role of plant growth-promoting rhizobacteria in integrated disease management and productivity of tomato

Bacterial population composition in 49 day-old tomato roots inoculated with Bacillus subtilis strain MBI600, B.. Bacterial population composition on 56 day-old tomato roots inoculated wi

ROLE OF PLANT GROWTH-PROMOTING RHIZOBACTERIA IN INTEGRATED

DISEASE MANAGEMENT AND PRODUCTIVITY OF TOMATO

UMI Number: 3197883

3197883 2006

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ABSTRACT

The ability of three rifampicin-resistant strains of Bacillus spp (B subtilis MI600,

B subtilis GBO3 and B amyloliquefaciens IN937) to improve fresh market

tomato growth and productivity and reduce the intensity of diseases caused by

Xanthomonas euvesicatoria, Alternaria solani, Septoria lycopersici and

Pseudomonas cichorii was evaluated under greenhouse, field, and controlled environmental conditions Rifampicin-resistant mutants of each strain were generated and used to monitor colonization of tomato roots There were no differences in colony morphology, endospore production or ERIC fingerprint pattern between wild-type and rifampicin resistant mutants of MBI600 and IN937 The rifampicin-resistant mutant of GBO3 had the same colony morphology and ERIC-fingerprint pattern as the wild-type, but produced significantly fewer endospores Under greenhouse conditions, no growth increase or bacterial leaf

spot (X euvesicatoria) suppression was observed in tomato plants inoculated with any of the Bacillus spp evaluated in this study Under field conditions,

MBI600 and GB03+IN937 were integrated in an intensive tomato management program that included mulch, drip irrigation and a forecasted fungicide spray

program Bacillus spp population densities were 104 - 106 CFU g-1 during the

seedling stage and dropped to less than 103 CFU g-1 during the flowering and

fruiting stages Significant increases in plant height were observed in all inoculated tomato plants, however, foliar diseases incited by A solani and S

Bacillus-lycopersici were not reduced GBO3+IN937-inoculated plants were more

susceptible to bacterial stem rot caused by Pseudomonas cichorii than those inoculated with MBI600 or non-inoculated control plants In controlled environmental studies, Bacillus spp population densities ranged from 101-105CFU g-1 tomato root Neither nitrogen concentration in the nutrient solution nor activation of the systemic acquired resistance defense mechanism had a significant effect on population density of MBI600, GBO3 or IN937 In a separate

experiment under controlled environmental conditions, Bacillus spp strains

MBI600, and GBO3+IN937 did not increase nitrogen concentration in tomato

leaves Lesions induced by Pseudomonas cichorii on tomato stems tended to be

longer in GBO3+IN937 inoculated plants than those inoculated with MBI600 or a water-treated control

DEDICATION

To my parents

Maria Cristina Diaz Lozano

Jose Guadalupe Nava Bernal

reri

ACKNOWLEDGMENTS

The author would like to express his deepest gratitude to all members of his advisory committee, Dr Sally A Miller, Dr Michael A Ellis, Dr Douglas J Doohan, and Dr Matthew D Kleinhenz for their various inputs during course and laboratory work as well as guidance in this research

The author would like to thank Ms Melanie L Lewis Ivey and Dr Annette L Wszelaki for their important inputs and guidance during his stay at The Ohio State University Department of Plant Pathology

The author would like to acknowledge the economical support of the National Council for Science and Technology (CONACyT) Mexico and the Ohio State University

The author would like to acknowledge Mr Bert Bishop, Dr Larry Madden, and

Dr Paul Pierce for patient support in statistical analysis

The author would like to acknowledge the support of Dr Fulya Baysal Tustas The author would like to thank the help and support of Emilia Gabriela Briceno Montero

The author acknowledges the support of Fatthy and Samia Abdelalim

The author acknowledges the support of friends: Katia A Figueroa R., Mizuho Nita, Angel Rebollar Alviter, Jhony Mera, Carilyn Perry and Santiago X Mideros

