylene films with or without cabbage residue incorporated into the soil at a rate of 89 tons/ha for test 2, and 81 tons/ha for test 3. Controls consisted of a nontarped treatment and a tarped treatment (white-on-black film) fumigated with a 67:33 formulafion of methyl bromide plus chloropicrin at 39.2 g/m^. At sites 1 and 2 and for test 1 at site 3, each replicate plot consisted of one raised, 0.20-m X 0.90-m X 20-m bed, prepared according to standard commercial production practices; row orientation was north/south. Preplant fertilizer was broadcast into the beds at 212 kg of N, 65 kg of P, and 212 kg of K/ha, and drip irrigation tubing was placed 5 cm beneath the soil on sites 1 and 2. At site 3 for test 1, fertilizer was applied at 196 kg of N, 26 kg of P, and 163 kg of K/ha. Drip irrigafion tubing was laid on the surface of the bed prior to covering with plasfic. At site 3 for tests 2 and 3, each replicate plot consisted of one raised, 0.20-m X 0.90-m X 4.0-m bed prepared according to standard commercial

PAGE 28

19 production practices; row orientation was north/south. No fertilizer was applied and no irrigation tubing was placed on the beds. Cabbage was grown and harvested in the plots at sites 1 and 2, and the residue was spread over the plots on 23 and 24 May 1994. Beds were prepared, the nylon envelopes containing the inoculum were placed in the soil, and fumigant and plastic were applied on 3 June 1994 at site 1. The solarization period was terminated on 22 July 1994, after 49 days, and the samples were removed for determination of survival. At site 2, beds were prepared, the nylon envelopes containing the inoculum were buried, and fumigant and plastic were applied on 15 June 1994; the solarization period was terminated on 2 August 1994, after 48 days, when the samples were removed. For the first test at site 3, cabbage was grown in plots near the experimental plots, harvested, and the head and wrapper leaves incorporated into the plots on 19 May 1995. Beds were prepared, the inoculum bags were buried, and fumigant and plastic applied on 26 May 1995. The solarization period was terminated on 20 July 1995, after 55 days, when the samples were removed. For tests 2 and 3 at site 3, cabbage was harvested fi-om a commercial field in Hastings, FL; head and wrapper leaves were incorporated into the plots on 12 June for test 2, and on 19 June 1996 for test 3. Beds were prepared, two sets of nylon envelopes containing the inoculum were placed in each plot, and fumigant and plastic were applied on 21 June and 28 June 1996, respectively. The solarization period was terminated with the removal of the last samples on 6 and 13 August 1996, after 45 days, for tests 2 and 3, respectively. Daily ambient temperature data were obtained from a weather station at the North Florida Research and Educational Center, in Quincy, located approximately 25 km from sites 1 and 2, while daily precipitation amounts were recorded at each site. In 1994, soil temperatures were monitored in site 2 at depths of 10 and 25 cm using thermocouple sensors connected to an electronic data logger (Omnidata International, Logan, UT). For all three tests at site 3, daily rainfall and ambient temperature data were recorded at the

PAGE 29

20 site. For test 1 at site 3, hourly temperature changes were monitored for 33 days at an external test location approximately 500 m from test 1 ; however, temperatures were still recorded at 10 and 25 cm depths in the soil of the nontarped and the clear, gas impermeable treatments with thermocouple sensors cormected to an electronic data logger (Campbell Scientific, Logan, UT). In 1996, temperature changes were monitored in test 2, at 10 and 25 cm depths in the soil. Statistical Analysis All data of survival of each pathogen were log-transformed (ln[ppg+l]) prior to analysis of variance. Analysis of variance was performed with PROC MIXED of SAS (SAS Institute, Cary, NC; release 6. 11 for personal computers), considering that the environmental effects on the inoculum due to the depth factor could be correlated. Survival of P. nicotianae and P. capsici was compared using Tukey's Honestly Significant Difference procedure. Results Environmental Conditions Environmental conditions varied from year to year, with 1994 being characterized by above average precipitation and below average temperatures (Figure 2.1). In 1995 both precipitation and temperatures were normal, and in 1996 precipitation was below average and temperatures were normal. The heating process of the soil was interrupted by precipitation during 61% of the days of the solarization period at site 1; 65% of the days at site 2; 30% of the days in test 1 at site 3; 42% of the days in test 2 at site 3; and 47% of the days in test 3 at site 3 (Table 2.2).

PAGE 30

Figure 2.1: Maximum and minimum air temperature and precipitation recorded at each site during the solarization period, from 1994 to 1996. A. Solid arrows (^) indicate beginning and end of solarization at Site 1 ; open arrows (^) indicate beginning and end of solarization at Site 2. B. Solid arrows (^) indicate beginning and end of solarization in test 1 at Site 3. C. Solid arrows {^) indicate beginning and end of solarization in test 2 at Site 3; open arrows (^) indicate beginning and end of solarization in test 3 at Site 3.

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22 CO <+-! o Ui (U 1 I Al u, -4— » (U & e .9 o o « o e o '5 S .2 c .9S a. c 03 O o h-1 iy5 o 0^ o On ON o O NO On ^ —< ^ CN 00 00 ON ON T3 O ON On o c On On o c NO NO ON ON o c 3 o E a s a (N m --» -4— » (A c/l
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23 Soil moisture conditions at the beginning of the solarization treatments varied from experiment to experiment and ranged from -5 to -50 kPa (Table 2.1). Even though there were several rain events during the solarization period in all experiments, it is possible that soil moisture was not replenished under the plastic films used due to the structure of the sandy soils studied; the high proportion of large size pores in these soils does not favor the lateral movement of water. During the 48 days of solarization at site 2, there were 2 days in which temperatures exceeded 41°C at the 10-cm soil depth in the nontarped treatment; 41°C was exceeded on 1 5 days in the clear plastic treatment and on 20 days in the gas impermeable treatment (Table 2.3). Temperatures exceeded 44°C at the 10-cm depth on 9 days under the clear plastic and on 1 3 days under the gas impermeable film. Temperatures were above 47°C for 1 day and 3 days under the clear and the gas impermeable films, respectively. At the 25-cm depth, temperatures never reached the 41°C threshold under any of the treatments. Maximum temperatures attained in site 2 at the 10-cm depth under nontarped soil, clear low density polyethylene plastic, and clear, gas impermeable plastic were 41.5°, 47.3°, and 48.5°C, respectively. At the 25-cm depth, maximum temperatures were 34.6°, 37.9°, and 38°C under nontarped soil, clear low density polyethylene plastic, and clear, gas impermeable plastic, respectively. The average soil temperature change during the day is illustrated in Figure 2.2.A. Temperature was not monitored in site 1. At site 3 for test 1, temperatures above 41°C were recorded at the 10-cm depth under the clear low density polyethylene plastic on 30 days, and under the nontarped treatment on 5 days; 44°C was reached on 20 days and 47°C was reached on 6 days during the 33 days over which temperatures were monitored under the clear low density polyethylene film. At the 25-cm depth, temperatures never reached the 41°C threshold (Table 2.3). Maximum soil temperatures under nontarped soil and clear low density polyethylene plastic at the 10-cm depth were 42.4° and 48.7°C, respectively; at the 25-cm

PAGE 33

24 T3 C S H r -*-» --» cJ5 ^ o o m CM S o — o o o O m o m O m ^ CM ^ CM — CM fS c75 --» c?5 o i-< cn o d o g <^ O -o — C o -o CO m -o .s c ; <^ On 4J = .S ^ G O S >^ St X! 1o t2 -Is u c N CO 'C g ^ ^""^ *— » -o ilP in § a CJ -a S N o ^ c O 3 I 3 C Co I ^ E U. ^ 3 D 2 ^ E H o s o. c/1 ca c ea N •c i2 "o GO O CA T3 CM Tt+-• c/} E a. ca E a. P CO 00 c °c 3 -a -a E 3 c« CO ca 00 ca ii^ 3 e k. o
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25 —9 — Nontarped 10 cm — • — Nontarped 25 cm —AWhite-on-black 1 0 cm —AWhite-onblack 25 cm ® Clear 10 cm — O— Clear 25 cm • -GllOcm -BGI25cm 1994 1995 B 1996 .D--H. .0' 8 12 Time of the day 16 20 24 Figure 2.2: Average temperature change during a 24 hour period at 10 and 25 cm of depth. Temperatures were measured in the untreated soil (nontarped); under the white-on-black, low density polyethylene film (White-on-black); under the clear, low density polyethylene film (Clear);and under the clear, gas impermeable plastic film (GI). A. Site 2 (John Allen Smith farm, Gadsden County); B. Site 3 (North Florida Research and Educational Center in Gadsden County) test 1; C. Site 3 test 2.

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26 depth maximum temperatures were 35.1° and 40.3°C. The average soil temperature change during the day is illustrated in Figure 2.2.B. During the 45 days of solarization at site 3 for test 2, maximum temperatures attained at the 10-cm depth under nontarped soil, white-on-black plastic, and clear, gas impermeable plastic were 41.1°, 39.0° and 49.1°C, respectively. At the 25-cm depth, maximum temperatures were 37.4°, 37.5°, and 41.4°C under nontarped soil, white-onblack plastic, and clear, gas impermeable plastic, respectively. Under the clear, gas impermeable plastic, at the 10-cm depth, temperatures were above 41 °C for 32 days, above 44°C for 24 days, and above 47°C for 10 days; while at the 25-cm depth, 41°C was reached on only 1 day. Temperatures in the nontarped treatment reached 41 °C only once during the solarization period, and under the white-on-black plastic temperatures never reached that threshold. The average temperature change during the day is illustrated in Figure 2.2.C. Soil temperature changes for test 3 at site 3 were monitored using thermocouples placed in test 2 at site 3, which was located a few meters away. Maximum temperatures attained at the 10-cm depth under nontarped soil, white-on-black plastic, and clear, gas impermeable plastic were 41.1°, 44.2° and 49.1°C, respectively. At the 25-cm depth, maximum temperatures were 37.4°, 37.5°, and 41.4°C under nontarped soil, white-onblack plastic, and clear, gas impermeable plastic, respectively. Under the clear, gas impermeable plastic, at the 10-cm depth, temperatures were above 41 °C for 30 days, above 44°C for 22 days, and above 47°C for 8 days. Temperatures never reached these thresholds at the 25-cm depth. Temperatures in the nontarped treatment and in the whiteon-black plastic never reached the 41°C threshold. The highest accumulation of hours in which soil temperatures were greater than 41°, 44° and 47°C was observed at the 10-cm depth in test 2 at site 3, followed by test 3 at site 3. The lowest accumulation of hours was observed at site 2 (Table 2.3). At site 2 it was possible to compare the accumulation of hours above the given thresholds under the

PAGE 36

27 solarization films. Under the gas impermeable film, temperatures were above 41°, 44° and 47°C for 82, 42 and 7 hours, respectively; while under the clear low density polyethylene plastic the accumulation was of 63, 25, and 2 hours, respectively. In the nontarped treatment and at the 25-cm depth of all treatments, temperatures never reached the 44°C threshold. Survival of Phytophthora nicotianae Recovery of P. nicotianae varied fi-om site to site and fi-om year to year. At site 1 in 1994, no propagules were recovered fi-om the nontarped or the methyl bromide treatments, or fi-om the 10-cm depth in the gas impermeable treatment, with or without cabbage, after 49 days of solarization (Table 2.4). The highest survival was observed under the white-on-black polyethylene film at 10 cm of depth with or without cabbage amendment. At the 25-cm depth under the gas impermeable film, propagules were recovered at the same levels as in the white-on-black film treatment. At site 2 in 1 994, Burkholderia cepacia, a contaminant from the soil, covered the selective medium plates in such a way that very few propagules of P. nicotianae were recovered, and only from soil under the clear low density polyethylene at 10-cm of depth (data not shown). The omission of hymexazol in the selective medium for the determination of survival of P. nicotianae from site 3 for test 1, made it impossible to recover the pathogen from the soil because other microorganisms, especially Pythium spp., colonized the organic substratum (shredded wheat seeds) and may have inhibited P. nicotianae in the soil or on the dilution plates. Propagules of P. nicotianae were recovered from all treatments in test 2 at site 3, except from soil treated with methyl bromide; however, there were no statistically significant differences among the treatments (Table 2.4).

PAGE 37

28 On O O -SP 3) CO O. O c/5 On On O O (U da ao cc o. o a, Q c s -*-» U H O O i ON oo m ir» ;;; o O ; ^ JO o r-^ O 00 c<3 On m cd ^ cO cO O On 00 ; O O : On VT) O CN O _o CO CO cO cO (U U U V £ S E S U I(U 0) flj lU a. a. o. a. Vl Vl VI Kfi cO cO CO cO o o o o a 2 iS ^ i o 3. x> — ' c« Vo u ,o ca c "g u •T3 c 3 w u ^ 60 35 1 53 ^ re (u « -= s X i 00 3 3.^ S ^ as jD re 00 J3 -a I 3 o o 00 re 60 re :§ re a t= 2 o 60 5 i 60 o re •a re T3 O o VI a, 3 •a cn en( day: iffer ON Q .*-• s dfo ays) fica u N •a 'c ;olari: tly Sig I/] rgiai (U 'C onesi o u _re a (SO v. nty Cou Coui toT ii en 60 C 'eca dsd^ ardi Q re o c a o re

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29 Trends similar to those observed in site 1 were observed in test 3 at site 3 (Table 2.4). Only methyl bromide eliminated the pathogen from the soil. The gas impermeable film was the next best treatment at the 1 0-cm depth, regardless of the cabbage amendment, for the reduction of the population of the pathogen; however, at the 25-cm depth the gas impermeable film was not effective, and more than 200 propagules survived in each gram of soil. These values were not statistically different from those observed under the white-on-black film, with or without cabbage amendment, or in the nontarped treatment. Survival in the nontarped treatments in tests 2 and 3, in contrast to no survival in Site 1 could have been due to the change in the procedure for soil infestation from that used for site 1 ; chlamydospores were already present at the beginning of the experiments in the soil prepared for tests 2 and 3, but not for test 1. Samples removed after 17 days of solarization in tests 2 and 3 at site 3 demonstrated that the pathogen was present in the samples, except in the methyl bromide treatment, and that the populations declined with time of exposure to the solarization process (Table 2.5). Increases in population were observed in test 3, mostly at the 25-cm depth, in the nontarped, gas impermeable, and white-on-black treatments after the 45 days of solarization. Statistically significant differences were observed at the 10-cm depth between the two sampling dates under the gas impermeable film; the population of P. nicotianae dropped to levels comparable to those in the methyl bromide treatment. Survival of Phytophthora capsici Oospores of Phytophthora capsici do not germinate readily in vitro, and this was clearly noted during the field experiments. Although the presence of oospores was verified in the inoculum before the soil was infested, there was not a corresponding high recovery

PAGE 39

30 Table 2.5. The effect of time of exposure to solarization on the survival of Phytophthora nicotianae at Site 3 . Treatment Propagules per gram of soil Depth Test 2 Test 3 (cm) 1 7 day< 45 days 1 7 days 45 days Gas Impermeable 10 2.0'' a^ 0.4a 544.6b 4.4a Gas Impermeable 25 4.1 a 2.1a 168.0b 202.3b Gas Impermeable + Cabbage 10 2.7 a 0.9a 52.5b 1.0a Gas Impermeable + Cabbage 25 0.7 a 2.3a 491.3b 362.0b White-on-black 10 22.8 a 4.0a 196.6b 733.9b White-on-black 25 16.3 a 0.9a 186.4b 697.8b White-on-black + Cabbage 10 101.9 a 14.8a 830.5b 825.3b White-on-black + Cabbage 25 25.0 a 12.3a 608.3b 479.5b Methyl Bromide 10 0.0 a 0.0a 0.0a 0.0a Methyl Bromide 25 0.0 a 0.0a 0.0a 0.0a Nontarped 10 21.6 a 14.9a 1138.7b 700.7b Nontarped 25 6.2 a 4.5a 311.8b 687.6b Treatments consisted of untreated soil (Nontarped); soil fiamigated with methyl bromide plus chloropicrin at 39.2 g/m^ (Methyl bromide); soil solarized under a 30-nm-thick, coextruded white-on-black, low density polyethylene film (White-on-black); or soil solarized under a 30-|im-thick, clear, gas impermeable plastic film (Gas Impermeable). Soil was either nonamended or amended with cabbage (+ Cabbage) at 89 and 81 tons/ha for tests 2 and 3, respectively. Weighted means ([Exp {mean}]-l). ^ Main effect means at each date for each test followed by the same letter do not differ according to Tukey's Honestly Significant Difference procedure (P< 0.05); data transformed to (ln[ppg+l]) prior to analysis.

PAGE 40

31 of the pathogen, either before or after the solarization treatments. Recovery of the pathogen on the selective medium was erratic. At site 1, no propagules were recovered at 10-cm depth after treatment with methyl bromide, the gas impermeable plastic, or the gas impermeable plastic combined with cabbage (Table 2.6). Low levels of survival at the 25-cm depth under the methyl bromide treatment, the gas impermeable plastic, and the gas impermeable plastic combined with cabbage were not statistically different from the same treatments at the 10-cm depth. The highest survival was observed under the white-on-black plastic at the 10-cm depth, regardless of cabbage amendment, and in the nontarped treatment. At site 2, no statistical differences were observed in the survival of P. capsici under the clear low density polyethylene film, clear low density polyethylene film plus cabbage, and the gas impermeable plastic at the 10-cm depth, or under the methyl bromide and the gas impermeable plastic combined with cabbage at both depths (Table 2.6). Survival was highest in the nontarped treatment at both depths, and populations were not significantly different from those at the 25-cm depth in all other treatments, except treatments with methyl bromide and gas impermeable plastic combined with cabbage. The lack of hymexazol in the selective medium for the determination of survival of P. capsici from test 1 at site 3 made it impossible to recover the pathogen from the soil because other microorganisms colonized the organic substratum (shredded wheat seeds) and grew faster than the pathogen on the dilution plates. Not even the addition of 100 mg of lecithin per liter of medium to stimulate the germination of oospores of the pathogen could compensate for the lack of hymexazol. Fewer oospores were observed in the inoculum used for tests 2 and 3 at site 3, than in previous experiments, and very few colonies were observed on the selective medium at the end of the experiments. No valid analysis could be performed and the data are not presented.

PAGE 41

32 Table 2.6. Survival of Phytophthora capsici after soil solarization in 1994. Treatment" Depth Site r Site T ( cxn) vjds iinpciiiicduic 10 0 O'' a'' 0.0a Oaa lllipci IllCaUiC 1 5 ab 7.0ab llllUCl lllCdUlC ' \^ci\J\ja.^\Z 10 0,0 0.4a 25 1.2 ab 0.5a Clear LDPE 10 nt^ 0.0a Clear LDPE 25 nt 11.2ab Clear LDPE + Cabbage 10 nt 0.2a Clear T DPE + Cahhaee 25 nt 7.9ab \A/Ti 1 1 p n n h 1 p r W vv iiiiv KJii \ji(i\^r%. 10 84.6 c nt White-on-black 25 47.4 c nt White-on-black + Cabbage 10 70.8 c nt White-on-black + Cabbage 25 16.3 be nt Methyl Bromide 10 0.0 a 0.2a Methyl Bromide 25 1.1 ab 0.9a Non-tarped 10 33.4 c 33.9b Non-tarped 25 63.0 c 25.6b " Treatments consisted of untreated soil (Non-tarped); soil fumigated with methyl bromide plus chloropicrin at 39.2 g/m^ (Methyl bromide); soil solarized under a 30-^m-thick, coextruded white-on-black, low density polyethylene film (White-on-black); or soil solarized under a 30|im-thick, clear, low density polyethylene film (Clear LDPE); or a 30fxm-thick, clear, gas impermeable plastic film (Gas Impermeable). Soil was either nonamended or amended with cabbage (+ Cabbage) at 80 and 68 tons/ha at sites 1 and 2, respectively. " Site 1 = Naples Tomato Growers-Gargiulo farm number 4 in Decatur County, Georgia (49 days of solarization). ^ Site 2 = John Allen Smith farm located in Gadsden County, Florida (48 days of solarization). " Weighted means ([Exp {mean}]-!). ^ Main effect means followed by the same letter do not differ according to Tukey's Honestly Significant Difference procedure (P< 0.05); data were transformed to ln(ppg+l) for analysis. ^ nt = not tested.

PAGE 42

33 Discussion Temperature Changes and Soil Moisture Content Soil solarization is a pasteurization process dependent on solar energy to heat the soil and kill pathogens. Effective pasteurization occurs when soil sustains temperatures that are lethal to the fungal propagules infesting the soil. In arid climates, soil temperatures between 48° and 54°C have been reported as effective for the control of Rhizoctonia solani, Pythium spp., Thielaviopsis basicola (Pullman et ai, 1979), Verticillium dahliae (Grinstein et al, 1979b; Pullman et al, 1979), Pyrenochaeta terrestris, Fusarium spp. (Katan et al, 1980) and Sclerotium rolfsii (Grinstein et al, 1979a). However, in climates where cloud cover and rainfall interrupt the heating process of the soil, maximum temperatures recorded were below 50°C (Chellemi et al., 1994). Temperature changes were monitored in site 2 for the experiments in sites 1 and 2, and in test 2 at site 3 for both tests 2 and 3. Inferences from one site in relation to the other have to be made with caution since soil color, proportion of sand, silt and clay content, and soil moisture were different. Each one of these factors can affect the temperature accumulation in soil, and when taken together could cause significant deviations in temperatures from the patterns observed at the site where temperatures were actually measured. These differences may hold for sites that are several kilometers apart (sites 1 and 2), as well as for sites a few meters apart, as was the case in site 3, between tests 2 and 3. Because tests 2 and 3 at site 3 were only a few meters apart, with the same type of soil, any differences in temperature could be explained best by the change in moisture content. As shown by Mahrer et al. (1984), the higher the water content of the soil, the higher the maximum temperature will be.