VITAE

1971………….…Born, Mexico city

1994………….…Research Assistant, Phytopathogenic Fungi

1995………… BSc Agricultural Parasitology, Autonomous Chapingo University 1997………… M.S Phytopathology, Postgraduate College

1997……… Technician, Plant Parasitic Nematodes

2001………….…Research Assistant, Integrated Disease Management

2002 – present…Student Research Assistant, The Ohio State University

PUBLICATIONS

Nava-Diaz, C., Abdelalim, F., Kleinhenz, M.D., Doohan, D.J., Lewis Ivey M.L.,

and Miller, S.A 2005 Effect of mulch, irrigation, fungicide program and

Bacillus spp on fresh market tomato Phytopathology 95: s73

Lewis-Ivey, M.L., Nava-Diaz, C., and Miller, S.A 2004 Identification and

management of Colletotrichum acutatum on immature bell peppers Plant

Disease 88: 1198-1204

Nava-Diaz, C., Kleinhenz, M.D., Doohan, D.J., Lewis-Ivey, M.L and Miller, S.A

2004 Bacillus spp with potential as biological control agents

Phytopathology 84(6): s74

Teliz, O.D and Nava, D.C 2001 Integrated pest management: bases and

philosophy 15-22 (8p.) Teliz, O D 2001 Integrated Pest Management Simposio Annual Congress (ISBN 968-5284-07-5) The Postgraduate College, Mexican Entomological Society and Mexican Phytopathological Society Queretaro, México July 2001: 129p

FIELDS OF STUDY

Plant Pathology

TABLE OF CONTENTS

ABSTRACT ii

DEDICATION iv

ACKNOWLEDGMENTS v

VITAE vi

LIST OF TABLES ix

LIST OF FIGURES xx

LIST OF ABBREVIATIONS xxv

INTRODUCTION 1

CHAPTER 1 28

INTRODUCTION 28

MATERIALS AND METHODS 34

RESULTS 39

DISCUSSION 49

CONCLUSIONS 53

LIST OF REFERENCES 54

CHAPTER 2 60

INTRODUCTION 60

MATERIALS AND METHODS 65

RESULTS 71

DISCUSSION 91

CONCLUSIONS 97

LIST OF REFERENCES 98

CHAPTER 3 105

INTRODUCTION 105

MATERIALS AND METHODS 110

RESULTS 120

DISCUSSION 142

CONCLUSIONS 147

LIST OF REFERENCES 148

CHAPTER 4 153

INTRODUCTION 153

MATERIALS AND METHODS 158

RESULTS 167

DISCUSSION 181

CONCLUSIONS 185

LIST OF REFERENCES 186

CHAPTER 5 192

INTRODUCTION 192

MATERIALS AND METHODS 197

RESULTS 206

DISCUSSION 214

CONCLUSIONS 217

LIST OF REFERENCES 218

CONCLUSIONS 220

LIST OF REFERENCES 223

LIST OF TABLES

Table 1.1 Analysis of variance of endospore production by Bacillus subtilis

(MBI600 and GBO3) and B amyloliquefaciens (IN937) on endospore-forming

medium after 14 days incubation Means followed by the same letter are not significantly different (LSD 0.05) 42

Table 1.2 Mathematical models that best describe Bacillus subtilis (MBI600 and GBO3) and B amyloliquefaciens (IN937) endospore formation over time 46

Table 2.1 Composition of nutrient solution used to fertilize tomato seedlings 67 Table 2.2 Bacterial population composition in 49 day-old tomato roots inoculated

with Bacillus subtilis strain MBI600, B subtilis strain GBO3 and B

amyloliquefaciens strain IN937 Colonies were isolated from roots on amended medium Identification was based on colony morphology and rep-PCR with ERIC primers carried out on three colonies per sample (Experiment I) 74

rifampicin-Table 2.3 Analysis of variance of Bacillus subtilis strain MBI600, B subtilis strain GBO3 and B amyloliquefaciens strain IN937 population densities on 49 day-old

tomato roots Colonies were isolated from roots on rifampicin-amended medium Identification was based on colony morphology and rep-PCR with ERIC primers carried out on three colonies per sample (Experiment I) Barlett’s p-value 1.0 Normality assumed 74

Table 2.4 Bacterial population composition on 56 day-old tomato roots

inoculated with Bacillus subtilis strain MBI600, B subtilis strain GBO3 and B

amyloliquefaciens strain IN937 Colonies were isolated from roots on amended medium Identification was based on colony morphology and rep-PCR with ERIC primers carried out on three colonies per sample (Experiment II) 75

rifampicin-Table 2.5 Analysis of variance of Bacillus subtilis strain MBI600, B subtilis strain GBO3 and B amyloliquefaciens strain IN937 population densities on 56 day-old

tomato roots Colonies were isolated from roots on rifampicin-amended medium Identification was based on colony morphology and rep-PCR with ERIC primers carried out on three colonies per sample (Experiment II) Barlett’s p-value 1.0 Normality assumed 75

Table 2.6 Growth promotion and induced resistance against Xanthomonas

euvesicatoria in three varieties of tomato inoculated with rifampicin-resistant

mutants of Bacillus subtilis strain MBI600, B subtilis strain GBO3 and B

amyloliquefaciens strain IN937 under greenhouse conditions (Experiment I) Plant height was evaluated over time The area under the plant height curve was used for statistical analysis Root length, fresh weight, dry weight and bacterial leaf spot were evaluated at the end of the experiment Tomato varieties

‘Mountain Fresh’ and ‘Mountain Spring’ are abbreviated as ‘Mfresh’ and

‘Mspring’, respectively Inoculation and non inoculation with X euvesicatoria are

abbreviated as ‘xc’ and ‘xo’, respectively Levene’s p-values were 0.493, 0.359, 0.063, 0.032, and 0.000 for plant height, root length, fresh weight, dry weight and bacterial leaf spot density, respectively Normality was assumed 80

Table 2.7 Growth promotion and induced resistance against Xanthomonas

euvesicatoria on four varieties of tomato inoculated with rifampicin-resistant

mutants of Bacillus subtilis strain MBI600, B subtilis strain GBO3 and B

amyloliquefaciens strain IN937 under greenhouse conditions (Experiment II) Plant height was evaluated over time The area under the plant height curve was used for statistical analysis Root length, fresh weight, dry weight and bacterial leaf spot were evaluated at the end of the experiment Tomato varieties

‘Mountain Fresh’ and ‘Mountain Spring’ are abbreviated as ‘Mfresh’ and

‘Mspring’, respectively Inoculation and non inoculation with X euvesicatoria are

abbreviated as ‘xc’ and ‘xo’, respectively Levene’s p-values were 1.000, 0.504, 0.169, 0.045, and 0.009 for plant height, root length, fresh weight, dry weight and bacterial leaf spot density, respectively Normality was assumed 85 Table 3.1 Bacterial population composition on tomato roots inoculated with

Bacillus subtilis strain MBI600, B subtilis strain GBO3 and B amyloliquefaciens

strain IN937 Colonies were isolated from roots on rifampicin-amended medium Identification was based on colony morphology and rep-PCR with ERIC primers carried out on three colonies per sample Seedling (29 day old), vegetative (49

day old), flowering (102 day old) and fruiting (132 day old) stages (Experiment I) 123 Table 3.2 Bacterial population composition in tomato roots inoculated with

Bacillus subtilis strain MBI600, B subtilis strain GBO3 and B amyloliquefaciens

strain IN937 Colonies were isolated from roots on rifampicin-amended medium Identification was based on colony morphology and rep-PCR with ERIC primers carried out on three colonies per sample Seedling (36 day old), vegetative (60 day old), flowering (114 day old) and fruiting (143 day old) stages (Experiment II) 124 Table 3.3 Growth promotion of ‘Mountain Spring’ tomato inoculated with

rifampicin-resistant mutants of Bacillus subtilis strain MBI600, B subtilis strain GBO3 and B amyloliquefaciens strain IN937 under field conditions (Experiment