PAGE 43

34 Survival of Phvtophthora nicotianae Phytophthora nicotianae is believed to survive in root fragments and organic matter in the soil or as chlamydospores free in the soil (Erwin and Ribeiro, 1996). Infested wheat seeds were selected for inoculum to favor the formation of chlamydospores in soil and thus simulate natural spore formation for field survival. It is possible that lower temperatures during the first days of solarization allowed the formation of chlamydospores in the soil, and, therefore, a higher recovery was observed at the end of some experiments. Soil moisture also may have contributed to the variability in recovery of P. nicotianae from test to test and site to site. In test 2, the plastic treatments were laid down after a week of frequent rains, with soil moisture very uniform throughout the soil profile. In test 3, the week preceding the application of the plastic treatments was hot and dry, and irrigation was required to provide adequate moisture for the solarization. These factors were reflected in the results observed for the tests, especially in test 3, where higher numbers of surviving propagules were observed at lower depths, possibly indicating that temperatures did not rise as much as in test 2 due to the lack of moisture. According to Mahrer et al. (1984) temperature maxima of the soil increase with increasing soil moisture content. A 10-fold difference in the water status was observed between tests 2 and 3, which may have led to differences in survival of P. nicotianae at lower depths. The absence of chlamydospores in the wheat seeds at the time of preparation of the inoculum and a subsequent lack of survival of P. nicotianae in the nontarped treatments in 1994 and 1995 prompted a search for methods that would augment the production of this type of inoculum. loannou and Grogan (1985) indicated that chlamydospores were formed abundantly when mycelial mats in liquid cultures were exposed to an environment containing 10% COj and 2% Oj. This procedure was evaluated for the wheat seed inoculum, but increased chlamydospore production did not

PAGE 44

35 occur (Appendix A). However, when soil was infested and incubated at 18° or 25°C, large numbers of chlamydospores were formed (Appendix A). Since the highest number of chlamydospores was observed in the pasteurized soil from site 1 after incubation at 18°C for 10 days, inoculum was prepared in 1996 by infesting microwaved soil from site 1 and incubating it at 18°C for 10 days before burial in the field. The clear, low density polyethylene and the gas impermeable films were effective in reducing populations of P. nicotianae at the 10-cm depth. At this depth control was similar to that achieved by fiimigation with methyl bromide. However, at the 25 -cm depth, no control was observed, and the populations recovered on the dilution plates were similar to those in the nontarped or the white-on-black plastic controls. Clearly, solarization alone cannot be relied upon to control P. nicotianae in soil under conditions encountered in these tests. Chellemi et al. (1994) found that soil solarization reduced populations of P. nicotianae to undetectable levels at a depth of 15 cm; however, at the 25 -cm depth, no differences in survival were observed between the solarized and the control treatments in two sites. Survival of Phytophthora capsici Although the same two isolates were used in all crosses to produce oospores for all tests, the numbers of oospores observed in wheat seeds were not consistent from year to year. Another problem associated with the evaluation of survival of P. capsici is the germination of the propagules in the selective medium. In preliminary studies it was found that approximately 1% of the oospores germinated in PARPH, which was confirmed by the work of Larkin et al. (1995). Phytophthora capsici survived better in the nontarped treatments and under the white-on-black plastic, regardless of the cabbage amendment. Control of the pathogen was most effective with methyl bromide, followed by the gas impermeable and clear low density polyethylene film at the 10-cm depth. In

PAGE 45

36 1995 and 1996, fewer oospores were produced in the wheat seeds and the recovery of propagules after solarization was inconsistent, which precluded analysis of the results. Effect of Cabbage Amendment Cabbage did not affect the survival of either pathogen. This finding is contrary to that observed by other researchers (Keinath, 1996; RamirezVillapudua and Munnecke, 1987, 1988). It is possible that the differences observed are due to the preparation of the cabbage amendment and its incorporation into the soil. For all of the field plots, cabbage was either chopped with a machete (1994) or shredded in a shredder (1995 and 1996). In either case, the fragments were relatively large, and incorporation was done by disking the residue into the soil at the same time that the raised beds were being prepared, which may have led to an uneven distribution of the amendment in the soil profile. In contrast, RamirezVillapudua and Munnecke (1988) air dried the cabbage and ground it with a Wiley mill to a fine powder. This residue was incorporated into small amounts of soil, which were placed in plastic bags to be solarized. Further work by these authors (RamirezVillapudua and Munnecke, 1987) indicated that populations ofFusarium oxysporum f.sp. conglutinans were reduced by the incorporation of cabbage into the soil at rates that were higher than those used in the experiments reported here. Keinath (1996) found that gummy stem blight of watermelon was reduced in areas that received cabbage amendment and solarization; however, no information about the amount of residue incorporated into the soil was given. It may be that the effectiveness of cabbage as a soil amendment is influenced by the drying process, and, during the experiments reported here, rainfall during the drying time may have had a detrimental effect.

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CHAPTER 3 THERMAL INACTIVATION OF PHYTOPHTHORA NICOTIANAE Introduction Studies of thermal inactivation of plant pathogens can yield important information on survival of specific types of propagules, and, using simulated conditions, can provide information for the estimation of time constraints for effective soil solarization. JuarezPalacios et a/. (1991) found that determination of the heat sensitivity of isolates of Phytophthom spp. in the laboratory closely reflected their inactivation in solarized soil and indicated the possible use of soil solarization for management of those pathogens. These authors found that a high-temperature isolate of P. megasperma survived exposure for 30 minutes at 45°C in a heat sensitivity study, and that leaf discs used as bait were infected by the pathogen after solarization for 4 weeks; however, P. cinnamomi and a low-temperature isolate off. megasperma did not survive exposure to 45°C for 20 minutes. In similar studies, BoUen (1985) observed that oospores of P. capsici were more thermotolerant than mycelium from either mating type used to produce the oospores. The temperature difference for tolerance was more than 5°C, with oospores surviving at 50°C for 30 minutes. However, the system used did not reflect field conditions, since the continuously flooded soil employed in the experiment would rarely be attained in any field. BoUen (1985) did not analyze the relationship of time to varying temperature, and the standard temperature of 50°C used in the laboratory tests is rarely obtained at soil depths below 10 cm. One interesting aspect of this study was the use of cucumber seedlings for a plant disease assay, which was more sensitive than soil dilution plating on 37

PAGE 47

38 potato-dextrose agar (PDA, pH 5.6) for the detection of residual populations of the pathogen. Pullman et al. (1981a) observed a logarithmic relationship between time and temperature in the inactivation of propagules of four soilbome plant pathogens. These authors found that the exposure time required to reduce the population by 90% (LDgo) for oospores of Pythium ultimum was 30 minutes at 50°C, 110 minutes at 47°C, 8 hours at 45°C, 42 hours at 42°C, 13 days at 38.5°C and 26 days at 37°C, respectively. This study was performed using soil moisture adjusted to field capacity, and it closely reflected common temperature profiles in soil solarization tests. Studies of thermal inactivation can be complemented to simulate other conditions in the field, such as the addition of organic amendments to the soil prior to solarization (Gamliel and Stapleton, 1993a, 1993b; RamirezVillapudua and Munnecke, 1987, 1988). Cabbage residue associated with solarization was used successfully to control cabbage yellows, caused by Fusarium oxysporum f sp. conglutinans, by RamirezVillapudua and Munnecke (1987, 1988). The addition of cabbage alone was only partially effective for the control of cabbage yellows, but, when cabbage residue was heated during solarization, there was significant control of the pathogen. Due to the demands on time and equipment, thermal inactivation studies very seldom explore extensive combinations of both time and temperature ranges. Thus, the fiall benefits that can be derived fi^om such experiments are often not realized. The objectives of this study were to evaluate the effects of both constant and pulsing temperatures, cabbage amendment, soil water matric potential, and three, pasteurized and nonpasteurized soils on the survival of Phytophthora nicotianae.

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39 Materials and Methods Production of Chlamvdospore Inoculum of Phvtophthora nicotianae Chlamydospores of Phytophthora nicotianae were produced in V8 broth, as described by Mitchell and Kannwischer-Mitchell (1992). Four, 5-mm-diameter V8 juiceagar plugs of a culture with actively growing mycelium of isolate Pn21 were transferred to a 325-ml prescription bottle containing 25 ml of clarified V8 broth. After incubation at 25°C in the dark for 24 hours, the bottle was shaken vigorously to fi-agment the mycelial mats. The hyphal fragments adhering to the walls were resuspended by slowly rotating the bottle. The bottle was incubated horizontally as a stationary culture at 25°C for 6 days. One himdred milliliters of sterile deionized water were added to submerge the mycelial mat, and the culture was further incubated at 18°C, vertically, for a minimum of 3 weeks. The mycelial mats were rinsed in deionized water on a 400-mesh sieve, transferred to a blender with enough water to make a slurry, and blended on high for 1 minute. The resulting slurry was ground about 30 times in a glass mortar and pestle and then subjected to two, 30-second cycles of sonication at 100 W (Model 450 Sonifier, Branson Ultrasonics Corporation, Danbury, CT 06810). The total number of chlamydospores in the suspension was estimated with a hemacytometer, and the suspension was immediately used to infest soil to a density of 500 chlamydospores per gram of soil. Effect of Constant Temperature on the Inactivation of Chlamydospores of Phvtophthora nicotianae One-kilogram lots of moist soil fi'om Site 1 (Naples Tomato Growers-Gargiulo farm number 4 in Decatur County, Georgia) were pasteurized in a microwave at 700 W

PAGE 49

40 for 4 minutes in a plastic bag. After pasteurization soil moisture was adjusted to 6% (-15 kPa) with sterile water. Thirty grams of infested soil were dispensed into a test tube, which was loosely closed with a plastic cap to allow exchange of air. A set of test tubes was placed in each of eight water baths held at each of the following constant temperatures with circulation heaters: 25°, 35°, 38°, 41°, 44°, 47°, 50° and 53°C. The time of exposure at each temperature varied from 5 to 120 minutes or from 2 to 480 hours. Three tubes were removed at each time interval and part of the soil (15 g) was diluted with soft agar (2.5 g of Difco agar per liter of deionized water) and plated on a medium selective for pythiaceous fimgi within 24 hours. The selective medium (PARP) consisted of 17 g of commeal agar (Difco) in 1 liter of deionized water amended with 5 mg of pimaricin, 250 mg of ampicillin, 10 mg of rifampicin, and 100 mg of pentachloronitrobenzene (Mitchell and Kannwischer-Mitchell, 1992). The soil overlay was removed after 48 hours by gently washing the agar surface with tap water. The total number of colom'es formed on PARP after 72 hours of incubation was recorded as an estimation of the number of chlamydospores surviving the heat treatment. The other part of the soil (15 g) was transferred to a Petri plate and covered with approximately 10 ml of sterile water to provide a thin layer of water over the soil. Three, 3-day-old tomato seedlings, cultivar solar set, were placed on the soil in each plate and incubated in a growth chamber at 27°C for 3 days. The seedlings were rinsed twice in sterile water, blotted dry and plated on PARP to evaluate the colonization of tissues by P. nicotianae. The number of seedlings with fimgal colonization was recorded. The experiment with times of exposure ranging from 5 to 120 minutes was repeated once, while the experiment with times of exposure ranging from 2 to 480 hours was repeated twice.

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41 The Effect of Cabbage Amendments on the Thermal Inactivation of Chlamvdospores of Phvtophthora nicotianae In order to determine the effect of cabbage amendments on the inactivation of chlamydospores of P. nicotianae, experiments similar to the ones described previously were conducted, but temperatures were restricted to 35°, 38°, and 41 °C, and the time was limited from 2 to 480 hours. Cabbage leaves {Brassica oleracea var. capitata) were air dried in a greenhouse and ground in a Wiley mill with a 20-mesh screen. Pasteurized soil was amended with dry, ground cabbage leaves at 0, 0.125, 0.25 or 0.5% (w/w). Preliminary work indicated that concentrations above 0.5% were phytotoxic to tomato seedlings. Thirty grams of amended soil were dispensed into a test tube, a disk of filter paper was laid on top of the soil, and then thirty grams of amended and infested soil were dispensed on the top of the first layer. The tube was sealed with a gas impermeable plastic film (Bromotec film, Lawson Mardon Packaging, United Kingdom), and a plastic cap was placed over the tube to reduce the exchange of air and trap the volatiles released by heating the cabbage amended soil. The test tubes were placed in water baths held at constant temperatures with circulation heaters. Three tubes were removed at each time interval, and part of the soil (15 g) was diluted with soft agar and plated on PARP within 24 hours, as previously described. The soil overlay was removed after 48 hours by gently washing the agar surface with tap water, and the number of colonies was recorded. Fifteen grams of soil were transferred to Petri plates and covered with sterile water, and three, 3-day-old tomato seedlings were incubated on the soil slurry for 3 days. After incubation the seedlings were rinsed twice in sterile water, blotted dry, and plated on PARP. The number of colonized seedlings was recorded. Another test was done to determine if the bottom layer was necessary to ensure adequate concentration of volatiles in the test tube. Soil infested with the pathogen was

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42 dispensed into empty test tubes or into tubes containing a bottom layer of cabbageamended, noninfested soil. All test tubes were placed in the water baths at constant temperatures, as described above, and incubated for 1 week. The tubes were removed from the water baths, and the number of propagules were estimated using procedures previously described. No significant effects of the soil layering were observed when the data were analyzed. For this reason all other tests were done with only a single layer of infested, cabbage-amended soil. The Effect of Cycling Temperatures and Cabbage Amendments on the Thermal Inactivation of Chlamvdospores of Phvtophthora nicotianae A more critical analysis of the effects of daily temperature fluctuation, as observed during soil solarization, on the survival of the pathogen was performed in this experiment. Two temperatures, representing average thresholds at 10 and 25 cm of depth, were selected, and the diu^ation of the daily exposure at each temperature was determined by an analysis of published data from north Florida (Chellemi et ai, 1994). The daily regimes selected were 5 hours at 41 °C and 8 hours at 35°C, with a baseline temperature of 25°C during the rest of the day. These temperatures were maintained with circulation heaters in water baths. After preliminary tests indicated a high percentage of the spores survived the heat freatment, a third temperature, 44°C for 1.5 hours daily, was added. The duration of the daily exposure at 44°C was based both on the results of previous tests with constant temperature and on the analysis of temperature profiles from field experiments in 1994 (Chellemi et al., 1994). Microwaved and infested soil was adjusted to a final moisture of 6% with sterile water. Half of the infested soil was amended with dry, ground cabbage leaves at 0.125% (w/w). Thirty grams of soil were dispensed into a test tube, which was sealed with a piece

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43 of the gas impermeable plastic film, and a plastic cap was placed over the tube to reduce the exchange of air and trap the volatiles released by heating the cabbage-amended soil. The test tubes were placed in the water baths at 25°C until the temperature of the soil and the water were equilibrated, and then the cycling was initiated. Temperatures were monitored using a CRIO datalogger (Campbell Scientific, Inc., Logan, Utah 84321). Three tubes were removed after 1, 2, and 3 days, and every 3 days thereafter during a period of 24 days. Part of the soil (15 g) was diluted with soft agar and plated on PARP within 24 hours, as previously described. The number of colonies was recorded. The other part of the soil was used in a plant disease assay. At the end of the thermocycling experiment, all samples were transferred to 50ml, tripour plastic beakers and one, 1 -month-old tomato seedling was transplanted into the soil in each beaker. A small amount of vermiculite was poured on the top of the soil to prevent the roots from desiccating. All plants were kept in an growth chamber at 27°C for 30 days. As plants died, the root systems were rinsed in water, surface disinfested for 30 seconds in 70% ethanol, rinsed twice in sterile water, and plated on PARP. At the end of the experiment, all plants were cut and the root systems plated on PARP for determination of infection. The use of average temperatures achieved during soil solarization for the temperature cycling experiments was not sufficient to completely inactivate chlamydospores of P. nicotianae; therefore, an additional experiment was conducted using temperature regimes achieved during an optimum solarization day at the NFRECQuincy in 1995. Temperature regimes used were 47°C for 3 hours, 44°C for 5 hours, and35°C for 8 hours daily. Throughout this experiment a baseline temperature of 25°C was maintained during the rest of the day. The duration of each experiment was 15 days, and the experiment was repeated once. The procedures for production of chlamydospores, treatment of soil, and estimation of soil populations of P. nicotianae were the same as described previously. At

PAGE 53

44 the end of the temperature cycUng experiments, a plant disease assay was performed for 4 weeks with 1 -month-old tomato seedlings, as described previously. All dead plants were plated on PARP to confirm the presence of myceUum typical of Phytophthora spp., and at the end of the experiment all surviving plants were plated to determine if any infection had occurred. The Effect of Soil Water Matric Potential. Temperature Regimes, and Cabbage Amendments on the Thermal Inactivation of Chlamvdospores of Phytophthora nicotianae The effect the soil water matric potential in association with temperature regimes that simulate soil solarization and cabbage amendments was examined. The soil water matric potentials used were 0 kPa, -10 kPa and -30 kPa. These potentials were selected due to their importance on the Ufe cycle of the pathogen. The temperature regimes used were 1.5 hours at 44°C, 5 hours at 41°C and 8 hours at 35°C, or 3 hours at 47°C, 5 hours at 44°C and 8 hours at 35°C, with a baseline temperature of 25°C during the rest of the day. These temperatures were maintained with circulation heaters in water baths. The experiment was repeated once. The procedures for production of chlamydospores, treatment of soil, and estimation of surviving populations of P. nicotianae were the same as described previously. At the end of the temperature cycling experiments, a plant disease assay was performed for 4 weeks with 1 -month-old tomato seedlings, as described previously. All dead plants were plated on PARP to confirm the presence of mycelium typical of Phytophthora spp., and at the end of the experiment all surviving plants were plated to determine if any infection had occurred.

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45 The Effect of Three Nonpasteurized Soils. Temperature Regimes and Cabbage Amendments on the Thermal Inactivation of Chlamydospores of Phytophthora nicotianae The effects of nonpasteurized soils and cabbage amendments were examined using soil from the three field sites where soil solarization was evaluated from 1994 to 1996 (see Chapter 2 for a description of the soil types). The soil water matric potential of all soils was adjusted to -10 kPa, and the following temperature regimes were evaluated in one test, with three replicates per combination of soil, pasteurization, temperature and exposure time: 47°C for 3 hours, 44°C for 5 hours, 44°C for 1.5 hours, 41°C for 5 hours, and 35°C for 8 hours. The experiment was repeated once. The procedures for production of chlamydospores, treatment of soil, and estimation of surviving populations of P. nicotianae were the same as described previously. At the end of the temperature cycling experiments, a plant disease assay was performed for 4 weeks with 1 -month-old tomato seedlings, as described before. This assay was done with the soil from temperature regimes of 4 1 °C and above, since all previous experiments indicated that all of the plants were infected at the lower temperature regimes after 30 days. All dead plants were plated on PARP, amended with 50 mg of hymexazol per liter of medium to confirm the presence of mycelium typical of Phytophthora spp., and at the end of the experiment all surviving plants were plated to determine if infection had occurred. Statistical Analysis The results of each experiment were analyzed individually. Whenever statistical analysis of the residues indicated that the results could be pooled due to the lack of variation, a final analysis was done with the pooled data. The response surfaces were analyzed using the procedure PROC NLIN of S AS (SAS Institute, Cary, NC; release 6. 1 1 for personal computers). Survival data were

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46 transformed using In (ppg+1) prior to analysis. Comparisons between the qualitative and quantitative methods used to determine the number of chlamydospores surviving the heat treatment were made. The analyses of the effect of temperature cycling and cabbage amendment on the survival of chlamydospores of P. nicotianae were performed with Statgraphics Plus, version 2.1 (Manugistics, Inc., Rockville, MD), for the convenience of comparing several linearization models at once. The experiments on the determination of the effects of soil water matric potential and the effects of different soils on survival of P. nicotianae were analyzed using PROC GLM of SAS for the analysis of variance. The determination of the effect of each individual factor was calculated as the average of that factor across all other factors; the analysis of each interaction was done by calculating the average for the secondary factor within the main factor across all other factors. Results Effect of Constant Temperature on the Inactivation of Chlamvdospores of Phvtophthora nicotianae Phytophthora nicotianae was consistently recovered throughout the 2-hour experiment when temperatures were below 47°C (Table 3.1). At 44°C, a sharp decrease was observed after 30 minutes, but 10% of the initial population (500 ppg) survived to the end of the experiment. After 75 minutes of heat treatment at 47°C, population levels dropped below 1 propagule per gram of soil, and after 2 hours populations had declined to levels undetectable by the procedure used to quantify the inoculum in the soil. At 50° and 53°C, populations were very low after only 5 minutes of exposure.