I) Plant height, number of leaves, leaf length, leaf width, nitrate concentration, foliar fresh and dry weights and leaf area were evaluated three times, on 81, 95, and 116 day-old plants The area under the curve was used for statistical

analysis Averages are shown Irrigation, fungicide, and PGPR treatment are abbreviated as ‘irrig’, ‘fungi’, and ‘treat’, respectively Levene’s p-values were 0.950, 0.893, 0.996, 0.998, 0.983, 0.999, 1.000, and 0.999 for plant height,

number of leaves, leaf length, leaf width, nitrate concentration, foliar fresh and dry weights and leaf area, respectively Normality was assumed 131

Table 3.4 Growth promotion of ‘Mountain Spring’ tomato inoculated with

rifampicin-resistant mutants of Bacillus subtilis strain MBI600, B subtilis strain GBO3 and B amyloliquefaciens strain IN937 under field conditions (Experiment

II) Plant height, number of leaves, leaf length, leaf width, nitrate concentration, foliar fresh and dry weights and leaf area were evaluated three times on 101,

118, and 141 day-old plants The area under the curve was used for statistical analysis Averages are shown Irrigation, fungicide, and PGPR treatment are abbreviated as ‘irrig’, ‘fungi’, and ‘treat’, respectively Levene’s p-values were 0.926, 0.991, 0.994, 0.610, 0.999, 0.603, 0.573, and 0.879 for plant height,

number of leaves, leaf length, leaf width, nitrate concentration, foliar fresh and dry weights and leaf area, respectively Normality was assumed 133 Table 4.1 Composition of nutrient solutions used to fertilize tomato seedlings 161

Table 4.2 Characteristics of Bacillus spp inoculum suspensions drenched on 14

and 21 day-old tomato seedlings, variety ‘Mountain Spring’ (Experiment I / II).163 Table 4.3 Bacterial population composition in 49 day-old tomato roots inoculated

with rifampicin-resistant Bacillus subtilis strains MBI600, B subtilis strain GBO3 and B amyloliquefaciens strain IN937 Colonies were isolated from roots on

rifampicin-amended medium Identification was based on colony morphology and

rep-PCR with ERIC primers carried out on one colony per sample (Experiments I and II) 168

Table 4.4 Analysis of variance of rifampicin-resistant Bacillus subtilis strains MBI600 and GBO3 and B amyloliquefaciens strain IN937 population densities

on 49 day-old tomato roots Colonies were isolated from roots on amended medium Identification was based on colony morphology and rep-PCR with ERIC primers carried out on one colony per sample Levene’s p-value were 0.814 and 0.953 for Experiment I and II, respectively Normality was assumed 169 Table 4.5 Analysis of variance of the effect of nitrogen concentration on

rifampicin-rifampicin-resistant Bacillus subtilis strains MBI600 and GBO3 and B

amyloliquefaciens strain IN937 population densities on 49 day-old tomato roots Colonies were isolated from roots of on rifampicin-amended medium

Identification was based on colony morphology and rep-PCR with ERIC primers carried out on one colony per sample Levene’s p-value were 0.814 and 0.953 for Experiment I and II, respectively Normality was assumed 169 Table 4.6 Analysis of variance of the effect of acibenzolar-S-methyl on

rifampicin-resistant Bacillus subtilis strain MBI600 and GBO3 and B

amyloliquefaciens strain IN937 population densities on 49 day-old tomato roots Colonies were isolated from roots of on rifampicin-amended medium