PAGE 56

47 o 3 C s > o •a > O o o <+O S « 00 CO SO a. ' o ' u a. <+-. o ' (U < X) S 3 3 g 53 H o 'O m rn t~o CO ^ OS On oo r-i o 00 On o 00 ^ " t~On — Vi 0> Qo' o °g 00 'O 00 O 3i <^ ^ ^2 ON oo m d ^ Tjm CN On O r-) ^ ^ ^ ^ o ^ ^ ^ CN ^ ro o ON o d m d ON ON ^. CN d oo 00 oo d CN 00 00 cs d (N ON CN O d CS 00 in 2 ^ r— d d NO cs >> in cs NO in o m d 00 NO 00 00 o 00 NO m cs d c o u > o o u C5 a. en 4) u. O O. C/5 O s -2 "o o o in T3 -*-» C/3 ,1>

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48 The decline in survival of the chlamydospores of P. nicotianae in soil exposed to constant heat above 38°C for 5 to 120 minutes was best described by the equation /«(ppg + 1) = (6.2 0.00108* time^ >)^g-o.02208*temperature ^ ^^^^ ^j^^ comparison of the plotting of Observed , Predicted Values, Residuals X Time, and Residuals X Temperature, and on the R^^^j = 0.8234, in which ppg is the number of propagules recovered per gram of soil. The use of tomato seedlings as bait for the detection of survival of P. nicotianae indicated that survival was affected by the heat treatment only after 90% of the initial population of chlamydospores had been inactivated, which occurred generally only after exposure to 47°C or higher (Tables 3.1 and 3.2). The detection of survival with the use of seedlings was not as sensitive as the detection with the soil dilution procedure used. For example, at 53°C no seedlings were infected after the 3-day incubation period (Table 3.2); in contrast, the soil dilution procedure indicated that 0.1 propagule per gram of soil were still alive after 15 minutes of treatment (Table 3.1). Exposure of chlamydospores of P. nicotianae to heat for 480 hours indicated that the pathogen can survive in the at 35°C for a long period (Table 3.3). Populations declined to levels below 1 propagule per gram of soil when temperatures were maintained at 38°C for more than 288 hours. After 96 hours of exposure at 41 °C, or 48 hours at 44°C, no propagules were recovered fi-om the soil. Exposure to 47°C reduced the populations to residual levels after 4 hours. The decline in survival of the chlamydospores of P. nicotianae in soil exposed to constant temperatures above 38°C for 2 to 480 hours was best described by the equation /«(ppg + 1) = 6.2*e(-*^°*^'^^*^'"^-°°'^^*^^'"P^^^^^^) , based on the comparison of the plotting of Observed X Predicted Values, Residuals X Time, and Residuals X Temperature, and on the R\dj = 0.6773, in which ppg is the number of propagules recovered per gram of soil.

PAGE 58

49 4—* 3 C > o c 1) CO 0) -t3 N '2 _o o o so CO 4-» c o aQ. E o o o o o „ o o o o o ^ o o o o o o o o o o o o o o o _ o o o o o ;i. O O O O O o o o o o ^ o o o o o ^ O O O O O I I o o o o o _ O O O O O ^ O 1 o o o o o o o o O O O w o o o o o o o o o o o o o g g o o o ir\ IT) oo
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50 o oo 00 00 00 E > o T3 o o o o e oc 2: o fN c^ ^ 00 o — ^ — o o ^" C-~ ro O ^ O o 1 s i u> • — ^ o -4-* tn '!« C (/I T3 — o o o
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51 Generally, the use of the tomato seedling assay provided similar results to the soil dilution plating procedure, except when only residual inoculum (0.1 propagule per gram of soil) was left (Tables 3.3, 3.4). When residual inoculum survived the heat treatment, baiting the soil with seedlings allowed detection of the fimgus in two cases that were not detected by soil plating, but failed to detect the pathogen in three other instances that were detected by the plating procedure. All tomato seedlings were colonized by P. nicotianae whenever more than 50 propagules per gram of soil were recovered in the soil dilution plates with PARP; in one case 100% infection was observed when 10 propagules per gram of soil were recovered. The Effect of Cabbage Amendments on the Thermal Inactivation of Chlamvdospores of Phytophthora nicotianae The addition of dried, ground cabbage leaves changed the response of the chlamydospores to heat (Table 3.5). As concentration of cabbage increased, inactivation also increased; thus, less time was required to inactivate spores in cabbage amended soil. At 35°C chlamydospores survived in the soil for 480 hours when no cabbage amendment was present (Table 3.5). The addition of 0.125% cabbage to the soil reduced the population to residual levels after 192 hours. Ninety six hours were required with 0.25%) cabbage to reduce population counts below one propagule per gram of soil; similar population reductions were attained after 48 hours when 0.5%) cabbage was added to the soil. The addition of cabbage to soils incubated at 38°C reduced pathogen populations in the following manner: the time required to reduce the inoculum to below 1 propagule per gram of soil dropped fi-om 1 92 hours without cabbage amendment to 48 hours with 0.125% or 0.25%o amendment, and to 24 hours with 0.5% amendment added to the soil. Survival of the chlamydospores in soil treated at 41°C declined to undetectable levels

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52 3 O o 00 o a c B ts u >> CO CO c« £>0 C CO x; X) c o '-4-» o (U 4-* o -4— t C CO N 'S _o o a CO c« C o 3 13 (D O. o o o o o o 2 2 2 o o o o 2 r2 2 t-^ o o 2 2 \0 oo ^ o o o o _ o o o ^ o o o o _ o o o ^ o o o o o o o o o o o o o o o o o o ^ o o o o o o o o o o o o o o o o o ^ c« c o o a(u I 5

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53 o 00 3 O E oo 00 0) > o 0) > o o o (N eC 30 9> O. (N i/l 3 a o a. o u. 0) Q. O 0) o !-< >N £ o CO D in " T3 ^ « .23 , '3 o *r: C ^ D J2 O CO O ttJ Z

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54 after 96 hours in nonamended soil, 48 hours in soil amended with 0.125% cabbage, and 24 hours in soil amended with 0.25% cabbage (Table 3.5). Only 16 hours were required to reduce the population to 0.2 propagule per gram of soil in soil amended with 0.5% cabbage. The decline in survival of the chlamydospores of P. nicotianae in soil amended with cabbage residue and exposed to constant temperature was best described by the linear-power model presented in Table 3.6. According to the model used for comparisons of survival of P. nicotianae, the equation describing the inactivation of chlamydospores in the absence of cabbage amendment is significantly different from the others (P<0.05); the equations generated for amendment with 0.125% and 0.25% cabbage are not different from each other, and the equation generated for amendment with 0.5% cabbage is different from all other equations. Detection of survival of P. nicotianae with tomato seedlings as baits followed similar trends as those observed with the soil dilution plating procedure (Tables 3.5, 3.7). As the number of propagules recovered in the soil plates decreased from 500 to 100 propagules per gram of soil, the percentage of colonized seedlings was relatively constant. Further decreases in the propagule counts generally reflected a decrease in the percentage of colonized seedlings. Of the 41 cases in which no propagules were detected by the soil dilution plating procedure, the survival of the pathogen was detected in 17 cases by the baiting technique. Generally, this discrepancy was observed right after the number of propagules recovered in the selective medium had dropped below 1 propagule per gram of soil.

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55 Table 3.6. Equations of models that describe the thermal inactivation of chlamydospores of Phytophthora nicotianae as a factor of constant temperature between 35°C and 41°C, time of exposure ranging from 2 to 480 hours, and cabbage amendment concentrations. Cabbage (%) Equation 0 /w(ppg^ + l): = (6.2 0.0141* Time)*e-^°°^^*^''"P"^"^ 0.5062 0.125 /«(ppg + l) = (6.20.0181* Time)*e-*^°^^'*'^"^'"P"^'"^" 0.5474 0.25 /«(ppg + 1) = (6.20.0183* Time)*e-°°2^2*^''"P"^'"'' 0.6124 0.5 /n(ppg + l) = (6.20.01 87* Time)* 0282. Temperature 0.6928 ^ ppg = propagules per gram of soil.

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56 o 00 00 CO 3 o 4^ > O G o cn O CM T3 N "E o o o ao c o 00 00 x> u 3 a. °^ S < «^ cn oo oo (N tM ^ 00 c^ vo fN c^ '<3r4 CN — o o o o o o o o o o o o o o o o o o 2222S2oo«-ix>^ o o O O O O :0 o '^r 2 2 2 2 2 2 2 S ^ 2 o5^ S ^ o S ^ S 0-=^ o o ° o ^' inir>u-iu-ioooooooo— ' — — ' s: o CO D c o a. l-l (U 4) Ic (L> s 'C 0) Q. x lU o o CO o c o o c -a o g o

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57 The Effect of Cycling Temperatures and Cabbage Amendments on the Thermal Inactivation of Chlamvdospores of Phvtophthora nicotianae Generally, the use of pulsing temperatures increased the time required to inactivate the chlamydospores of P. nicotianae in comparison with the constant temperature treatment (Tables 3.5, 3.8). After 24 days at 35°C for 8 hours daily, 40% of the initial infestation level of spores (500 chlamydospores per gram of soil) was still recovered from the nonamended soil in the selective medium. The addition of 0.125% cabbage to the soil reduced the population to levels below 10% after 18 days. The effect of cabbage was evident from the very first cycles. Cycling temperatvu-es at 41°C for 5 hours daily reduced the survival of the spores by more than 90% of the initial infestation level after 24 days; when cabbage was added, less than 5% of the propagules were recovered from the soil at the end of the experiment. Survival was below 5% of the imtial infestation level after 21 days of exposure to 1.5 hours at 44°C daily; with cabbage amendment, detection was lower than that observed in the nonamended soil. Exposure to temperature regimes simulating optimum solarization periods reduced the populations to very low levels (Table 3.8). Five hours daily at 44°C for 15 days reduced the population to 0.4 propagule per gram of soil. The use of cabbage amendment reduced populations to the same levels after 6 days. Only 3 days of exposure at 47° for 3 hours daily were required to reduce populations to levels below 1 propagule per gram of soil; with cabbage amendment populations were reduced to levels below 1 propagule per gram of soil after 2 days. Populations were reduced to levels below detection only at the two highest temperature regimes (44°C for 5 hours daily and 47°C for 3 hours daily) after 9 days of treatment. Soil amendment with cabbage appeared to have a greater impact at lower temperatures, where the heat alone was not sufficient to inactivate the chlamydospores.

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58 Table 3.8. The effect of temperature regime, cabbage amendment, and time on the survival of chlamydospores of Phytophthora nicotianae. Temperature Cabbage Number of propagules per grain of soil recovered over time (days) Regime^ (%) 1 2 3 6 9 12 15 1 0 18 1 1 21 24 35-8 0 321.9 336.4 304.1 334.6 342.2 314.1 324.4 256.7 234.0 199.3 35-8 0.125 114.5 121.2 129.4 130.5 108.8 84.0 80.8 41.7 32.9 30.9 41-5 0 251.4 329.1 359.0 : 353.0 250.2 247.2 155.9 80.7 51.9 41.3 41-5 . 0.125 47.8 58.2 42.6 41.3 37.2: 22.2 24.5 18.6 21.5 18.7 44-1.5 0 98.0 93.8 91.5 75.5 41.0 36.8 33.2 25.3 22.0 19.8 44-1.5 0.125 31.4 33.1 35.0 24.0 20.0 16.8 15.6 16.3 12.6 14.4 44-5 0 47.2 34.0 26,0 :/ 5:2 2.5^ 1.7 0.4 44-5 0.125 34.4 14.6 2.7 0.4 0.0 0.0 0.0 47-3 0 3.2 1.2 0.3 0.1 0.2 0.0 0.1 47-3 0.125 2.2 0.1 0.0 0.1 0.0 0.0 0.0 " Soil initially infested with 500 chlamydospores per gram. ^ Temperature regimes that simulated solarization consisted of temperatures increased daily to 35°, 41°, 44°, 44°, or 47°C for 8, 5, 1.5, 5 or 3 hours, respectively; the remainder of each day was maintained at 25°C. ^ Not tested.

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59 The reduction in survival of P. nicotianae was modeled using linear regression. The model selected for the comparisons among temperature regimes and cabbage amendment was ln(Y + l) = a + b*X,in which Y is the number of propagules recovered per gram of soil and X is the number of days of exposure to the temperature regime. A lack of fit of the model to the data was observed at 35°C, regardless of the cabbage amendment (Table 3.9). As temperature increased a better fit of the model was observed, but at the highest temperature regime tested a lower fit was obtained again. The plant disease assay verifying the pathogenicity of the surviving populations of P. nicotianae indicated that temperature regimes below 44°C for 5 hours daily did not reduce the infection of the tomato seedlings (Table 3.10). At 44°C for 5 hours, infection was reduced in the soil amended with cabbage after 3 days. After 3 days of heat treatment at 47°C for 3 hours daily, no infection of seedlings was observed. Mortality of the seedlings was observed in the lower temperatiire regimes (Table 3.1 1); however, when the soil was treated at 44°C for 5 hours daily no seedlings died, except after 2 or 3 days in soil amended with cabbage. No seedlings died when the soil was heated to 47°C for 3 hours daily. The Effect of Soil Water Matric Potential Temperature Regimes, and Cabbage Amendments on the Thermal Inactivation of Chlamvdospores of Phvtophthora nicotianae The temperature regimes evaluated in these tests simulated either average daily periods or optimum daily temperature periods observed during solarization. Generally, as temperature within the regimes increased, the number of chlamydospores recovered in the soil dilution plates decreased (Tables 3.12, 3.13, 3.14). Propagule survival in the two temperature regimes simulating optimum solarization periods (47°C for 3 hours and 44°C

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60 Table 3.9. Regression coefBcients of the linear regressions of temperature regimes and survival of chlamydospores of Phytophthora nicotianae, the model used for the analysis was In (ppg+1) = a + b*days. Temperature Cabbage Parameters P-Value^ Regime" % slope intercept 35-8 0 -0.01203 5.717 0.0267 0.062 35-8 0.125 -0.05508 4.808 0.2800 0.001 41-5 0 -O.09034 6.049 0.6679 OOOl 41-5 0.125 -0.05091 3.812 0.2195 0.001 44-1.5 0 -0.07680 4.631 0.7264 0.001 44-1.5 0.125 -0.04426 3.454 0.2856 0.001 44-5 0 -0 26132 3.886 0.8684 0.001 44-5 0.125 -0.21618 2.531 L: 0.5631 0.001 47-3 0 -0.05692 0.718 0.2422 0.001 47-3 0.125 -0.04026 0.455 0.2272 0.001 " Temperature regimes that simulated solarization consisted of temperatures increased daily to 35°, 41°, 44°^ 44°^ or 47°C for 8, 5, 1.5, 5 or 3 hours, respectively; the remainder of each day was maintained at 25°C. ^ Significance associated to the coefficient of determination (R").

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61 Table 3.10. Infection of tomato seedlings after incubation in soil amended with cabbage, infested with Phytophthora nicotianae, and heat treated over time. Temperature Cabbage Percent infection of tomato seedlings over time (days) Regime (%) 1 2 i o Q y 1 0 1 <: 1\ £• i. 24 35-8 0 100^ 100 100 100 100 100 100 100 100 1U(J 35-8 0.125 100 100 100 100 100 100 100 100 100 100 41-5 0 100 100 1(X) 100 100 100 100 100 100 100 41-5 0.125 100 100 100 100 100 100 100 100 100 100 44-1.5 0 100 100 100 100 100 100 100 100 100 100 44-1.5 0.125 100 100 100 100 100 100 100 100 100 100 44-5 0 100 100 100 100 100 100 83 7. 44-5 0.125 100 100 67 67 50 67 53 47-3 0 100 100 0 0 0 0 0 47-3 0.125 100 83 0 0 0 0 0 " Temperature regimes that simulated soiarization consisted of temperatures increased daily to 35°, 41°, 44°^ 44°^ or 47°C for 8, 5, 1.5, 5 or 3 hours, respectively; the remainder of each day was maintained at 25°C. ^ Each data point consists of the average of two experiments with three repetitions each. ^ Not tested.

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62 Table 3.11. Mortality of tomato seedlings after incubation in soil amended with cabbage, infested with Phytophthora nicotianae, and heat treated over time. Temperature Cabbage Percent mortality of tomato seedlings over tiine (days) Regime" (%) 1 2 3 6 9 12 15 18 21 24 35-8 0 33y 27 27 27 33 33 33 33 44 44 35-8 0.125 33 27 20 40 27 40 33 44 44 33 41-5 0 33 33 33 44 ^;:^;33":^^ 33 44 56 41-5 0-123 ^ 44 33 44 33 , 44 33 33 44 44 22 44-1.5 0 44 44 33 44 33 22 33 11 22 22 44-1.5 0.125 33 33 33 33 33 45 0 22 11 11 44-5 0 0 0 0 0 :;:o 0 0 44-5 0.125 0 16 33 0 0 0 0 47-3 0 0 0 0 0 0 0 0 47-3 0.125 0 0 0 0 0 0 0 " Temperature regimes that simulated solarization consisted of temperatures increased daily to 35°, 41°, 44°^ 44°^ or 47°C for 8, 5, 1.5, 5 or 3 hours, respectively; the remainder of each day was maintained at 25°C. ^ Each data point consists of the average of two experiments with three repetitions each. ^ Not tested.

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63 Table 3 .12. The effect of temperature regimes that simulate daily solarization periods and cabbage amendment, at a soil water matric potential of 0 kPa, on the survival of chlamydospores of Phytophthora nicotianae, and on the percentages of infection and Temperature Cabbage Test 1 Test 2 Regime" (%) Days" PPG* r PPG T 1 M (%) (%) (%) (%) 35-8 0 1 223.08 e' 100 0 308.82 e 100 33 35-8 0 2 433.85 f 100 0 540.86 e 100 33 35-8 0 3 413.47 f 100 0 409.76 e 100 33 35-8 0 6 672.84 f 100 0 213.86 de 100 100 35-8 0.125 1 236.46 e 100 0 91.48 cde 100 33 35-8 0.125 2 226.47 ef 100 0 226.47 de 100 0 35-8 0.125 .J 169.72 c 100 0 110.16 cde 100 ^^17 \ 35-8 0.125 6 133.47 e 100 0 67.58 cde 100 iOQ 41-5 0 1 150.41 e 100 0 214.51 de 100 33 41-5 0 3 248.39 ef 100 0 104.64 cde 100 33 41-5 0 6 126.74 e 100 0 62.37 bed 100 67 41-5 0.125 1 90:84de 100 0 37.78 bed 100 0 41-5 0.125 62.43 de 100 0 1 1.06 b 100 100 41-5 0,125 6 34.52 c 100 0 27.73 be 100 33 44-1.5 0 1 34.16 cd 100 0 29.14 be 100 33 44-1.5 0 3 19.29 bed 100 0 15.61 b 100 67 44-1.5 0 6 14.80 bed 100 0 10.32 b 100 67 44-1.5 0.125 1 49.91 de 100 0 42.90 bed 100 44-1.5 0.125 ::;::3 54.15 de 100 0 12;46b 100 67 0,125 6 20.98 cd 100 33 15 01 b 100 67 44-5 0 1 14.80 bed 100 0 18.39 be 100 0 44-5 0 2 7.76 bed 100 0 100 0 44-5 0 3 6.54 bed 100 0 9.32 b 100 0 44-5 o:i25 1 6 17 be 100 0 10.94 b too 0 44-5 0.125 7"2 5.42 be 100 0 6.61 b 100 ^ 0;; ;;; 44-5 0.125 3 3.62 be 100 0 6.24 b 100 0 47-3 0 1 0.62 a 100 0 0.78 a 100 0 47-3 0 2 0.00 a 67 0 0.00 a 100 0 47-3 0 3 0.00 a 67 0 0.00 a 100 0 47-3 0.125 1 0.52 a 100 0 0.92 a 100 :0: :\ ; 47-3 0.125 2 0.00 a J.) 0 0.00 a 100 0 47-3 0.125 3 0 00 a 50 0 0.00 a 100 0 " Temperature regimes that simulated daily solarization periods consisted of temperatures increased daily to 35°, 41°, 44°C, 44°C or 47°C for 8, 5, 1.5, 5 or 3 hours, respectively; the temperature for the remainder of each day was maintained at 25°C. " Days = duration of temperatiu^e regime. * PPG = propagules per gram of soil; initial population was 500 chlamydospores per gram of soil. " I = infection. ^ M = mortality. ^ Main eflFect means followed by the same letter in each test do not differ according to Tukey's Honestly Significant Dtfiference procedure (P< 0.05); data were transformed to ln(ppg+l) for analysis and presented as weighted means ([Exp {mean}]-!).

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64 Table 3.13. The effect of temperature regimes that simulate daily solarization periods and cabbage amendment, at a soil water matric potential of -10 kPa, on the survival of chlamydospores of Phytophthora nicotianae, and on the percentages of infection and Temperature Cabbage Test 1 Test 2 Regime" (%) Days' PPG" r PPG 1 M (%) (%) (%) (%) 35-8 0 1 181.18 e 100 17 202.16 cd 100 0 jjij 0 2 247.14 e 100 0 287.59 d 100 0 35-X 0 3 168.02 e 100 0 147.41 cd 100 17 35-8 0 6 191.48 e 100 0 115.63 cd 100 67 35-8 01 25 1 325.69 e IQO 0 218.42 d 100 50 35-8 0.125 " 2 274.06 e 100 67 274.06 d 100 0 35-8 0.125 3 200.34 e 100 0 115.51 cd 100 17 35-8 0.125 6 107.85 de 100 0 65.02 cd 100 33 41-5 6 1 449.34 e 100 0 258.82 d 100 33 41-5 0 3 261.43 e 100 0 109.50 cd 100 100 41-5 0 6 209.61 e 100 0 73.22 cd 100 67 41-5 0.125 1 215.16 e 100 0 146.67 cd 100 33 41-5 0.125 3 127:77 e : 100 0 77.89 cd 100 67 :::::: 0.125 6 97 JO de 100 0 55.15 cd 100 67 44-1.5 0 1 44.15 cd 100 0 14.26 b 100 33 44-1.5 0 3 19.70 cd 100 0 6.24 b 100 33 44-1.5 0 6 14.03 c 100 0 8.73 b 100 33 "44-1.5 0. 125 I 70:95 de lOO 0 74.19 cd 100 0 44-1.5 0.125 3 59;95d iOO 0 78.44 cd 100 33 44-1.5 0.125 6 45.34 cd I GO 33 39.77 cd 100 33 44-5 0 1 16.10 c 100 33 23.83 b 100 0 44-5 0 2 10. 10 be 100 0 lo.yy b 0 44-5 0 3 4.64 b 100 0 6.92 b 100 0 44-5 0.125 1 8.38 be 100 0 16.37 b 100 0 44-5 0.125 1 3.65 b 100 0 12.38 b 100 0 44-5 0.125 3 1:80 ab 100 0 8.48 b 100 0 47-3 0 1 2.24 b 100 0 3 .13 ab 100 0 47-3 0 2 0.30 a 100 0 0.25 a 100 0 47-3 0 3 0.09 a 100 0 0.00 a 100 0 47-3 0.125 1 1:70 ab 100 0 5.75 b 100 0 47-3 0.125 2 0.52 a 100 '\ o 0.45 a 100 0 47-3 0.125 3 0.45 a 100 0 0.28 a 100 0 " Temperatm-e regimes that simulated daily solarization periods consisted of temperatures increased daily to 35°, 41°, 44°C, 44°C or 47°C for 8, 5, 1.5, 5 or 3 hoiu-s, respectively; the temperature for the remainder of each day was maintained at 25°C. " Days = duration of temperature regime. * PPG = propagules per gram of soil; initial population was 500 chlamydospores per gram of soil. " 1 = infection. ^ M = mortality. ^ Main effect means followed by the same letter in each test do not differ according to Tukey's Honestly Significant Difference procedure (P< 0.05); data were transfonned to ln(ppg+l) for analysis and presented as weighted means ([E.xp {mean}]-l).

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65 Table 3.14. The effect of temperature regimes that simulate daily solarization periods and cabbage amendment, at a soil water matric potential of -30 kPa, on the survival of chlamydospores of Phytophthora nicotianae, and on the percentages of infection and Temperature Cabbage Test 1 Test 2 Regime" (%) Days' PPG" r M'' PPG I M (%) (%) (%) (%) 35-8 0 1 225.11 e 100 50 90.81 d 100 33 35-8 0 2 190.71 e 100 0 221.96 d 100 0 35-8 0 3 185.98 e 100 17 161.71 d 100 33 35-8 0 6 207.30 e 100 33 114.93 d 100 0 35-8 0:125 1 349.37 e 100 0 137.80 d 100 17 35-8 0.125 2 385,45 e 100 0 385.45 d 100 0 35-S 0. 125 230.60 e 100 17 107.96 d 100 50 Jw'.'*0 ft 175 6 133.29 de 100 0 63.07 cd 100 100 4T-5 *T L J 0 1 183.93 e 100 0 288.17 d 100 33 41-5 0 3 253.68 e 100 0 153.93 d 100 33 41-5 0 6 240.29 e 100 0 82.18 cd 100 33 41-5 0.125 1 371.78 e 100 0 116.20 d 100 67 41-5 0.125 3 124.21 dc 100 0 80.29 cd 100 67 ::: •:41-5. 0.125 : 6 85.57 de 100 0 56.34 cd 100 33 44-1.5 0 1 65.75 d 100 0 25.21 c 100 33 44-1.5 0 3 19.41 c 100 0 6.43 b 100 0 44-1.5 0 6 14.77 be 100 0 6.55 b 100 33 44-1.5 0.125 1 86.01 de 100 0 95.06 d 100 44-1.5 0.125 3 53.27 cd 100 0 30.88 c 100 67 44-1.5 0.125 6 42.73 cd: ::: 100 33 ^ 28.11 c 100 100 44-5 0 1 10.67 be 100 0 9.86 be 100 0 44-5 0 2 9.52 be 100 0 /.J J DC 1 nn 0 44-5 0 3 5.83 b 100 33 4.80 b 100 0 44-5 0.125 1 9.31 be 100 0 14.25 be 100 0 44-5 0.125 2 6 01 b 100 0 8.55 be 100 0 44-5 0.125 3 3.23 b 100 0 7.66 be 100 0 47-3 0 1 1.90 ab 100 0 1.44 ab 100 0 47-3 0 2 0.09 a 100 0 0.10 a 100 0 47-3 0 3 0.09 a 100 0 0.28 a 100 0 0.125 1 2 79 b 100 0 4.07 b 100 0 : 47-3: 0.125 2 0.79 a 100 0 1.15 ab 100 0 47-3 0.125 3 0.91 a 100 0 0.86 ab 100 0 " Temperature regimes that simulated daily solarization periods consisted of temperatures increased daily to 35°, 41°, 44°C, 44°C or 47°C for 8, 5, 1.5, 5 or 3 hours, respectively; the temperature for the remainder of each day was maintained at 25°C. " Days = duration of temperature regime. * PPG = propagules per gram of soil; initial population was 500 chlamydospores per gram of soil. " I = infection. ^ M = mortality. ^ Main effect means followed by the same letter in each test do not differ according to Tukey's Honestly Significant Difference procedure (P< 0.05); data were transformed to ln(ppg+l) for analysis and presented as weighted means ([E.xp {mean}]-l).

PAGE 75

66 for 5 hours daily) were significantly lower than those in the base temperature regime of 35°C for 8 hours daily, and generally lower than those treated at 41°C for 5 hours daily, regardless of the soil water matric potential (P<0.05). The use of a plant disease assay to confirm the pathogenicity of the surviving population of P. nicotianae indicated that, when the soil was maintained at 0 kPa and treated at 47°C for 3 hours daily, there was a reduction in infection of the seedlings in test 1 only (Table 3.12). In all other treatments under all three matric potentials, all of the seedlings were infected by P. nicotianae (Tables 3.12, 3.13, 3.14). A higher proportion of mortality of seedlings was observed in the second experiment than in the first experiment; however, little or no mortality occurred at any soil water matric potential when the soil was treated at 44°C for 5 hours daily or at 47°C for 3 hours daily. Over all treatments, in both tests, soil water matric potential had a significant effect in reducing the populations of P. nicotianae in the soil (P<0.05) (Table 3.15; Appendix D). In both tests the lowest survival was observed in saturated soils (0 kPa) (Tables 3.12, 3.13, 3.14; Appendix D). No differences were observed in the survival of chlamydospores in soils maintained at either -10 kPa or -30 kPa in test 1; however, in test 2 survival at -10 kPa was higher than at -30 kPa. As temperature increased, survival of P. nicotianae decreased (Table 3.15; Appendix D), and survival at each temperature regime was significantly different fi-om that in all others (P<0.05). Survival was lowest in soil maintained at 47°C for 3 hours daily (Appendix D). The incorporation of cabbage amendment into soil significantly reduced the populations of P. nicotianae in test 1, while no effect on the survival of the pathogen was observed in test 2 (P<0.05) (Table 3.15, Appendix D). However, differences in populations between amended and nonamended soil exposed to specific temperature regimes were not evident fi-om the analysis (Tables 3.12, 3.13, 3.14).

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67 Table 3.15. Analysis of variance of the effect of temperature regimes that simulate solarization periods, cabbage amendment, and soil water matric potential on the survival of chlamydospores of Phytophthora nicotianae Source of Variation Test 1 Test 2 df MS F P MS F P T^rf^^A** TV^f^^*^**^ V^J^^An'A'l o 1 ^ oOli waier luainc poicnnai T i. L.J J J 16 6$t 0 0001 A Tfi4 «07 31 14 74 0 0001 710 227 \.\J .imi* 1 1782.67 0.0001 V — d U <-l £^ ^ 1 0.924 10.87 0.0001 0.001 0.01 0.9149 I line I J A 7/10 U.UUUl 1 1 72'; 1 1. 1 LJ QQ X'X X Temperature Q O i.uyo 1 T CO U.UUUl l.JOO 1 1 1 l.jy U.UUU 1 !fm X Cabbage 2 1.827 21.48 0.0001 6.789 57.57 0.0001 If™ X Time 6 0.375 4.41 0.0003 0.133 1.13 0.3442 Temperature x Cabbage 4 4.658 54.77 0.0001 8.255 70.01 0.0001 Temperature x Time 8 0.873 10.26 0.0001 2.024 17.16 0.0001 Cabbage x Time 3 0.643 7.57 0.0001 0.036 0.30 0.8230 Temperature x 'P^ x Cabbage x 54 0.225 2.65 0.0001 0.265 2.25 0.0001 Time Residual 228 0.085 0.118 ^ Soil water matric potential adjusted to 0, -10, or -30 kPa. ^ Temperature regimes that simulated daily solarization periods consisted of temperatures increased daily to 35°, 41°, 44°C, 44°C or 47°C for 8, 5, 1.5, 5 or 3 hours, respectively; the temperature for the remainder of each day was maintained at 25°C. ^ Soil amended or not amended with dry, ground cabbage at a rate of 0. 125% (w/w). ^ Time = number of days of exposure to heat treatment at a given temperature regime.

PAGE 77

68 Generally, the longer the soil infested with chlamydospores of P. nicotianae was exposed to the heat treatments, the lower the survival of the pathogen (Tables 3.12, 3.13, 3.14, 3.15). However, the effect of time has to be analyzed with care because of the different durations of exposure to the temperature regimes. Because the time required to kill chlamydospores is inversely proportional to temperature, shorter sampling times (1, 2, and 3 days) were used at the higher temperatiu-e regimes (47°C for 3 hours daily and 44°C for 5 hours daily) and longer sampling times (1, 3, and 6 days) were used at the average temperature regimes (44°C for 1.5 hours daily and 41 °C for 5 hours daily); the base temperature (35°C for 8 hours daily) was sampled at all time intervals (1, 2, 3, and 6 days). Therefore, the analysis of the effect of time across all other factors is biased in the following manner: the data for 2 days of exposure was obtained only from the temperature regimes that simulate optimum solarization periods and the base temperature regime; after 3 days of heat treatment, a final sample was collected from the optimum temperature regimes, and intermediate samples were collected from the average temperature regimes; after 6 days only the average temperature regimes and the base temperature regime were evaluated. Consequently, artificially higher survival rates were generated at 6 days as compared to 2 or 3 days because the lowest survival rates under the optimum temperatxu-e regimes had not been determined (Appendix D). The interactions of soil water matric potential or cabbage with time reflect the same bias, and the responses followed trends similar to those described here for time alone. Although these two interactions were significant (Table 3.15), the biased results did not allow insight into the roles of soil water matric potential or cabbage amendment in relation to time of exposure on the survival of the pathogen. Within each of the soil water matric potentials evaluated, each of the temperature regimes significantly reduced the populations of P. nicotianae in relation to the base temperature (35°C for 8 hours daily) (P<0.05) (Tables 3.12, 3.13, 3. 14, 3.15; Appendix

PAGE 78

69 D). Survival of P. nicotianae at each temperature regime was significantly lower than that at all other preceding temperature regimes. Although there was a significant interaction of soil water matric potential and cabbage amendment, the effect of the amendment on survival of P. nicotianae was not consistent throughout the range of soil water matric potentials evaluated (P<0.05) (Tables 3.12, 3.13, 3.14, 3.15; Appendix D). Cabbage amendment reduced the survival of propagules in soil maintained at 0 kPa (Appendix D). However, when the soil was maintained at -30 kPa, higher survival of the pathogen was observed in soil amended with cabbage than in nonamended soil. At -10 kPa, the addition of cabbage to the soil had no impact on the survival of P. nicotianae in test 1 , while in test 2 survival was greater in soil amended with cabbage than in nonamended soil. The effect of cabbage amendment on survival of P. nicotianae within each temperature regime was significant, but not consistent (P<0.05) (Table 3.15; Appendix D). At the two lower temperature regimes (35°C for 8 hours and 41°C for 5 hours daily) the addition of cabbage to the soil reduced the number of propagules of P. nicotianae recovered on the selective medium (Tables 3.12, 3.13, 3.14). At 44°C for 1.5 hours, and 47°C for 3 hours daily, a higher proportion of the population of P. nicotianae survived in soil that was amended with cabbage than in nonamended soil. In test 1, at 44°C for 5 hours daily the number of propagules of the pathogen recovered was lower in the soil amended with cabbage than in nonamended soil; in contrast, in test 2 there were no differences in the survival of P. nicotianae in soils amended or nonamended with cabbage. Generally, longer exposure of the infested soil to each temperature resulted in lower recovery of propagules (Tables 3.12, 3.13, 3.14, 3.15; Appendix D). At the base temperature regime of 35°C for 8 hours daily, the lowest survival was observed after 3 or 6 days. In test 1 no differences in survival were observed between 3 and 6 days or between 1 and 2 days of exposure to the heat treatment. In contrast, in test 2, the highest

PAGE 79

70 survival was observed after 2 days of exposure, followed by 1 and 3 days of exposure to the heat treatment. When average temperature regimes were employed, the reduction in survival was not as consistent as with the optimum temperature regimes. At 41°C for 5 hours daily, and 44°C for 1.5 hours, survival decreased with increasing length of exposure in test 1 ; however, in test 2 no differences were observed between 3 and 6 days of exposure, in which survival was lower than after exposing the soil to 1 day of heat treatment. As time progressed, lower recovery was observed at 44°C for 5 hours daily. Exposing the chlamydospore-infested soil to either 2 or 3 days at 47°C for 3 hours daily resulted in lower survival as compared with a 1-day exposure. The Effect of Three Nonpasteurized Soils. Temperature Regimes, and Cabbage Amendments on the Thermal Inactivation of Chlamydospores of Phytophthora nicotianae Soils from the three sites where soil solarization was tested were used in this study. In all three soils the two higher temperature regimes (44°C for 5 hours and 47°C for 3 hours daily) were generally more effective in the inactivation of chlamydospores of P. nicotianae than the two lower regimes (35°C for 8 hours and 41°C for 5 hours daily) (Tables 3.16, 3.17, 3.18). Very few propagules were recovered fi'om soil fi"om Site 1 after 9 days at the temperature regime of 47°C for 3 hours daily, and no propagules were recovered from the other soils after this treatment. Few tomato seedlings died after 30 days of incubation in the treated soils (Table 3.16, 3.17, 3.18). However, almost all seedlings were infected in all treatments, except at the highest temperature regime of 47°C for 3 hours daily. At the highest temperature regime, infection was observed in the second test, even in treatments where no propagules were recovered. In contrast, in the first test, no infection of the root system

PAGE 80

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PAGE 82

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74 was observed in the highest temperature regime, even in treatments from which a few propagules had been recovered. The numbers of propagules recovered from the soil from site 2 were generally lower than the numbers recovered from the other two soils in both tests (Tables 3.16, 3.17, 3.18, 3.19; Appendix E). No differences in survival were observed between sites 1 and 3 in test 1, while survival was higher in soils from site 3 than in soils from site 1 in test 2 (P<0.05). The effect of pasteurization of the soil on the survival of P. nicotianae was significant in test 1; however, no differences in survival were observed in test 2 (Table 3.19). Populations were lower in pasteurized soil than in nonpasteurized soil in test 1 (P<0.05) (Appendix E). Lower numbers of propagules were recovered from the soils as temperature regimes increased (Tables 3.16, 3.17, 3.18, 3.19; Appendix E). Temperature regimes of 44°C for 1.5 hours or higher were more effective than 35°C for 8 hours or 41°C for 5 hours at reducing the populations of P. nicotianae in test 1 (P<0.05) (Appendix E). In test 2, survival under each temperature regime was significantly lower than that at each preceding regime. The incorporation of cabbage amendment into soil significantly reduced the populations of P. nicotianae in the soil (Table 3.19; Appendix E). However, differences in populations between amended and nonamended soil exposed to specific temperature regimes were not evident from the analysis (Tables 3.16, 3.17, 3.18). The duration of the heat treatment significantly affected survival of P. nicotianae in test 1, but not in test 2 (Table 3.19). Significantly fewer propagules were recovered in the selective medium after 9 days of heat treatment than after 3 days in test 1 (P<0.05) (Appendix E); in contrast, no differences in survival between the two sampling dates were observed in test 2.

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75 Table 3.19. Analysis of variance of the effects of three soils, pasteurization, temperature regimes that simulate daily solarization periods, cabbage amendment, and time on the survival of chlamydospores of Phytophthora nicotianae. Source of variation Test 1 Test 2 df MS F P MS F P Soir 2 3.88 19.64 0.0001 36.18 267.87 0.0001 Pasteurization* 1 3.33 16.81 0.0001 0.35 2.59 0.1086 Temperature" 4 190.82 964.25 0.0001 249.99 1850.83 0.0001 Cabbage^ 1 36.19 182.88 0.0001 38.69 286.47 0.0001 Time' 1 10.74 54.27 0.0001 0.44 3.25 0.0725 Soil X Pasteurization 2 4.09 20.66 0.0001 14.05 104.04 0.0001 Soil X Temperature g 2.50 12.65 0.0001 5.75 42.58 0.0001 Soil X Cabbage 2 1.71 8.64 0.0001 0.16 1.18 0.3105 Soil X Time 2 0.75 3.77 0.0245 2.73 20.20 0.0001 Temperature x Cabbage 4 6.91 34.94 0.0001 7.58 56.09 0.0001 Temperature x Time 4 1.61 8.16 0.0001 5.36 39.69 0.0001 Cabbage x Time 1 0.34 1.74 0.1889 1.29 9.58 0.0022 Soil x Pasteurization x Temperature x Cabbage x Time 87 1.01 5.12 0.0001 1.19 8.78 0.0001 Residual 240 0.20 0.13 " Soils used were from Site 1 (Naples Tomato Grovvers-Gargiulo farm number 4 in Decatur County, Georgia), Site 2 (John Allen Smith farm located in Gadsden County, Florida), and Site 3 (North Florida Research and Educational Center in Gadsden Coimty, Florida). " Soils (1-kg lots) were either nonpasteiuized or pasteurized in a microwave oven at 700 W for 4 minutes, after moisture had been adjusted to 5% (w/w). " Temperature regimes that simulated solarization consisted of temperatures increased daily to 35°, 41°, 44°, 44°, or 47°C for 8, 5, 1.5, 5 or 3 hours, respectively; the temperature for the remainder of each day was maintained at 25°C. ^ Soil amended or not amended with dry, ground cabbage at a rate of 0. 125% (w/w). ^ Time = number of days of exposure to heat treatment at a given temperature regime.

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76 A significant interaction between soils and pasteurization was observed in both tests (Table 3.19). In test 1, no differences in survival of P. nicotianae were observed between pasteurized and nonpasteurized soils from sites 2 and 3, and survival was higher in nonpasteurized than pasteurized soil from site 1 (P<0.05) (Appendix E). In test 2, survival was higher in nonpasteurized than pasteurized soil from sites 1 and 2, and lower in pasteurized soil from site 3. The interaction of soils with temperature regimes was significant in both tests (Table 3.19). Generally, survival of P. nicotianae decreased with increasing temperatures in regimes in each of the three soils tested (Tables 3.16, 3.17, 3.18; Appendix E). The effect of cabbage amendment on the survival of P. nicotianae within each soil was significant in test 1, but not in test 2 (P<0.05) (Table 3.19). Within each soil the addition of cabbage amendment lowered the number of propagules recovered from the soils (Appendix E). The effects of interactions of soils with duration of heat freatment on the survival of populafions of P. nicotianae were significant (Table 3.19). In test 1, lower survival was found after 9 days of heat freatment than after 3 days in soils from sites 1 and 3; no differences in survival between the two sampling dates were observed in soils from site 2 (P<0.05) (Appendix E). Lower populafion levels were detected after 9 days of heat treatment than after 3 days in soils from site 3 in test 2; no differences in survival were observed between the two sampling dates in soils from either site 1 or from site 2 in test 2. The effect of cabbage amendment on survival of P. nicotianae within each temperature regime was significant (Table 3.19). In test 1, cabbage amendment reduced the survival of P. nicotianae, except at 47°C for 3 hours (P<0.05) (Appendix E). In test 2, no differences in survival were found at 35°C for 8 hours or 47°C for 3 hours daily; at all other temperature regimes the cabbage amendment reduced the populations of P. nicotianae.

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77 The influence of the interaction of temperature regimes and time on survival was significant for both tests (Table 3.19). No propagules were recovered after either sampling time at 47°C for 3 hours daily in both tests (P<0.05) (Appendix E). Lower recovery of P. nicotianae was found after 9 days than after 3 days of heat treatment at 44°C for 1.5 hours and at 44°C for 5 hours in test 1; no differences in survival between the sampling times were observed at 35°C for 8 hours, 41 °C for 5 hours or 47°C for 3 hours. In test 2, significantly fewer spores germinated in the selective medium after 9 days of heat treatment than after 3 days in all temperature regimes, except at 44°C for 1.5 hours. The interaction of cabbage amendment and time in reducing populations of P. nicotianae was significant only in test 2 (Table 3.19). Within each cabbage concentration fewer spores were recovered in test 1 after 9 days than after 3 days (P<0.05) (Appendix E). In contrast, no differences were observed when cabbage was added to the soils in test 2. Discussion The findings of this study are in general agreement with related work on the thermal inactivation of spores of Phytophthora spp. (Barbercheck and Von Broembsen, 1986; Benson, 1978; Bollen, 1985; Juarez-Palacios et al, 1991). BoUen (1985) reported that a soil culture of P. cryptogea required 30 minutes at 45 °C to be completely inactivated, and a soil culture of P. capsici had to be heated to 50°C for 30 minutes before oospores was killed. Benson (1978) found that culture disks of P. cinnamomi containing chlamydospores were killed after 90 minutes at 39°C or 4.5 minutes at 44°C; in contrast, Barbercheck and Von Broembsen (1986) noted that a suspension of chlamydospores of P. cinnamomi in water was inactivated after 10 minutes at 44°C. The differential heat sensitivity of isolates of the same species was demonstrated by Juarez-Palacios et al.

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78 (1991). These authors found that chlamydospores of P. cinnamomi or oospores of a lowtemperature isolate of P. megasperma added to soil did not survive 20 minutes at 45°C; in contrast, a high-temperature isolate of P. megasperma survived more than 30 minutes at the same temperature. The discrepancies among the values reported in the literature could be related to the different types of substratum used to produce the spores and to the different media used for the heat treatment in each study. It is expected that spore suspensions in water would be inactivated faster than soil cultures saturated with water, which in turn would die faster than spores added to soil at a lower soil water matric potential. These assumptions are based on the differential transmission of heat throughout the substrata, the formation of air pockets, or simply due to the nature of the spores formed in each substratum, as noted by Myers et al. (1983) and Katan (1981). The use of heat to inactivate spores of microorganisms in diverse media began as early as 1920 (Bigelow and Esty, 1920). Soon after that the advantages of the logarithmic transformation for the analysis of the data also were realized (Bigelow, 1921; Smith, 1923). The logarithmic transformation indicated that a constant proportion of the spore population was killed per unit of time, and also served the purpose of linearizing the data. Simple linear regressions, then, could be used for the analysis of the effect of time on the survival of spores at each temperature employed; or, coefficients for lethal dosages (LD50 or LD90) could be interpolated from the data. Pullman et al. (1981a) demonstrated that the logarithmic relationship between time and temperature on the survival of four plant pathogens was maintained at temperatures below 50°C. However, Anderson et al. (1996) and Cole et al. (1993) pointed out that the use of the 'log-linear' model assumes that all spores in a population have equal heat sensitivity and that death of an individual is dependent on it receiving sufficient heat. Deviations from this basic assumption have been observed and a vitalistic model has been proposed by these authors. The concept in this theory is that individuals in a population do not have identical heat resistances and that these differences are permanent. It is possible that a better understanding of the

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79 survival of P. nicotianae would be achieved by using the vitalistic model described by Co\q etal. (1993). In the present study the relationship of time and temperature on the survival of P. nicotianae was evaluated over a series of temperatures and times (35° to 53°C for 5 to 120 minutes, and 35° to 47° for 2 to 480 hours), for which a surface response approach allowed the simultaneous analysis of the effects of both time and temperature on the survival of the pathogen. The models that best described survival of P. nicotianae were linear-exponential or exponential. The analysis of the effect of temperature regimes on the survival of P. nicotianae indicated that a single model is not adequate to describe the changes in survival over time as temperature increased. At the base temperature regime (35°C for 8 hours daily), none of the models accurately described survival, which was little affected by the temperature regime. As temperature increased, the change in survival was best explained by other models, such as an exponential model, then a model of square root of either time or survival, then a logarithmic model of time, and finally a model of the reciprocal of time. The changes in spore survival that resulted in these models may have occurred as constant numbers of spores died per unit time at lower temperatures, a constant proportion of the population was killed per unit time at intermediate temperatures, and larger proportions of populations were inactivated early during the exposure to the highest temperature. The use of tomato seedlings as baits to detect survival of P. nicotianae in the treated soil demonstrated that this method can be as sensitive as the soil dilution plating procedure, except when residual populations are present (less than 1 propagule per gram of soil). The reasoning for using the baiting test was that heat treatment at temperatures that did not eliminate the pathogen from the soil could possibly have affected the ability of the surviving population to infect and colonize a susceptible host, or to germinate in the presence of an external stimulus, such as root extracts. However, it is clear from these

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80 tests that if P. nicotianae is present in the soil, even in residual levels, infection of a tomato seedling can occur. The baiting technique has the following disadvantages: population of the pathogen are not quantified, extensive amounts of time and space may be required to grow the seedlings and incubate the baited soil, and the soil samples may be cross contaminated due to handling procedures. Amending infested soils with dried, ground cabbage leaves and exposing it to heat treatment generally reduced survival of P. nicotianae in the present study. The effectiveness of cabbage amendment could be observed in two ways: first, at a given temperature, less time was required to achieve the same reduction in population as cabbage concentration increased; second, with increasing concentrations of cabbage amendment, lower temperatures were required to achieve the same reduction in populations of the pathogen. Populations of P. nicotianae were eliminated after 24 hours at 4rC or after 12 days at 38° and 35°C in soil amended with 0.5% cabbage; after 4 days at 41 °C or after 20 days at 35°C only residual populations of the pathogen were detected. After 12 days at 38°C no propagules were detected in soils amended with 0.125% or 0.25% cabbage. These findings contrast with the results obtained in the field work, where no additional reductions in populations of P. nicotianae were achieved with cabbage amendments. The amount of cabbage incorporated into the soil in the field experiments ranged fi-om 0.125% to 0.25%; however, leaf pieces were much larger and during the drying period may have been rained on, which could have started a decay process with subsequent loss of volatiles. Furthermore, incorporation of the amendment into the field soil was done by disking, and it could not be determined if uniform distribution throughout the soil profile was attained. The use of cabbage amendment without any additional soil heating has been shown to be effective for the control of Aphanomyces euteiches (Lewis and Papavizas, 1971), or inconsistent for control of Fusarium oxysporum f sp. conglutinans (RamirezVillapudua and Munnecke, 1987, 1988). Heating cabbage amended soils induces the

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81 volatilization of certain compounds that are fungicidal, as demonstrated by Gamliel and Stapleton (1993b). These authors determined that heating soil amended with cabbage released volatile compounds, such as methanol, isothiocyanates and aldehydes, which could be directly correlated with the inactivation of the spores of Pythium ultimum and sclerotia of Sclerotium rolfisii. The effect of cabbage amendment appears to be directly proportional to its concentration in the soil, as observed by RamirezVillapudua and Munnecke (1988). These authors found that increasing the concentration of cabbage from 0.25% to 2% reduced the population of F. oxysporum f sp. conglutinans to undetectable levels in 12 days instead of the 30 days required at the lower concentration. Other crucifers, such as kale {Brassica oleracea var. viridis) and mustard {B. nigra) were also effective for the control F. oxysporum f sp. conglutinans. Another factor that may determine the effectiveness of the crucifer amendment is the concentration of glucosinolates present in the crop (Mayton et al., 1996). These authors found that some cultivars of Brassica spp. were more effective than others in the control of F. sambucinum. Even though RamirezVillapudua and Munnecke (1988) demonstrated that dried cabbage residue is more effective than fresh residue for the confrol of F. oxysporum f. sp. conglutinans, other Brassica spp. may be more effective as fresh amendment, as demonstrated by Subbarao and Hubbard (1996) with broccoli (B. oleracea var. botrytis) and Verticillium dahliae. One major benefit of the use of intermittent heat in thermal inactivation studies is the provision of estimations of the effectiveness of soil solarization to control plant pathogens. However, cycling temperatures have not been employed routinely, possibly due to the compounded difficulties of establishing appropriate temperature regimes, the daily requirement of adjusting each temperature, and the longer time required to reach desirable control of the organism under study. Examples of the use of pulsing temperatures are provided by Gamliel and Stapleton (1993b), Porter and Merriman (1983), Tjamos and Fravel (1995) and Wicks (1988).

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82 Wicks (1988) analyzed the effect of intermittent heat on the survival of mycelium of Phytophthora cambivora. None of the isolates tested survived after 1 day at a regime of 45°C for 6 hours and 20°C for 18 hours daily. The response of the isolates was variable and inconclusive at either 40° or 35°C for 6 hours daily during 4 days. How^ever, mycelium is not the most likely survival structure of P. cambivora in the soil. Tjamos and Fravel (1995) evaluated intermittent heat on the survival of a suspension of microsclerotia of Verticillium dahliae over 4 days. The following daily temperature regimes were used: base temperature of 31°C for 10 hours and high temperature at 35°C for 14 hours, base temperature of 33°C for 10 hours and high temperature at 36°C for 14 hours, and base temperature of 35°C for 10 hours and high temperature at 38°C for 14 hours. After 4 days at the highest temperature regime, less than 1% of the sclerotia germinated. In their study the use of sclerotium suspensions in water negated the insulating effect of air pockets in the soil, and the duration of the high temperature in each regime was longer than that which would normally occur under field conditions. A more comprehensive study was done by Porter and Merriman (1983) with several soilbome plant pathogens. These authors used infested soil held at field capacity, and over 15 days evaluated cycles of low temperature at 25°C for 18 hours, followed by 6 hours daily at a supplemental temperature of 25°, 30°, 35°, 40°, 45° or 50°C. Survival of each pathogen depended on the heat sensitivity of the type of propagule being evaluated. For example, V. dahliae did not survive for 1 5 days when the high temperature was above 40°C; in contrast, Pythium irregulare was recovered at SxlO'' propagules per gram of soil at 50°C, even at the end of the experiment. Gamliel and Stapleton (1993b) selected two temperature regimes similar to those found in the San Joaquin Valley in California to evaluate the effectiveness of the regimes and cabbage amendment on the control of P. ultimum and S. rolfsii. Both pathogens v/ere eliminated after 4 days in the cabbage amended soil at either temperature regime of 38°C

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83 or 45°C for 4 hours daily plus 20 hours at 30°C; however, propagules of these pathogens could be recovered from the nonamended soil after 4 days at the same temperature regimes. Comparisons between studies are further complicated due to the differences in length of the experiments, amplitude between low and high temperatures in each regime, and the duration of the high temperature. If a given in vitro experiment is to be compared with soil solarization in the field, then the duration of the in vitro study should be similar to the solarization trial, and the temperature regimes should simulate those observed during solarization. In the present study populations of P. nicotianae decreased to residual levels after 15 days only when temperature regimes simulating optimum solarization conditions (47°C for 3 hours daily) were used; under these circumstances no infection of tomato seedlings was observed after as little as 3 days of heat treatment. The use of 44°C for 5 hours daily also reduced populations to levels below 1 propagule per gram of soil; however, infection of tomato seedlings exposed to this regime was observed throughout the experiment. The use of temperature regimes simulating average field temperature regimes reduced the populations to at least 40% of the initial infestation level, but all seedlings were colonized at these regimes. Athough the effect of soil moisture on reproduction and dispersal of Phytophthora spp. has been researched extensively (Browne and Mircetich, 1988; Ferrin and Mitchell, 1986; Lutz and Menge, 1991; Mcintosh, 1972; Neher and Duniway, 1992; Ristaino et al, 1992; Sidebottom and Shew, 1985a, 1985b), the effect of soil moisture and heat on the inactivation of spores of this genus has not been addressed before. Survival of chlamydospores of P. nicotianae was lower in saturated soil (0 kPa) than at the two other soil water matric potentials evaluated (-10 and -30 kPa). The two lower soil water matric potentials (-10 and -30 kPa) used for this study are close to what is normally considered field capacity of a soil and they represent an optimum for thermal inactivation studies.

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84 However, in field studies, these conditions are very difficult to maintain for the time required for soil solarization without supplemental irrigation. Porter et al. (1991) evaluated the effect of two moisture contents (field capacity and 10% of field capacity) and constant temperature on survival of Plasmodiophora brassicae in two soils. Only 15 days were required to kill all spores of P. brassicae in either soil at 40°C or above when the soil moisture was at field capacity. In dry soil, however, inactivation was observed only at 50° or 55°C. The effect of soil water matric potential on the thermal inactivation process is twofold. First, temperature maxima of the soil increase with increasing soil moisture content (Mahrer et al, 1984). This principle is important in field experiments, but is not operative in an in vitro system as used in the present study. The second effect of moisture is the increase in heat transfer or conduction in the soil, with a subsequent reduction of air pockets that could provide an insulation for the spores (DeVay, 1991a; Stapleton and DeVay, 1986). One of the key biological principles of soil solarization is that, due to the relatively low temperatures of solar heating, stimulation of microbiological activities in solarized soils may lead to biological control of plant pathogens (Katan, 1985). This indirect effect of soil solarization may be related to a differential thermal sensitivity of other soil microbiota, such as actinomycetes and certain bacteria, or to a greater saprophytic competitive ability of these microorganisms in relation to pathogens once the soil has cooled down (Stapleton and DeVay, 1986). Several researchers have enumerated some of the groups of microorganisms that may be involved in the eventual biological control of plant pathogens in solarized soils (DeVay, 1991; DeVay and Katan, 1991; Gamliel and Katan, 1993; Gamliel and Stapleton, 1993a; Keinath, 1996; RamirezVillapudua and Munnecke, 1988; Stapleton, 1991; Stapleton and DeVay, 1984). GamUel and Stapleton (1993a) determined the microbial activity in the soil using an indirect measure of respiration with fluorescein diacetate. Liitially, heated soils had less microbial

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85 activity than nonheated control soil, but after 2 weeks the activity was similar in both soils. Keinath (1996) determined populations of thermotolerant fungi, fluorescent Pseudomonas spp.. Bacillus spp., and actinomycetes by soil dilution plating. Populations of thermotolerant fungi increased during solarization and declined in the months following solarization, but remained higher than in nonsolarized soils. Fluorescent Pseudomonas spp. were not found in the soil immediately after solarization; populations of actinomycetes were not affected by solarization; and populations oi Bacillus spp. increased both in solarized and nonsolarized soils. All previous work on thermal inactivation of various plant pathogens has been done with spores in water suspension (Benson, 1978; Smith, 1923; Tjamos and Fravel, 1995), with pasteurized soil (Myers et al., 1983), or with namrally infested soil (Bollen, 1985; Juarez-Palacios et al., 1991; Kulkami et ai, 1992; Pullman et al., 1981a). One objective of the present research was to determine if lower survival of P. nicotianae occurred in nonpasteurized soils than in pasteurized soils. Two of the soils evaluated (Site 1 and Site 2) were collected from fields where tomato was commercially grown and soil fumigation with methyl bromide was routinely used. In these soils, survival of P. nicotianae was either higher in nonpasteurized soils or similar in nonpasteurized and pasteurized soil. In soil from Site 3, which had been weed fallow for several years, survival of P. nicotianae was either lower in nonpasteurized soil or similar imder both soil treatments. Soils that have been fallow for some time may have a greater diversity of microorganisms that could inhibit, compete with, or lyse the structures of plant pathogens, such as P. nicotianae, thus decreasing their survival. In confrast, soils that have been routinely used for commercial production of a crop and have been fumigated with methyl bromide regularly, may provide a biological vacuum after each fumigation, allowing the pathogen to reestablish in the soil and colonize it (Maloy, 1993). Possibly the duration of the tests was too short (9 days) to observe any definite trends in relation to the effects of natural soils and temperature regimes on the inactivation of chlamydospores

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86 of P. nicotianae. Although there were Umitations in time of exposure in this study, this the first attempt to determine the biological effects of three nonpasteurized soils on survival of P. nicotianae in an in vitro system.

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CHAPTER 4 SUMMARY AND CONCLUSIONS The effects of soil solarization and cabbage amendment on the survival of Phytophthora spp. were evaluated in North Florida. Soil solarization was applied as a potential soil disinfestation technique in mid-summer of 1994, 1995, and 1996, in five field trials at three sites for 49, 48, 55, 45, and 45 days. During the field trials precipitation occurred on 30, 31, 17, 19, and 21 days, respectively. Ambient air temperature exceeded 35°C only in 1995 and 1996. Soil temperatures exceeded temperature thresholds of 44° and 47°C only under clear, low density polyethylene film and under clear, gas impermeable plastic. Soil solarization using clear, low density polyethylene film, or with clear, gas impermeable plastic film was an effective strategy for the reduction of populations of Phytophthora nicotianae and P. capsici in the top 10-cm layer of soil fi"om Site 1 or Site 3. However, at the 25-cm depth, survival of P. nicotianae was similar to that in the whiteon-black low density polyethylene, and survival of P. capsici was similar to that in the nontarped treatment. The incorporation of 70 to 90 metric tons of cabbage into the soil did not affect survival of either pathogen. Phytophthora nicotianae could not be quantified in the selective medium for any of the treatments in Site 2 (1994) due to bacterial contamination by Burkholderia cepacia, or from the first test in Site 3 (1995). Phytophthora capsici was not recovered fi-om any of the trials in Site 3 in 1995 or 1996. In 1996 no oospores of P. capsici were observed in the inoculum at the time of soil infestation, and this might have contributed to the lack of survival of this pathogen. Overall condifions for solarization were excellent in 1995; the soil was at field capacity (87

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88 5 kPa), air temperature exceeded 35°C six times, precipitation occurred only on 17 days, and solarization was carried on for 55 days. During the solarization period soil temperatures under the clear, low density polyethylene film exceeded 44° and 47°C for 20 and 6 days respectively. All these factors coupled with the potentially diverse soil microbiota present in a field that had been fallow for several years might account for the lack of survival of the Phytophthora spp. in any of the treatments, even the controls. Some of the possible changes to allow better evaluation of the effectiveness of soil solarization in combination with amendment for the control of Phytophthora spp. include: disking the cabbage residue into the soil before making the beds for solarization, and thus promoting a better distribution of the amendment throughout the soil profile; irrigating the field for a few days to ensure that soil moisture is optimum dovra to 30 cm of depth; and making sure that the appropriate survival structures are present in the inoculum before it is taken to the field. The temporal analysis of survival of P. nicotianae in soil fi"om Site 3 indicated that populations of the pathogen declined with time at the 1 0-cm depth in plots that were solarized fi-om 17 to 45 days. At the 25-cm depth and in the nontarped treatment, populations of P. nicotianae were not affected. No propagules of the pathogen were recovered at either depth or sampling date from soil treated with methyl bromide. Further studies following the course of inactivation of survival structures, such as chlamydospores or oospores, during soil solarization would help to determine the minimum time required for soil solarization to effectively control Phytophthora spp. in the soil. The time required to eliminate P. nicotianae fi-om the soil in laboratory thermal inactivation studies decreased with increasing temperatures. Populations of the pathogens were reduced fi-om the infestation level of 500 chlamydospores per gram of soil at the beginning of the tests to 0.2 propagule per gram of soil after 10 minutes at 53°C, 45 minutes at 50°C, 4 hours at 47°C, 12 hours at 44°C, 4 days at 41°C, and 16 days at 38°C.

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89 More than 10% of the initial population survived for 20 days at 35°C . The incorporation of cabbage amendment into the soil reduced the time required to inactivate the chlamydospores of P. nicotianae at all temperatures tested. Populations of the pathogen dropped below a detectable level after 4 days in soil amended with 0.5% cabbage and heated to 35°C. Populations also declined below a detectable level in soils amended with 0.125, 0.25, and 0.5% cabbage and heated at 38°C. Similar reductions in populations of the pathogen were not observed in the field experiment, possibly due to the different procedures in the preparation and incorporation of cabbage into the soil. For the laboratory experiments, cabbage wrapper leaves were air dried in a greenhouse, ground in a Wiley mill with a 20-mesh screen; in contrast, for the field test, cabbage heads and wrapper leaves were chopped with a machete, or shredded in a mechanical shredder, air dried for 1 week on the soil in the plots and disked into the soil as the solarization beds were prepared. These differences may have resulted in a loss of potential volatiles during the drying period and uneven incorporation of the amendment throughout the soil profile, which may have prevented the release of volatiles at the lower depths of the soil (25 cm). Although no propagules were detected by the soil dilution plating in soils maintained at 41 °C, the presence of the pathogen was detected by the tomato seedling baiting technique. Detection of P. nicotianae by the baiting technique provided similar results to the soil dilution plating procedure, except when residual populations were present in the soil (less than 0.2 propagule per gram of soil). At this low level the pathogen was more fi-equently detected by one or the other method due to the restricted volume of soil used in the assays. The use of temperature regimes that simulated solarization periods increased the time required to inactivate the chlamydospores of P. nicotianae in relation to the use of constant temperature. Of all temperature regimes evaluated, only those simulating optimum solarization periods (44°C for 5 hours daily and 47°C for 3 hours daily) eliminated the pathogen from the infested, amended soil and prevented mortality of

PAGE 99

90 tomato seedlings; infection was prevented only in soil treated at the highest temperature regime. All other temperature regimes (35°C for 8 hours daily, 41°C for 5 hours daily and 44°C for 1.5 hours daily) did not prevent infection or mortality of the seedlings. One of the few factors that can be controlled during soil solarization is the soil water matric potential through the use of irrigation. The analysis of the effect of soil water matric potential and temperature regimes on the inactivation of chlamydospores of P. nicotianae in cabbage amended soils indicated that survival was lowest in saturated soil; and as temperature increased, survival of the pathogen decreased at all soil water matric potentials evaluated (0,-10 and -30 kPa). The soil water matric potentials evaluated represent optimum levels for the study of thermal inactivation; however, under field conditions lower potentials may be found, as in the third trial in Site 3. Extending the range of soil water matric potentials to -50 kPa, or -100 kPa and the treatment time to 1 5 to 20 days would allow better comparisons with the field data. The study with different soils and soil pasteurization, coupled with cabbage amendment and heat simulating solarization periods indicated that soils with potentially greater microbiological activity can be more suppressive to populations of P. nicotianae. The duration of the tests (9 days) did not allow the full expression of any of the biological factors that may have been present. Further studies should be conducted on the effects of temperature regimes, such as 44°C for 1 .5 or 5 hours daily, on the inactivation of chlamydospores of P. nicotianae over time periods similar to those observed during solarization. Another promising area of study is the possibility of inducing suppressiveness of a soil after heat treatment. Such a study could be carried by heating nonpasteurized soils for 1 5 days, incubating it for another 15 days (average time between the end of a solarization period and transplanting the field with seedlings), and then adding the inoculum and fiirther incubating it to follow the dynamics of the population of the pathogen.

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91 Another area that needs clarification is the relationship of inoculum density of P. nicotianae to disease incidence in tomato. The studies in this dissertation indicated that very low levels of the pathogen could cause infection of 1 -month-old tomato seedlings, even after the stress of the heat treatments, ft is not known how closely the disease responses described here would relate to root infection of tomato with P. nicotianae or P. capsici and subsequent disease under field conditions.

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APPENDIX A PRODUCTION OF CHLAMYDOSPORES OF PHYTOPHTHORA NICOTIANAE Introduction Chlamydospores, the primary inoculum of Phytophthora nicotianae, overwinter in the soil or on plant tissue, such as roots, or other organic material. Production of chlamydospores in controlled environments has been limited by the requirements of the fungus for specific conditions that may favor the formation of one type of spore over other forms. As an example, chlamydospore production is favored by low temperatures and saturated soils, and sporangium production is favored by high temperatures and high soil moisture, but not saturation. The most commonly used procedure for producing chlamydospores for quantitative studies was described by Tsao in 1971 (Mitchell and Kannwischer-Mitchell, 1992), and it is still being used today. loannou and Grogan (1985) described the use of controlled atmosphere for the formation of chlamydospores of P. nicotianae. These authors found that whenever the concentration of Oj was kept between 10 and 12% and the concentration of COj was raised to 6 to 8%, chlamydospores were the only spores formed in liquid culture. The advantages of the method described by loannou and Grogan (1985) were the reduced time for the formation of spores, 2 weeks instead of 4, and the use of a growth chamber at 20°C, which alleviated the need to change temperatures for the selective production of chlamydospores. Studies on the formation of chlamydospores in solid organic media have yet to be reported. The objectives of this study were to determine the effect of a controlled atmosphere environment on the formation of chlamydospores in a solid organic medium, 92

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93 wheat seeds, and the effect of the soil on the induction of chlamydospore formation in the seeds. Materials and Methods Isolate Pn21 of Phytophthora nicotianae was maintained in V8-juice agar cultures for use in all experiments. For the studies in liquid media, four plugs from actively growing cultures were transferred to petri plates containing 20 ml of sterile V8-juice broth. After 2 days of incubation at 25°C, the medium was removed by suction; the mycelial mats were then rinsed with sterile distilled water three times at 5-minute intervals. Fifteen milliliters of water were added to the plate, and each plate was placed in ajar maintained with a confrolled atmosphere. Controls consisted of plates prepared in the same manner and left outside of the jar on the same shelf of an incubator in which the experiment was conducted. The cultures in the solid medium were prepared by autoclaving a flask containing 20 g of wheat seeds and 30 ml of distilled water on 2 consecutive days. Four plugs of actively growing mycelium were transferred to each flask and incubated in the dark at 25°C for 2 weeks. The flasks were shaken vigorously twice a week to ensure uniform colonization of the wheat seeds. Ten grams of colonized wheat seeds were transferred to a petri plate, which was placed in a jar for the controlled atmosphere experiment. Controls consisted of plates prepared in the same manner and left outside of the jar on the same shelf of the incubator in which the experiment was conducted The controlled atmosphere chamber was prepared with 10% O2, 6% CO2, and 84% N2 at a flow rate of 193 ml/min; the temperature was maintained at 20°C. The plates were incubated for 2 weeks in the growth chamber and then removed for determination of the relative number of chlamydospores formed.

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94 The presence of chlamydospores was determined by observing the Uquid cultures directly under the microscope, or by pressing the wheat seeds between a cover glass and a glass slide and examining them under the microscope. The number of spores produced in each treatment was estimated by grinding the cultures in 10 ml of water and counting the number of spores in five microscope fields with the combination of a 1 OX ocular and lOX objective of a microscope, which yielded a real viewfield of 2.5 mm". A second experiment on the effect of soil on the formation of chlamydospores in the solid medium was maintained in two incubators, one at 25°C and the other at 18°C. One gram of the wheat seed inoculum was mixed in 150 g of soil and placed in a 300-ml plastic container with a lid. Soils fi-om the following three sites where solarization was evaluated were either microwaved or left untreated and tested at the two temperatures: Site 1 at the Naples Tomato Growers-Gargiulo farm number 4 in Decatur County, South Georgia; Site 2 at the John Allen Smith farm located in Gadsden County, North Florida; and Site 3 at the North Florida Research and Educational Center in Gadsden County. After 2 weeks of incubation, the containers were removed from the incubators, the inoculum was retrieved by sieving, and the relative abundance of chlamydospores was determined as previously described. Comparisons among all treatments in each experiment were performed using the procedure PROC GLM of SAS (SAS Institute, Gary, NC; release 6.1 1 for personal computers) and the means were separated using Tukey's Honestly Significant Difference procedure.

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95 Results and Discussion The controlled atmosphere was very effective in the selective production of chlamydospores in the liquid cultures (P<0.05); in contrast, the plates in the normal atmosphere contained empty sporangia and few chlamydospores (Table 1). The results confirmed the findings of loaimou and Grogan (1985). However, very few spores were formed on the wheat seeds, although P. nicotianae grew on the plates in both the controlled atmosphere and the normal atmosphere. The average diameter of chlamydospores in the liquid medium was 34.5 \im, with a range of 15 to 50 ^m. In the second experiment, more chlamydospores were formed in raw soil at 25°C than in microwaved soil (P<0.05) (Table 2). However, at 18°C more spores were found in the microwaved soil. A comparison of relative abundance of chlamydospores formed in the soils indicated that the lower temperature (18°C) was more effective for the production of this type of spore. No chlamydospores were observed on wheat seeds incubated at 25 or 18°C, or on flooded wheat seeds maintained at 18°C. Furthermore, P. nicotianae grew in the flooded culture. It is possible that a depletion of nutrients is required before chlamydospores by P. nicotianae are formed, and that more time would be needed for the depletion of the nutrients in wheat seed cultures. Whenever the inoculum is added to soil, competition for nutrients takes place, and P. nicotianae apparently responds to the depletion of nutrients by forming chlamydospores. Lower temperatures may limit the intensity of the overall microbial activity and allow P. nicotianae to form more chlamydospores before all of the nutrients are depleted.

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96 Table A.l. Number of chlamydospores produced by Phytophthora nicotianae after 2 weeks of exposure to a controlled atmosphere. Medium Environment Spore type Number of spores" Average Standard Deviation Liquid 10% O2, 6% CO2. chlamydospores 13.00 a^ 0.74 Liquid 10% 0,, 6%C02. sporangia 0.04 c 0.04 Liquid air chlamydospores 6.00 b 0.51 Liquid air sporangia 6.00 b 0.44 wheat seeds 10% O2, 6%C02. chlamydospores 1.00 c 0.20 wheat seeds 10% O2, 6% CO2. sporangia 0.00 c 0.00 wheat seeds air chlamydospores 0.00 c 0.00 wheat seeds air sporangia 0.00 c 0.00 " Average of 27 optical fields observed with a combination of a 1 OX ocular and a lOX objective (2.5-mm^ field of view). ^ Main effect means followed by the same letter do not differ according to Tukey's Honestly Significant Difference procedure (P< 0.05).

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97 Table A.2. Number of chlamydospores produced by Phytophthora nicotianae in wheat seeds buried in three different soils for 2 weeks. soil iviicrowa.viiig 1 ernperdiure J Number of spores^ Site 1 YbS 1 o 60 a^ Site 1 YES 25 10 de Site 1 NO 1 o 15 d
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APPENDIX B DETERMINATION OF SOIL WATER MATRIC POTENTIAL Water relations are among the most important physical phenomena that affect the biology of soilbome plant pathogens. Duniway's (1979) review, "Water Relations of Water Molds," highlights the importance of water in every aspect of the life cycle of this group of organisms. In summary, sporangia of Phytophthora spp. are formed at soil water matric potentials ranging from -2.5 kPa to -60 kPa. While saturated soils (0 kPa) may inhibit the formation of sporangia, zoospore release is enhanced and may continue as the matric potential is maintained above -5 kPa. Similarly, zoospore motility has been detected in soils with matric potentials ranging from 0 to -5 kPa. The effects of soil water potentials on chlamydospores has been less extensively studied; however, it is known that water requirements for chlamydospore formation and germination by some species parallel those for sporangial formation. Generally, biological phenomena in the soil are more closely related to the soil water matric potential than to moisture content. Furthermore, the use of the soil water matric potential allows direct comparisons among soils. The soil water matric potentials of disturbed soil samples from the following sites were determined with a pressure plate apparatus equipped with a 3-bar ceramic plate (Soilmoisture Equipment Corp., Santa Barbara, CA): Site 1 was located at the Naples Tomato Growers-Gargiulo farm number 4 in Decatur County, Georgia; Site 2 at the John Allen Smith farm located in Gadsden County, Florida; and Site 3 at the North Florida Research and Educational Center in Quincy, Gadsden County. Water retention curves for soil from each of the three sites are presented. 98

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99 Figure B.l. Moisture retention curves for soils from Site 1 (Naples Tomato GrowersGargiulo farm number 4 in Decatur County, Georgia), Site 2 (John Allen Smith farm located in Gadsden County, Florida), and Site 3 (North Florida Research and Educational Center in Quincy, Gadsden County).

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APPENDIX C REGRESSION ANALYSES OF THE EFFECTS OF TEMPERATURE REGIMES AND CABBAGE AMENDMENT ON SURVIVAL OF PHYTOPHTHORA NICOTIANAE Regression models generated with Statgraphics Plus were used to characterize the effect of cycling temperatures and cabbage amendment on the inactivation of chlamydospores of Phytophthora nicotianae. Table C.l. Comparison of models used to describe the relationship of exposure time (days) to a temperature regime of 35°C for 8 hours daily in soil amended with 0% cabbage on the survival of Phytophthora nicotianae (propagules per gram of soil). Comparison of Alternative Models Model Correlation R-Squared Linear -0 2221 4 . 93% Square root-Y -0 1987 3 . 95% Square root-X -0 1919 3.68% Exponential -0 1620 2 .62% Logarithmic -X -0 1560 2 .43% Multiplicative -0 0987 0 . 97% Reciprocal-X 0 0937 0 .88% Scurve 0 0637 0.41% Reciprocal-Y 0 0575 0 .33% Double reciprocal -0 0165 0.03% Logistic Log probit 100

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101 Table C.1.1. Regression analysis with the model that best described the relationship of exposure time (days) to a temperature regime of 35°C for 8 hours daily in soil amended with 0% cabbage (T350C) on the survival of Phytophthora nicotianae (propagules per gram of soil). Regression Analysis Linear model: Y = a + b*X Dependent variable: T350C Independent variable : DAYS Parameter Standard Estimate Error T Statistic P -Value Intercept Slope 348.047 20.2675 -4.32383 1.66457 17 . 1726 -2 . 59756 0 . 0000 0 . 0105 Analysis of Variance Source Sum of Squares Df Mean Square FRatio P-Value Model Residual 131256.0 1 2.52891E6 130 131256 . 0 19453 .1 6.75 0.0105 Total (Corr ) 2.66016E6 131 Correlation Coefficient = -0.222129 R-squared = 4.93415 percent Standard Error of Est. = 13 9.4 74

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102 Table C.l .2. Model used to compare the relationship of exposure time (days) to a temperature regime of 35°C for 8 hours daily in soil amended with 0% cabbage (T350C) on the survival of Phytophthora nicotianae (propagules per gram of soil) with all other temperature regimes (data transformed as In [ppg+1]). Regression Analysis Linear model: Y = a + b*X Dependent variable: LOG(T350C+1) Independent variable : DAYS Parameter Standard T Estimate Error Statistic P-Value Intercept Slope 5.71761 0.0780225 73.2815 -0.0120329 0.006408 -1.87779 0 . 0000 0 . 0627 Analysis of Variance Source Sum of Squares Df Mean Square F-Ratio P-Value Model Residual 1.01654 1 1.01654 37.4776 130 0.28829 3 . 53 0 . 0627 Total (Corr ) 38.4942 131 Correlation Coefficient = -0.162504 R-squared = 2.64075 percent Standard Error of Est. = 0.536926 Table C.l. Comparison of models used to describe the relationship of exposure time (days) to a temperature regime of 35°C for 8 hours daily in soil amended with 0.125% cabbage on the survival of Phytophthora nicotianae (propagules per gram of soil). Comparison of Alternative Models Model Correlation R-Squared Exponential -0 5305 28 . 14% Reciprocal-Y 0 5286 27 . 94% Square root-Y -0 4753 22 .59% Multiplicative -0 4367 19 . 07% Linear -0 4137 17.12% Square root-X -0 3860 14 . 90% Logarithmic -X -0 3379 11 .42% Double reciprocal -0 3104 9.64% Scurve 0 3017 9 . 10% Reciprocal -X 0 2200 4 . 84% Logistic Log probit

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103 Table C.2.1. Regression analysis with the model that best described the relationship of exposure time (days) to a temperature regime of 35°C for 8 hours daily in soil amended with 0.125% cabbage (T35125C) on the survival of Phytophthora nicotianae (propagules per gram of soil). Regression Analysis Exponential model: Y = exp(a + b*X) Dependent variable: T35125C Independent variable : DAYS Parameter Standard Estimate Error T Statistic P-Value Intercept Slope 4.79999 0.0958888 -0.0561875 0.00787536 50 . 0579 -7 . 13459 0 0 . 0000 . 0000 Analysis of Variance Source Sum of Squares Df Mean Square F Ratio P-Value Model Residual 22.1647 1 56.6067 130 22 . 1647 0 . 435436 50.90 0.0000 Total (Corr ) 78.7714 131 Correlation Coefficient = -0.530453 R-squared = 28.138 percent Standard Error of Est. = 0.659876

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104 Table C.2.2. Model used to compare the relationship of exposure time (days) to a temperature regime of 35°C for 8 hours daily in soil amended with 0.125% cabbage (T35125C) on the survival of Phytophthora nicotianae (propagules per gram of soil) with all other temperature regimes (data transformed as In [ppg+1]). Regression Analysis Linear model: Y = a + b*X Dependent variable: LOG (T35125C+1) Independent variable : DAYS Parameter Standard Estimate Error T Statistic P-Value Intercept Slope 4.80793 0.0943055 -0.055084 0.00774532 50 . 9825 -7.1119 0 . 0000 0 . 0000 Analysis of Variance Source Sum of Squares Df Mean Square FRatio P -Value Model Residual 21.3027 1 54.7528 130 21 .3027 0 .421175 50 . 58 0 . 0000 Total (Corr.) 76.0555 131 Correlation Coefficient = -0.529239 R-squared = 28.0094 percent Standard Error of Est. = 0.64898 Table C.3. Comparison of models used to describe the relationship of exposure time (days) to a temperature regime of 41°C for 5 hours daily in soil amended with 0% cabbage on the survival of Phytophthora nicotianae (propagules per gram of soil). Comparison of Alternative Models Model Correlation R-Squared Exponential -0 8175 66.83% Reciprocal-Y 0 7844 61 . 53% Square root-Y -0 7778 60 . 50% Linear -0 7141 50 . 99% Square root-X -0 6673 44 .53% Multiplicative -0 6612 43 .72% Logarithmic-X -0 5804 33 .69% Scurve 0 4305 18 .53% Double reciprocal -0 4099 16 . 80% Reciprocal -X 0 3504 12 .28% Logistic Log probit

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105 Table C 3.1. Regression analysis with the model that best described the relationship of exposure time (days) to a temperature regime of 41°C for 5 hours daily in soil amended with 0% cabbage (T410C) on the survival of Phytophthora nicotianae (propagules per gram of soil). Regression Analysis Exponential model: Y = exp(a + b*X) Dependent variable: T410C Independent variable : DAYS Parameter Standard Estimate Error T Statistic P-Value Intercept Slope 6.05032 0.092978 -0.0912544 0.00685257 65 . 0725 -13 . 3168 0 0 . 0000 . 0000 Analysis of Variance Source Sum of Squares Df Mean Square F-Ratio P-Value Model Residual 45.6348 1 22.6453 88 45 . 6348 0 . 257333 177.34 0 . 0000 Total (Corr ) 68.2801 89 Correlation Coefficient = -0.817525 R-squared = 66.8347 percent Standard Error of Est. = 0.50728

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106 Table C.3.2. Model used to compare the relationship of exposure time (days) to a temperature regime of 41°C for 5 hours daily in soil amended with 0% cabbage (T410C) on the survival of Phytophthora nicotianae (propagules per gram of soil) with all other temperature regimes (data transformed as In [ppg+1]). Regression Analysis Linear model: Y = a + b*X Dependent variable: LOG(T410C+1) Independent variable : DAYS Parameter Standard Estimate Error T Statistic P-Value Intercept Slope 6.04972 0.0921267 -0.090339 0.00678983 65 .6674 -13 . 3051 0 . 0000 0.0000 Analysis of Variance Source Sum of Squares Df Mean Square F-Ratio P-Value Model Residual 44.7238 1 22.2325 88 44 . 7238 0 .252642 177 . 02 0 . 0000 Total (Corr.) 66.9563 89 Correlation Coefficient = -0.817285 R-squared = 66.7955 percent Standard Error of Est. = 0.502635 Table C.4. Comparison of models used to describe the relationship of exposure time (days) to a temperature regime of 41°C for 5 hours daily in soil amended with 0.125% cabbage on the survival of Phytophthora nicotianae (propagules per gram of soil). Comparison of Alternative Models Model Correlation R-Squared Square root-X -0 5100 26 . 01% Square root-Y -0 5019 25.20% Linear -0 4985 24.85% Logarithmic-X -0 4982 24 . 82% Exponential -0 4538 20.60% Multiplicative -0 4322 18 .68% Reciprocal-X 0 4025 16 .20% Scurve 0 3359 11.28% Double reciprocal -0 1266 1 .60% Reciprocal -Y Logistic Log probit Table C 4.1. Regression analysis with the model that best described the relationship of exposure time (days) to a temperature regime of 41°C for 5 hours daily in soil amended

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107 with 0.125% cabbage (T41 125C) on the survival of Phytophthora nicotianae (propagules per gram of soil). Regression Analysis Square root-X model; Y = a + b*sqrt(X) Dependent variable: T41125C Independent variable: DAYS Parameter Standard Estimate Error T Statistic P-Value Intercept Slope 62.46 5.70552 -9.52535 1.71251 10 . 9473 -5 .5622 0 . 0000 0.0000 Analysis of Variance Source Sum of Squares Df Mean Square F-Ratio P-Value Model Residual 13894.0 1 39519.9 88 13894 . 0 449 . 089 30 . 94 0 . 0000 Total (Corr ) 53413.8 89 Correlation Rsquared = Coefficient = -0.510019 26.0119 percent Standard Error of Est. = 21.1917

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108 Table C.4.2. Model used to compare the relationship of exposure time (days) to a temperature regime of 41°C for 5 hours daily in soil amended with 0.125% cabbage (T41 125C) on the survival of Phytophthora nicotianae (propagules per gram of soil) with all other temperature regimes (data transformed as In [ppg+1]). Regression Analysis Linear model: Y = a + b*X Dependent variable: LOG(T41125C+l) Independent variable : DAYS Parameter Standard T Estimate Error Statistic P-Value Intercept Slope 3.81191 0.13885 27.4534 -0.0509075 0.0102334 -4.97463 0 . 0000 0 . 0000 Analysis of Variance Source Sum of Squares Df Mean Square F-Ratio P-Value Model Residual 14.2021 1 14.2021 50.5024 88 0.573891 24.75 0 . 0000 Total (Corr ) 64.7045 89 Correlation Coefficient = -0.468499 R-squared = 21.9491 percent Standard Error of Est. = 0.757556 Table C.5. Comparison of models used to describe the relationship of exposure time (days) to a temperature regime of 44°C for 1.5 hours daily in soil amended with 0% cabbage on the survival of Phytophthora nicotianae (propagules per gram of soil). Comparison of Alternative Models Model Correlation R-Squared Square root-Y -0 8559 73 .25% Square root-X -0 8528 72 . 72% Exponential -0 8501 72 . 27% Logarithmic -X -0 8444 71 . 31% Linear -0 8256 68.16% Multiplicative -0 8089 G5 .43% Reciprocal-Y 0 7170 51.41% Reciprocal-X 0 7116 50 . 64% Scurve 0 6377 40 . 66% Double reciprocal -0 4657 21.69% Logistic Log probit Table C 5.1. Regression analysis with the model that best described the relationship of exposure time (days) to a temperature regime of 44°C for 1 .5 hours daily in soil amended

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109 with 0% cabbage (T440C) on the survival of Phytophthora nicotianae (propagules per gram of soil). Regression Analysis Square root-Y model: Y = (a + b*X) *2 Dependent variable: T440C Independent variable : DAYS Parameter Standard Estimate Error T Statistic P-Value Intercept Slope 9.81598 0.22684 -0.259534 0.0167183 43 .2728 -15.5239 0 . 0000 0 . 0000 Analysis of Variance Source Sum of Squares Of Mean Square FRatio P-Value Model Residual 369.127 1 134.789 88 369 . 127 1 . 5317 240 . 99 0 . 0000 Total (Corr.) 503.916 89 Correlation Coefficient = -0.855872 R-squared = 73.2516 percent Standard Error of Est. = 1.23762

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110 Table C.5.2. Model used to compare the relationship of exposure time (days) to a temperature regime of 44°C for 1.5 hours daily in soil amended with 0% cabbage (T440C) on the survival of Phytophthora nicotianae (propagules per gram of soil) with all other temperature regimes (data transformed as In [ppg+1]). Regression /Analysis Linear model: Y = a + b*X Dependent variable: LOG(T440C+1) Independent variable : DAYS Parameter Standard T Estimate Error Statistic P-Value Intercept Slope 4.63098 0.0681822 67.9207 -0.0768018 0.00502509 -15.2837 0 . 0000 0 . 0000 Analysis of Variance Source Sum of Squares Df Mean Square FRatio P-Value Model Residual 32.3245 1 32.3245 12.1775 88 0.138381 233 . 59 0 . 0000 Total (Corr ) 44.502 89 Correlation Coefficient = -0.852268 Rsquared = 72.S36 percent Standard Error of Est. = 0.371996 Table C.6. Comparison of models used to describe the relationship of exposure time (days) to a temperature regime of 44°C for 1.5 hours daily in soil amended with 0.125% cabbage on the survival of Phytophthora nicotianae (propagules per gram of soil). Comparison of Alternative Models Model Correlation R-Squared Square root-X -0 6224 38 . 74% Logarithmic -X -0 6151 37.83% Linear -0 6003 36 . 04% Square root-Y -0 5796 33 . 59% Reciprocal-X 0 5025 25 .25% Exponential Reciprocal-Y Double reciprocal Multiplicative Scurve Logistic Log probit

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Ill Table C 6.1. Regression analysis with the model that best described the relationship of exposure time (days) to a temperature regime of 44°C for 1 .5 hours daily in soil amended with 0.125% cabbage (T41 125C) on the survival of Phytophthora nicotianae (propagules per gram of soil). Regression Analysis Square root-X model: Y = a + b*sqrt(X) Dependent variable: T44125C Independent variable : DAYS Parameter Standard Estimate Error T Statistic P-Value Intercept Slope 39.6709 2.58388 -5.7855 0.775552 15.3532 -7.45985 0.0000 0 . 0000 Analysis of Variance Source Sum of Squares Df Mean Square FRatio P-Value Model Residual 5125.62 1 8105.3 88 5125.62 92 . 1057 55 .65 0 . 0000 Total (Corr.) 13230.9 89 Correlation Coefficient = -0.622412 R-squared = 38.7397 percent Standard Error of Est. = 9.59717

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112 Table C.6.2. Model used to compare the relationship of exposure time (days) to a temperature regime of 41°C for 1.5 hours daily in soil amended with 0.125% cabbage (T44125C) on the survival of Phytophthora nicotianae (propagules per gram of soil) with all other temperature regimes (data transformed as In [ppg+1]). Regression Analysis Linear model: Y = a + b*X Dependent variable: LOG (T44125C+1) Independent variable : DAYS Parameter Standard Estimate Error T Statistic P-Value Intercept Slope 3.45449 0.101241 -0.0442587 0.00746156 34 . 1214 -5 . 93156 0 . 0000 0 . 0000 Analysis of Variance Source Sum of Squares Df Mean Square F-Ratio P-Value Model Residual 10.7346 1 26.8491 88 10 . 7346 0.305104 35 . 18 0 . 0000 Total (Corr.) 37.5837 89 Correlation Coefficient = -0.534433 R-squared = 28.5618 percent Standard Error of Est. = 0.552362 Table C.7. Comparison of models used to describe the relationship of exposure time (days) to a temperature regime of 44°C for 5 hours daily in soil amended with 0% cabbage on the survival oi Phytophthora nicotianae (propagules per gram of soil). Comparison of Alternative Models Model Correlation R-Squared Logarithmic-X -0 . 9536 90 . 94% Reciprocal-X 0 . 9281 86.14% Square root-X -0 . 9133 83 .40% Linear -0 . 8525 72 .68% Exponential Reciprocal-Y Double reciprocal Multiplicative Square root-Y S curve Logistic Log probit Table C 7.1. Regression analysis with the model that best described the relationship of exposure time (days) to a temperature regime of 44°C for 5 hours daily in soil amended

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113 with 0% cabbage (T440C) on the survival of Phytophthora nicotianae (propagules per gram of soil). Regression Analysis Logarithmic-X model: Y = a + b*ln(X) Dependent variable: T440c Independent variable : DAYS Parameter Standard Estimate Error T Statistic P-Value Intercept Slope 45.5995 1.67579 -18.4265 0.919831 27 . 2107 -20 . 0325 0 . 0000 0 . 0000 Analysis of Variance Source Sum of Squares Df Mean Square FRatio P-Value Model Residual 12285.9 1 1224.61 40 12285 . 9 30 . 6153 401.30 0 . 0000 Total (Corr.) 13510.5 41 Correlation Coefficient = -0.953603 R-squared = 90.9359 percent Standard Error of Est. = 5.5331

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114 Table C.7.2. Model used to compare the relationship of exposure time (days) to a temperature regime of 44°C for 5 hours daily in soil amended with 0% cabbage (T440C) on the survival of Phytophthora nicotianae (propagules per gram of soil) with all other temperature regimes (data transformed as In [ppg+1]). Regression Analysis Linear model: Y = a + b*X Dependent variable: LOG(T440c+l) Independent variable : DAYS Parameter Standard Estimate Error T Statistic P-Value Intercept Slope 3.88569 0.135962 -0.261322 0.0160873 28 . 5792 -16 . 244 0 . 0000 0 . 0000 Analysis of Variance Source Sum of Squares Df Mean Square F-Ratio P-Value Model Residual 70.0062 1 10.6123 40 70 . 0062 0 .265308 263.87 0.0000 Total (Corr.) 80.6185 41 Correlation Coefficient = -0.93186 Rsquared = 86.8364 percent Standard Error of Est. = 0.515081 Table C.8. Comparison of models used to describe the relationship of exposure time (days) to a temperature regime of 44°C for 5 hours daily in soil amended with 0.125% cabbage on the survival of Phytophthora nicotianae (propagules per gram of soil). Comparison of Alternative Models Model Correlation R-Squared Reciprocal -X 0 . 9560 91 .39% Logarithmic -X -0 . 8441 71.25% Square root-X -0 . 7517 56 . 50% Linear -0. 6611 43 . 71% Exponential Reciprocal-Y Double reciprocal Multiplicative Square root-Y Scurve Logistic Log probit

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115 Table C 8.1. Regression analysis with the model that best described the relationship of exposure time (days) to a temperature regime of 44°C for 5 hours daily in soil amended with 0.125% cabbage (T44125C) on the survival of Phytophthora nicotianae (propagules per gram of soil). Regression Analysis Reciprocal-X model: Y = a + b/X Dependent variable: T44125C Independent variable : DAYS Parameter Standard Estimate Error T Statistic P-Value Intercept Slope -4.77879 0.825204 37.8509 1.83695 -5 . 79104 20 . 6053 0 . 0000 0 . 0000 Analysis of Variance Source Sum of Squares Df Mean Square FRatio PValue Model Residual 5864.72 1 552.52 40 5864 . 72 13 . 813 424 .58 0.0000 Total (Corr.) 6417.24 41 Correlation Coefficient = 0.955982 R-squared = 91.3901 percent Standard Error of Est. = 3.71659

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116 Table C.8.2. Model used to compare the relationship of exposure time (days) to a temperature regime of 44°C for 5 hours daily in soil amended with 0.125% cabbage (T44125C) on the survival of Phytophthora nicotianae (propagules per gram of soil) with all other temperature regimes (data transformed as In [ppg+1]). Regression Analysis Linear model: Y = a + b*X Dependent variable: LOG (T44125C+1) Independent variable : DAYS Parameter Standard Estimate Error T Statistic P-Value Intercept Slope 2.53127 0.254472 -0.216177 0.0301095 9 . 94715 -7 . 1797 0 . 0000 0 . 0000 Analysis of Variance Source Sum of Squares Of Mean Square P-Ratio P -Value Model Residual 47.9075 1 37.1751 40 47 . 9075 0 . 929377 51 . 55 0.0000 Total (Corr.) 85.0826 41 Correlation Coefficient = -0.75038 R-squared = 56.3071 percent Standard Error of Est. = 0.964042 Table C.9. Comparison of models used to describe the relationship of exposure time (days) to a temperature regime of 47°C for 3 hours daily in soil amended with 0% cabbage on the survival oi Phytophthora nicotianae (propagules per gram of soil). Comparison of Alternative Models Model Correlation R-Squared Reciprocal -X 0 . 6563 43 . 07% Logarithmic-X -0.5726 32 . 78% Square root-X -0 . 5082 25 . 82% Linear -0 . 4466 19 . 95% Exponential Reciprocal-Y Double reciprocal Multiplicative Square root-Y Scurve Logistic Log probit

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117 Table C 9.1. Regression analysis with the model that best described the relationship of exposure time (days) to a temperature regime of 47°C for 3 hours daily in soil amended with 0% cabbage (T470C) on the survival of Phytophthora nicotianae (propagules per gram of soil). Regression Analysis Reciprocal-X model: Y = a + b/X Dependent variable: T4 70C Independent variable : DAYS Parameter Standard Estimate Error T Statistic P-Value Intercept Slope -0.365705 0.279522 3.42285 0.622231 -1 . 30832 5 . 50092 0 . 1982 0 . 0000 Analysis of Variance Source Sum of Squares Df Mean Square F-Ratio P -Value Model Residual 47.9588 1 63.3954 40 47 . 9588 1 . 58489 30 . 26 0 . 0000 lotal (Corr.) 111.354 41 Correlation Coefficient = 0.656267 R-squared = 43.0687 percent Standard Error of Est. = 1.25892

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118 Table C.9.2. Model used to compare the relationship of exposure time (days) to a temperature regime of 47°C for 3 hours daily in soil amended with 0% cabbage (T470C) on the survival of Phytophthora nicotianae (propagules per gram of soil) with all other temperature regimes (data transformed as In [ppg+1]). Regression Analysis Linear model: Y = a + b*X Dependent variable: LOG(T470C+1) Independent variable : DAYS Parameter Standard Estimate Error T Statistic P-Value Intercept Slope 0.718247 0.134545 -0.0569253 0.0159196 5 . 33833 -3 . 5758 0 . 0000 0 . 0009 Analysis of Variance Source Sum of Squares Df Mean Square F-Ratio P-Value Model Residual 3.32196 1 10.3922 40 3 .32196 0 . 259806 12 . 79 0 . 0009 Total (Corr.) 13.7142 41 Correlation Coefficient = -0.492167 R-squared = 24.2228 percent Standard Error of Est. = 0.509712 Table C.IO. Comparison of models used to describe the relationship of exposure time (days) to a temperature regime of 47°C for 3 hours daily in soil amended with 0.125% cabbage on the survival oi Phytophthora nicotianae (propagules per gram of soil). Comparison of Alternative Models Model Correlation R-Squared Reciprocal -X 0 . 7557 57 . 10% Logarithmic-X -0 . 6016 36.20% Square root-X -0 . 5111 26.12% Linear -0 .4342 18 . 86% Exponential Reciprocal-Y Double reciprocal Multiplicative Square root-Y Scurve Logistic Log probit Table C 10.1. Regression analysis with the model that best described the relationship of exposure time (days) to a temperature regime of 47°C for 3 hours daily in soil amended

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119 with 0.125% cabbage (T47125C) on the survival of Phytophthora nicotianae (propagules per gram of soil). Regression Analysis Reciprocal-X model: Y = a + b/X Dependent variable: T47125C Independent variable : DAYS Parameter Standard Estimate Error T Statistic P-Value Intercept Slope -0.364854 0.136029 2.20952 0.302808 -2 .68218 7 .29677 0 . 0106 0 . 0000 Analysis of Variance Source Sum of Squares Df Mean Square F-Ratio P-Value Model Residual 19.9844 1 15.0138 40 19 . 9844 0.375344 53 .24 0 . 0000 Total (Corr.) 34.9982 41 Correlation Coefficient = 0.755654 R-squared = 57.1013 percent Standard Error of Est. = 0.612654

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120 Table C.10.2. Model used to compare the relationship of exposure time (days) to a temperature regime of 47°C for 3 hours daily in soil amended with 0.125% cabbage (T47125C) on the survival of Phytophthora nicotianae (propagules per gram of soil) with all other temperature regimes (data transformed as In [ppg+1]). Regression Analysis Linear model: Y = a + b*X Dependent variable: LOG (T47125C+1) Independent variable : DAYS Parameter Standard Estimate Error T Statistic P-Value Intercept Slope 0.455171 0.099209 -0.0402598 0.0117386 4 . 588 -3 .4297 0 .0000 0 . 0014 Analysis of Variance Source Sum of Squares Df Mean Square F-Ratio P-Value Model Residual 1.6616 1 5.65034 40 1.6616 0 . 141259 11.76 0.0014 Total (Corr.) 7.31195 41 Correlation Coefficient = -0.476702 R-squared = 22.7245 percent Standard Error of Est. = 0.375844

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APPENDIX D SUMMARY TABLE OF THE STATISTICAL ANALYSIS OF THE EFFECT OF SOIL WATER MATRIC POTENTIAL, TEMPERATURE REGIMES, AND CABBAGE AMENDMENTS ON THE THERMAL INACTIVATION OF CHLAMYDOSPORES OF PHYTOPHTHORA NICOTIAN AE Table D.l. Summary statistics for the effect of soil water matric potential, temperature regimes, and cabbage amendments on the survival of Phytophthora nicotianae r dcior Propagules per gram of soil Test 1 Test 2 Matric Potential (-kPa) 0 26.8^ a 21.3 a 10 31.4 b 28.3 c 30 32.9 b 25.0 b Temperature Regime 35-8 234.2 e 163.9 e 41-5 155.5 d 84.1 d 44-1.5 34.8 c 21.3 c 44-5 6.7 b 10.3 b 47-3 0.6 a 0.7 a Cabbage Amendment Non-amended 32.0 b 25.0 a Amended (0.125%) 29.0 a 24.4 a Time (days) 1 day 37.8 c 33.5 c 2 days 13.0 a 15.3 a 3 days 22.6 b 17.1 b 6 days 79.6 d 43.6 d (continued) ^ Main effect means followed by the same letter in each column do not differ according to the Tukey's Honestly Significant Difference procedure (P< 0.05); data were transformed to ln(ppg+l) for analysis and presented as weighted means ([Exp {mean}]-!). 121

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122 Table D. 1 . continued Factor Propagules per gram of soil Test 1 Test 2 Matric Potential x Temperature 280.4 e 197.2 e 0 X 41-5 V/ A. " 1 ^ 99.2 d 50.9 d 0 Y 44-1 5 28.9 c 18.5 c n X 446.8 b 9.8 b 0 X 47-3 0.2 a 0.2 a 10x35-8 202.3 e 161.0e 10x41-5 201.3 d 104.7 d 10x44-1.5 36.8 c 24.3 c 10x44-5 6.2 b 13.1 b 10x47-3 0.7 a 1.0 a 30x 35-8 226.4 e 138.7 e 30x41-5 188.4 d 111.6 d 30x44-1.5 39.6 c 21.5 c 30 X 44-5 7.0 b 8.3 b 30 X 47-3 v.y a i .u a Matric Potential x Amendment 0 X 0% 32.6 b 29.4 b 0x0.125% 22.0 a 15.4 a 10x0% 32.6 a 25.0 a 10x0.125% 30.4 a 32.1 b 30 X 0% 31.0a 21.3 a 30x0.125% 36.3 b 29.2 b Matric Potential x Time 0x1 11.1 c 26.6 c 0x2 12.3 a 14.0 a 0x3 22.2 b 14.8 b 0x6 73.3 d 40.2 d lOx 1 43.3 c 42.4 c 10x2 12.8 a 17.1 a 10x3 21.7b 18.6 b 10x6 81.0d 47.4 d 30 X 1 45.0 c 33.3 c 30x2 13.8 a 14.9 a 30x3 23.9 b 18.1 b 30x6 85.0 d 43.5 d (continued)

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123 Table D. 1 . continued Factor Propagules per gram of soil Test 1 Test 2 Temperature Regime x Amendment 35-8 X 0% 251.9 b 205.0 b 35-8x0.125% 217.7 a 131.1 a 41-5x0% 222.4 b 130.2 b 41-5x0.125% 108.8 a 54.3 a 44-1.5x0% 23.8 a 11.8 a 44-1.5 x0.125% 50.7 b 38.0 b 44-5 x 0% 8.9 b 10.8 a 44-5x0.125% 4.9 a 9.7 a 47-3 X 0% 0.4 a 0.5 a 47-3 x0.125% 0.7 b 1.0 b Temperature Regime x Time 35-8 X 1 250.2 b 158.3 b 35-8 X 2 280.9 b 306.5 c 35-8 X 3 216.3 a 154.3 b 35-8 X 6 197.7 a 96.3 a 41-5 X 1 212.3 c 147.9 b 41-5x3 154.4 b 71.1 a 41-5x6 111.1 a 56.5 a 44-1.5 X 1 56.0 c 38.6 b 44-1.5x3 33.2 b 16.7 a 44-1.5x6 22.5 a 14.8 a 44-5 X 1 10.6 c 15.0 c 44-5 X 2 6.7 b 10.0 b 44-5 X 3 4.0 a 7.1 a 47-3 X 1 1.5 b 2.3 b 47-3 X 2 0.3 a 0.3 a 47-3 X 3 0.2 a 0.2 a Amendment x Time " 0% x 1 37.5 c 33.3 c 0% X 2 13.6 a 15.4 a 0% X 3 23.4 b 17.5 b 0% X 6 94.6 d 45.1 d 0.125% X 1 38.1 c 33.7 c 0.125% X 2 12.4 a 15.1 a 0.125% X 3 21.8b 16.7 b 0.125% X 6 67.0 d 42.1 d

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APPENDIX E SUMMARY TABLE OF THE STATISTICAL ANALYSIS OF THE EFFECT OF THREE DIFFERENT SOILS, SOIL PASTEURIZATION, TEMPERATURE REGIMES, AND CABBAGE AMENDMENT ON THE THERMAL INACTIVATION OF CHLAMYDOSPORES OF PHYTOPHTHORA NICOTIANAE Table E.l. Summary statistics for the effect of three different soils, soil pasteurization, temperature regimes, and cabbage amendment on the survival of Phytophthora nicotianae. Factor Propagules per gram of soil Te-st 1 -1 Vol 1 Test J Soir Site 1 6.9^ b 12.0b Site 2 4.8 a 4.5a Sites 6.9 b 14.1c Pasteurization^ Pasteurized soil 5.5 a 9.6a Non-pasteurized soil 6.8 b 8.9a Temperature regime^ 35-8 31.2 d 80.2e 41-5 29.9 d 39.3d 44-1.5 10.2 c 13.9c 44-5 0.6 b 1.3b 47-3 0.0 a Oa Cabbage amendment Non-Amended 8.8 b 13.2b Amended (0.125%) 4.2 a 6.4a Time (days) 3 days 7.5 b 9.6a 9 days 5.0 a 8.9a (continued) 124

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125 Table E.l. continued Factor Propagules per gram of soil Test 1 Test 2 Soil X Pasteurization Site 1 Pasteurized soil 4.8 a 10.6 a Site 1 Non-pasteurized soil 9.8 b 13.6b Site 2 Pasteurized soil 4.7 a 3.4 a Site 2 Non-pasteurized soil 4.9 a 5.8 b Site 3 Pasteurized soil 7.2 a 21.9b Site 3 Non-pasteurized soil 6.6 a 8.9 a oOli X 1 eniperaiure reginie Cifo 1 V 1^ 8 j)iie 1 X jj-o JO. J Q 49 4 H 79 7 r /Z. / t. <5itp> 1 V AA 1 1 Ah 91 Q h Z 1 .7 U one 1 X H'i-J U.O d 9 6 a Z.O a oiie 1 X 't / -J n 1 Q U. 1 a U.U a oiie z X J j-o JV.y U Jj.y U 9 Y 41 ^ OllC Z A Hl-J 1 o.O L 14 7 H Cifp 9 Y 44 1 ^ oiie z X t't-i.j 7 9 V> /.ZD n r> D.U C Site 2 X 44-5 0.3 a 0.3 b Site 2 X 47-3 0.0 a 0.0 a Site 3 X 35-8 22.6 c 256.7 e Site 3 X 41-5 33.6 c 55.4 d Site 3x44-1.5 19.2 c 19.5 c Site 3 X 44-5 0.8 b 1.5 b Site 3 X 47-3 0.0 a 0.0 a Soil X Amendment Site 1 X 0% 8.5 b 16.4 b Site 1 X 0.125% 5.6 a 8.8 a Site 2 X 0% 7.7 b 6.7 b Site 2x0.125% 2.8 a 2.9 a Site 3 X 0% 10.2 b 20.5 b Site 3 X 0.125% 4.5 a 9.5 a Soil X Time Sitel X 3 days 8.8 b 11.8b Site! X 9 days 5.4 a 12.3 b Site2 X 3 days 5.3 a 4.0 a Site2 X 9 days 4.3 a 4.9 a Site3 X 3 days 8.8 b 17.5 b Site3 X 9 days 5.3 a 11.3 a (continued) Table E.l. continued

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126 Factor Propagules per gram of soil Test 1 Test 2 Temperature regime x Amendment 35-8 X 0% 37.8 b 79.0 a 35-8x0.125% 25.7 a 81.4 a 41-5 X 0% 68.5 b 63.6 b 41-5 X 0.125% 12.7 a 24.1 a 44-1.5x0% 15.3 b 30.3 b 44-1.5x0.125% 6.6 a U.W a 44-5 x 0% 1.0b A.H D 44-5x0.125% 0.3 a Kj.j a 47-3 X 0% 0.0 a U.U d 47-3 X 0.125% 0.0 a W.U a Temperature regime x Time 35-8 X 3 days 31.5 a 52.7 a 35-8 X 9 days 30.9 a 121.8b 41-5 X 3 days 37.0 b 47.7 b 41-5 X 9 days 24.1 a 32.4 a 44-1.5 X 3 days 14.7 b 15.1 a 44-1.5 X 9 days 6.9 a 19 7a 44-5 X 3 days 1.1 b 9 n h 44-5 X 9 days 0.2 a \J. 1 a 47-3 X 3 days 0.1 a U.U d 47-3 X 9 days 0.0 a 0.0 a Cabbage Amendment x Time 0% X 3 days 11.0 b 14.6 b 0% X 9 days 7.0 a 11.9a 0.125%, X 3 days 5.0 b 6.2 a 0.125% X 9 days 3.5 a 6.6 a " Site 1 = Naples Tomato Growers-Gargiulo farm number 4 in Decatur County, Georgia; Site 2 = John Allen Smith farm located in Gadsden County, Florida; Site 3 = North Florida Research and Educational Center in Gadsden County. " Main effect means followed by the same letter in each column do not differ according to the Tukey's Honestly Significant Difference procedure (P< 0.05); data were transformed to ln(ppg+l) for analysis and presented as weighted means ([Exp {mean}]-!). Soil (1-kg lots) was pasteurized in a microwave oven at 700 W for 4 minutes, after moisture had been adjusted to 5% (w/w). ^ Temperature regimes that simulated solarization consisted of temperatures increased daily to 35°, 41°, 44°, 44°, or47°C for 8, 5, 1.5, 5 or 3 hours, respectively; the temperature for the remainder of each day was maintained at 25°C.

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LIST OF REFERENCES Alfieri Jr., S. A., Langdon, K. R., Kimbrough, K. R., El-GhoU, N. E., and Wehlburg, C. 1994. Diseases and Disorders of Plants in Florida. Division of Plant Industry, Florida Department of Agriculture and Consumer Services. Anderson, W. A., McClure, P. J., Baird-Parker, A. C, and Cole, M. B. 1996. The application of a log-logistic model to describe the thermal inactivation of Clostridium botulinum 213B at temperatures below 121 . 1°C. Journal of Applied Bacteriology 80:283-290. Baker, K. F. and Cook, R. J. 1974. Biological Control of Plant Pathogens. W.H. Freeman and Co. San Francisco. 433pp. Barbercheck, M. E. and Von Broembsen, S. L. 1986. Effects of soil solarization on plantparasitic nematodes and Phytophthora cinnamomi in South Africa. Plant Disease 70:945-950. Benson, D. M.1978. Thermal inactivation of Phytophthora cinnamomi for control of Eraser Fir root rot. Phytopathology 68:1373-1376. Bernhardt, E. A. and Grogan, R. G. 1982. Effect of soil matric potential on the formation and indirect germination of sporangia of Phytophthora parasitica, P. capsici and P. cryptogea. Phytopathology 72:507-51 1. Bigelow, W. D.1921. The logarithmic nature of thermal death time curves. Journal of Infectious Diseases 29:528-536. Bigelow, W. D. and Esty, J. R. 1920. The thermal death point in relation to time of typical thermophilic organisms. Journal of Infectious Diseases 27:602-617. Bollen, G. J.1985. Lethal temperatures of soil fungi. Pages 191-193 in: Ecology and Management of Soilborne Plant Pathogens. Parker, C. A., Rovira, A. D., Moore, K. J., Wong, P. T. W., and Kollmorgen, J. F. (Eds.) APS Press. St. Paul, MN. Bowers, J. H. and Mitchell, D. J. 1990. Effect of soilwater matric potential and periodic flooding on mortality of pepper caused by Phytophthora capsici. Phytopathology 80:1447-1450. 127

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128 Bowers, J. H., Papavizas, G. C, and Johnston, S. A. 1990a. Effect of soil temperature and soil-water matric potential on the survival of Phytophthora capsici in natural soil. Plant Disease 1A:11\-111. Bowers, J. H., Sonoda, R. M., and Mitchell, D. J. 1990b. Path coefficient analysis of the effect of rainfall variables on the epidemiology of Phytophthora blight of pepper caused by Phytophthora capsici. Phytopathology 80:1439-1446. Browne, G. T. and Mircetich, S. M. 1988. Effects of flood duration on the development of Phytophthora root and crown rots of apple. Phytopathology 78:846-851. Chellemi, D. O., Olson, S. M., and Mitchell, D. J. 1994. Effects of soil solarization and fumigation on survival of soilbome pathogens of tomato in northern Florida. Plant Disease 78:1167-1172. Chellemi, D. O., Olson, S. M., Mitchell, D. J., Seeker, I., and McSorley, R. 1997. Adaptation of soil solarization to the integrated pest management of soilbome pests of tomato under humid conditions. Phytopathology 86:250-258. Cole, M. B., Davies, K. W., Munro, G., Holyoak, C. D., and Kilsby, D. C. 1993. A vitalistic model to describe the thermal inactivation of Listeria monocytogenes. Journal of Industrial Microbiology 12:232-239. DeVay, J. E. 199 la. Use of soil solarization for control of fungal and bacterial plant pathogens including biocontrol. Pages 79-93 in: Soil Solarization. DeVay, J.E., Stapleton, J. J., and Elmore, C.L (Eds.). FAO. Rome. DeVay, J. E. 199 lb. Historical review and principles of soil solarization. Pages 1-15 in: Soil Solarization. DeVay, J. E., Stapleton, J. J., and Ehnore, C. L. (Eds.) FAO. Rome. DeVay, J. E. and Katan, J. 1991. Mechanisms of pathogen control in solarized soils. Pages 87-101 in: Soil Solarization. Katan, J. and DeVay, J. E. (Eds.) CRC Press. Boca Raton. Duniway, J. M. 1979. Water relations of water molds. Annu. Rev. Phytopathol 17:431460. Erwin, D.C. and Ribeiro, O.K. 1996. Phytophthora Diseases Worldwide. APS Press, St. Paul, MN. 562pp. Ferrin, D. M. and Mitchell, D. J. 1986a. Influence of initial density and distribution of inoculum on the epidemiology of tobacco black shank. Phytopathology 76:1 153-1 158. Ferrin, D. M. and Mitchell, D. J. 1986b. Influence of soil water status on the epidemiology of tobacco black shank. Phytopathology 76:1213-1217.

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129 Gamliel, A., Hadar, E., and Katan, J. 1989. Soil solarization to improve yield of Gypsophila in monoculture systems. Acta Horticulturae 255:131-138. Gamliel, A., Hadar, E., and Katan, J. 1993. Improvement of growth and yield of Gypsophila paniculata by solarization or fumigation of soil or container medium in continuous cropping systems. Plant Disease 77:933-938. Gamliel, A. and Katan, J. 1993. Suppression of major and minor pathogens by fluorescent pseudomonads in solarized and nonsolarized soils. Phytopathology 83:6875. GamHel, A. and Stapleton, J. J. 1993a. Effect of chicken compost or ammonium phosphate and solarization on pathogen control, rhizosphere microorganisms, and lettuce grov^h. Plant Disease 77:886-891. GamHel, A. and Stapleton, J. J. 1993b. Characterization of antifungal volatile compounds evolved from solarized soil amended with cabbage residues. Phytopathology 83:899905. Ghini, R., Bettiol, W., Spadotto, C. A., Moraes, G. J., Paraiba, L. C, and Mineiro, J. L. C. 1993. Soil solarization for the control of tomato and eggplant Verticillium wilt and its effect on weed and micro-arthropod communities. Summa Phytopathologica 19:183-189. Grinstein, A., Katan, J., Razik, A. A., Zeydan, O., and Elad, Y. 1979a. Control of Sclerotium rolfsii and weeds in peanuts by solar heating of the soil. Plant Disease Reptr. 63:1056-1059. Grinstein, A., Orion, D., Greenberger, A., and Katan, J. 1979b. Solar heating of the soil for the control of Verticillium dahliae and Pratylenchus thornei in potatoes. Pages 431-438 in: Soil-borne Plant Pathogens. Schippers, B. and Gams, W. (Eds.) Academic Press. New York. Grooshevoy, S. E., Khudyna, 1. P., and Popova, A. A. 1941. Methods for disinfecting seed-bed soil by natural sources of heat. Rev. Appl. Mycol. 20:87-88. Hartz, T. K., DeVay, J. E., and Elmore, C. L. 1993. Solarization is an effective soil disinfestation technique for strawberry production. HortScience 28:104-106. Hord, M. J. and Ristaino, J. B. 1992. Effect of the matric component of soil water potential on infection of pepper seedlings in soil infested with oospores of Phytophthora capsici. Phytopathology 82:792-798. loannou, N. and Grogan, R. G. 1985. Formation and germination of chlamydospores of Phytophthora parasitica under various oxygen and carbon dioxide tensions. Plant Disease 69:400-403.

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130 Jacobsen, B. J., Greenberger, A., Katan, J., Levi, M., and Alon, H. 1980. Control of Egyptian broomrape (Orobanche aegyptiacd) and other weeds by means of solar heating of the soil by polyethylene mulching. Weed Science 28:312-316. Juarez-Palacios, C, Felix-Gastelum, R., Wakeman, R. J., Paplomatas, E. J., and DeVay, J. E. 1991. Thermal sensitivity of three species of Phytophthora and the effect of soil solarization on their survival. Plant Disease 75: 1 160-1 164. Kannwischer, M. E. and Mitchell, D. J. 1981. Relationships of numbers of spores of Phytophthora parasitica var. nicotianae to infection and mortality of tobacco. Phytopathology 71:69-73. Kassaby, F. Y.1985. Solar-heating soil for control of damping-off diseases. Soil Biol. Biochem. 17:429-434. Katan, J. 1980. Solar pasteurization of soils for disease control: status and prospects. Plant Disease 64:450-454. Katan, J. 1981. Solar heating (solarization) of soil for control of soilbome pests. Annu. Rev. Phytopathol. 19:211-236. Katan, J. 1985. Solar disinfestation of soils. Pages 274-278 in: Ecology and Management of Soilbome Plant Pathogens. Parker, C. A., Rovira, A. D., Moore, K. J., Wong, P. T. W., and Kollmorgen, J. F. (Eds.) APS Press. St. Paul, MN. Katan, J., Fishier, G., and Grinstein, A. 1983. Shortand long-term effects of soil solarization and crop sequence on Fusarium wilt and yield of cotton in Israel. Phytopathology 73:1215-1219. Katan, J., Greenberger, A., Alon, H., and Grinstein, A. 1976. Solar heating by polyethylene mulching for the control of diseases caused by soil-borne pathogens. Phytopathology 66:683-688. Katan, J., Grinstein, A., Greenberger, A., Yarden, O., and DeVay, J. E. 1987. The first decade (1976-1986) of soil solarization (solar heating): a chronological bibliography. Phytoparasitica 1 5 :229-255 . Katan, J., Rotem, I., Finkel, Y., and Daniel, J. 1980. Solar heating of the soil for the control of pink root and other soilbome diseases in onions. Phytoparasitica 8:39-50. Keinath, A. P. 1995. Reductions in inoculum density of Rhizoctonia solani and control of belly rot on pickling cucumber with solarization. Plant Disease 79:1213-1219.

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131 Keinath, A. P. 1996. Soil amendment with cabbage residue and crop rotation to reduce gummy stem blight and increase growth and yield of watermelon. Plant Disease 80:564-570. Kulkami, R. N., Kalra, A., and Ravindra, N. S. 1992. Integration of soil solarization with host resistance in the control of dieback and collar and root rot diseases of periwinkle. Tropical Agriculture 69:217-222. Larkin, R. P., Ristaino, J. B., and Campbell, C. L. 1995. Detection and quantification of Phytophthora capsici in soil. Phytopathology 85:1057-1063. Leonian, L. H.1922. Stem and fruit blight of peppers caused by Phytophthora capsici sp.nov. Phytopathology 912:401-408. Lewis, J. A. and Papavizas, G. C. 1971. Effect of sulfur-containing volatile compounds and vapors from cabbage decomposition on Aphanomyces euteiches. Phytopathology 61:208-214. Lewis, J. A. and Papavizas, G. C. 1974. Effect of volatiles from decomposing plant tissues on pigmentation, growth, and survival of Rhizoctonia solani. Soil Science 118:156-163. 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 Disease 75:173-179. Lutz, A. L., Menge, J. A., and Ferrin, D. M. 1991. Increased germination of propagules of Phytophthora parasitica by heating citrus soils sampled during winter. Phytopathology 81:865-872. Luz, E. D. M. N. and Mitchell, D. J. 1994. Influence of soil flooding on cacao root infection by Phytophthora spp. Agrotropica 6:53-60. Mahrer, Y., Naot, O., Rawitz, E., and Katan, J. 1984. Temperature and moisture regimes in soils mulched with transparent polyethylene. Soil Sc. Soc. Amer. J. 48:362-367. Malathrakis, N. E. and Loulakis, M. D. 1989. Effectiveness of the type of polyethylene sheet on soil solarization. Acta Horticulturae 255:235-241. Maloy, O. C. 1993. Plant Disease Control: Principles and Practice. John Wiley & Sons, Inc. New York. 346 pp. Mayton, H. S., Olivier, C, Vaughn, S. P., and Loria, R. 1996. Correlation of fungicidal activity of Brassica species with allyl isothiocyanate production in macerated leaf tissue. Phytopathology 86:267-271.

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132 McGovem, R.J. and Begeman, J.P. 1996. Reduction of Phytophthora blight of madagascar periwinkle in the landscape by soil solarization. Proc. Fla. State Hortic. Soc. 108:58-60. McGovem, R. J., Jones, J. P., Mitchell, D. J., Pluim, R. A., and Gilreath, P. R. 1993. Severe outbreak of Phytophthora blight and fruit rot of cucurbits in Florida. Phytopathology 83:1388 Mcintosh, D. L.1972. Effects of soil water suction, soil temperature, carbon and nitrogen amendments, and host rootlets on survival in soil of zoospores of Phytophthora cactorum. Can. J. Bot. 50:269-272. McSorley, R. and Parrado, J. L. 1986. Application of soil solarization to rockdale soils in a subtropical environment. Nematropica 16:125-140. Mitchell, D. J. 1978. Relationships of inoculum levels of several soilbome species of Phytophthora and Pythium to infection of several hosts. Phytopathology 68:17541759. Mitchell, D. J. and Kannwischer-Mitchell, M. E. 1992. Phytophthora. Pages 31-38 in: Methods for Research on Soilborne Phytopathogenic Fungi. Singleton, L. L., Mihail, J. D., and Rush, C. M. (Eds.) APS Press. St. Paul, MN. Moens, M. and Aicha, B. B. 1990. Control of pepper wilt in Tunisia. Parasitica 46:103109. Myers, D. P., Campell, R. N., and Greathead, A. S. 1983. Thermal inactivation of Plasmodiophora brassicae Woron. and its attempted control by solarization in the Salinas Valley of California. Crop Protection 2:325-333. Neher, D. and Duniway, J. M. 1991. Relationship between amount of Phytophthora parasitica added to field soil and the development of root rot in processing tomatoes. Phytopathology 8 1 : 1 1 24-1 129. Neher, D. and Duniway, J. M. 1992. Dispersal of Phytophthora parasitica in tomato fields by fiirrow irrigation. Plant Disease 76:582-586. Neher, D. A., McKeen, C. D., and Duniway, J. M. 1993. Relationships among Phytophthora root rot development, P. parasitica populations in soil, and yield of tomatoes under commercial field conditions. Plant Disease 77:1 106-1 111. Papavizas, G. C, Bowers, J. H., and Johnston, S. A. 1981. Selective isolation of Phytophthora capsici from soils. Phytopathology 71:129-133. Pinkas, Y., Kariv, A., and Katan, J. 1984. Soil solarization for the control of Phytophthora cinnamomi: thermal and biological effects. Phytopathology 74:796

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133 Porter, I. J. 1991. Factors which influence the effectiveness of solarization for control of soilbome fungal pathogens in South Eastern Australia. Ph.D. Dissertation, La Trobe University, Bundoora, Victoria, Australia. Porter, I. J. and Merriman, P. R. 1983. Effects of solarization of soil on nematode and fungal pathogens at two sites in Victoria. Soil Biol. Biochem. 15:39-44. Porter, I. J. and Merriman, P. R. 1985. Evaluation of soil solarization for control of root diseases of row crops in Victoria. Plant Pathology 34:108-118. Porter, I. J., Merriman, P. R., and Keane, P. J. 1991. Soil solarization combined with low rates of soil fumigants controls clubrot of cauliflowers, caused by Plasmodiophora brassicae Woron. Austral. J. Exper. Agr 31:843-851. Pullman, G. S., DeVay, J. E., and Garber, R. H. 1981a. Soil solarization and thermal death: a logarithmic relationship between time and temperature for four soilbome plant pathogens. Phytopathology 71:959-964. Pullman, G. S., DeVay, J. E., Garber, R. H., and Weinhold, A. R. 1979. Control of soilbome fungal pathogens by plastic tarping of soil. Pages 431-438 in: Soil-borne Plant Pathogens. Schippers, B. and Gams, W. (Eds.) Academic Press. New York. Pullman, G. S., DeVay, J. E., Garber, R. H., and Weinhold, A. R. 1981b. Soil solarization: effects on Verticillium wilt of cotton and soilbome populations of Verticillium dahliae, Pythium spp., Rhizoctonia solani, and Thielaviopsis basicola. Phytopathology 71:954-959. RamirezVillapudua, J. and Munnecke, D. E. 1987. Control of cabbage yellows {Fusarium oxysporum f sp. conglutinans) by solar heating of field soils amended with dry cabbage residues. Plant Disease 71:21 7-22 1 . RamirezVillapudua, J. and Munnecke, D. E. 1988. Effect of solar heating and soil amendments of cmciferous residues on Fusarium oxysporum f sp. conglutinans and other organisms. Phytopathology 78:289-295. Ristaino, J. B. 1991. Influence of rainfall, drip irrigation, and inoculum density on the development of Phytophthora root and crown rot epidemics and yield in bell pepper. Phytopathology 81:922-929. Ristaino, J. B., Duniway, J. M., and Marois, J. J. 1988. Influence of frequency and duration of furrow irrigation on the development of Phytophthora root rot and yield in processing tomatoes. Phytopathology 78:1701-1706.

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134 Ristaino, J. B., Hord, M. J., and Gumpertz, M. L. 1992. Population densities of Phytophthora capsici in field soils in relation to drip irrigation, rainfall, and disease incidence. Plant Disease 76:1017-1024. Schlub, R. L.1983. Epidemiology of Phytophthora capsici on bell pepper. /. Agric. Sci. 100:7-11. Shew, H. D.1983. Effects of soil matric potential on infection of tobacco by Phytophthora parasitica var. nicotianae. Phytopathology 73: 11 60-1 163. Sidebottom, J. R. and Shew, H. D. 1985a. Effects of soil texture and matric potential on sporangium production by Phytophthora parasitica var. nicotianae. Phytopathology 75:1435-1438. Sidebottom, J. R. and Shew, H. D. 1985b. Effect of soil type and matric potential on infection of tobacco by Phytophthora parasitica var. nicotianae. Phytopathology 75:1439-1443. Smith, J. H. 1923. The killing of Botrytis cinerea by heat, with a note on the determination of temperature coefficients. Ann. Appl. Biol. 10:335-347. Souza, N. L. 1994. SolarizafSo do solo. Summa Phytopathologica 20:3-15. Stapleton, J. J. 1991. Thermal inactivation of crop pests and pathogens and other soil changes caused by solarization. Pages 37-47 in: Soil Solarization. DeVay, J. E., Stapleton, J. J., and Elmore, C. L. (Eds.). FAO. Rome. Stapleton, J. J. and DeVay, J. E. 1984. Thermal components of soil solarization as related to changes in soil and root microflora and increased plant growth response. Phytopathology 74:255-259. Stapleton, J. J. and DeVay, J. E. 1986. Soil solarization: a non-chemical approach for management of plant pathogens and pests. Crop Protection 5:190-198. Stapleton, J. J. and Garza-Lopez, J. G. 1988. Mulching of soils with transparent (solarization) and black polyethylene films to increase growth of annual and perennial crops in southwestern Mexico. Tropical Agriculture 65:29-33. Stapleton, J. J., Duncan, R. A., and Thomassian, C. 1995. Antifungal activity of certain cruciferous amendments when combined with soil heating for biofumigation. Phytopathology 85:1042. Subbarao, K. V. and Hubbard, J. C. 1996. Interactive effects of broccoli residue and temperature on Verticillium dahliae microsclerotia in soil and on wilt in cauliflower. Phytopathology 86:1303-1310.

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135 Tjamos, E. C. and Fravel, D. R. 1995. Detrimental effects of sublethal heating and Talaromyces flavus on microsclerotia of Verticillium dahliae. Phytopathology 85:388392. Wicks, T. J. 1988. Effect of solarisation on the control of Phytophthora cambivora in almond and cherry. Austral. J. Exper. Agr. 28:539-545.

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BIOGRAPHICAL SKETCH Lisias Coelho was born on July 3, 1959, in Piumhy, State of Minas Gerais, Brazil. Lisias attended the Universidade Federal de Vi90sa in Vi90sa, MG, Brazil, between 1979 and 1983, where he earned a Bachelor of Science degree of Engenheiro Florestal. He began a master's program at the same institution immediately after the conclusion of the bachelor's degree. In 1985, he assumed the position of researcher in plant protection at CENIBRA Florestal S.A., in Ipatinga, MG, Brazil. In 1989, Lisias completed his Master of Science degree in plant pathology with a thesis titled "Physiological Variability of Puccinia psidii Winter The Eucalyptus Rust" and married Eliamar Cavaleiro de Moraes Coelho. In the Fall of 1990 Lisias was accepted at the University of Florida to pursue a doctoral degree in the Plant Pathology Department. 136

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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. Mitchell, 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. Daniel O. Chellemi Assistant Professor of Plant Pathology, NFREC, Quincy 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. Harold C. Kistler Associate 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. )bei^ J. McGov^rn Assistant Professor of Plant Pathology, GCREC, Bradenton 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. 6avid M. Sylvia ^ 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. May, 1997 Dean, College of Agriculture Dean, Graduate School