Identification was based on colony morphology and rep-PCR with ERIC primers carried out on one colony per sample Levene’s p-value were 0.814 and 0.953 for Experiment I and II, respectively Normality was assumed 170 Table 4.7 Analysis of variance of the effect of activation of systemic aquired resistance by acibenzolar-S-methyl before and after inoculation with rifampicin-

resistant Bacillus subtilis strains MBI600 and GBO3 and B amyloliquefaciens

strain IN937 on bacterial population densities on 49 day-old tomato roots

Colonies were isolated from roots of on rifampicin-amended medium

Identification was based on colony morphology and rep-PCR with ERIC primers carried out on one colony per sample Levene’s p-values were 0.814 and 0.953 for Experiments I and II, respectively Normality was assumed 171

Table 4.8 Analysis of variance of the effect of rifampicin-resistant Bacillus subtilis strain MBI600 and GBO3 and B amyloliquefaciens strain IN937 on nitrogen

absorption by tomato plants Area under the curve of nitrogen concentration in the effluent was used for statistical analysis Averages are shown Levene’s p-value were 0.220 and 0.665 for Experiments I and II, respectively Normality was assumed 172 Table 4.9 Analysis of variance of the effect of acibenzolar-S-methyl on nitrogen absorption by tomato plants Area under the curve of nitrogen concentration in the effluent was used for statistical analysis Averages are shown Levene’s p-

values were 0.220 and 0.665 for Experiments I and II, respectively Normality was assumed 173 Table 4.10 Analysis of variance of the effect of nitrogen concentration supplied

on nitrogen absorption by tomato plants Area under the curve of nitrogen

concentration in the effluent was used for statistical analysis Averages are

shown Levene’s p-values were 0.220 and 0.665 for Experiments I and II,

respectively Normality was assumed 173

Table 4.11 Analysis of variance of the effect of rifampicin-resistant Bacillus

subtilis strains MBI600 and GBO3 and B amyloliquefaciens strain IN937 on

biomass of 49 day-old tomato plants Area under the curve of plant height, stem diameter and number of leaves was used for statistical analysis Averages are shown Levene’s p-values for plant height, stem diameter, number of leaves, total fresh weight, total dry weight and root length were 0.981, 0.864, 0.972, 0.414, 0.284, 0.901 (Experiment I) and 1.000, 0.961, 0.966, 0.988, 0.991, 0.982

(Experiment II), respectively Normality was assumed 176 Table 4.12 Analysis of variance of the effect of acibenzolar-S-methyl on biomass

of 49 day-old tomato plants Area under the curve of plant height, stem diameter and number of leaves was used for statistical analysis Averages are shown Levene’s p-values for plant height, stem diameter, number of leaves, total fresh weight, total dry weight and root length were 0.981, 0.864, 0.972, 0.414, 0.284,

0.901 (Experiment I) and 1.000, 0.961, 0.966, 0.988, 0.991, 0.982 (Experiment II), respectively Normality was assumed 177 Table 4.13 Analysis of variance of the effect of nitrogen concentration supplied

on biomass of 49 day-old tomato plants Area under the curve of plant height, stem diameter and number of leaves was used for statistical analysis Averages are shown Levene’s p-values for plant height, stem diameter, number of leaves, total fresh weight, total dry weight and root length were 0.981, 0.864, 0.972, 0.414, 0.284, 0.901 (Experiment I) and 1.000, 0.961, 0.966, 0.988, 0.991, 0.982 (Experiment II), respectively Normality was assumed 178 Table 4.14 Analysis of variance of the effect of the interaction of nitrogen

concentration and acibenzolar-S-methyl on biomass of 49 day-old tomato plants Averages are shown Levene’s p-values for total fresh weight and total dry weight were 0.414, 0.284 (Experiment I) and 0.988, 0.991 (Experiment II), respectively Normality was assumed 179 Table 4.15 Analysis of variance of the effect of the interaction of acibenzolar-S-methyl and plant growth-promoting rhizobacteria on total fresh weight of 49 day-old tomato seedlings Averages are shown Levene’s p-values for total fresh weight were 0.414 (Experiment I) and 0.988 (Experiment II) Normality was

assumed 180 Table 5.1 Composition of nutrient solution used to fertilize tomato seedlings 200

Table 5.2 Bacterial population composition in 60 day-old tomato roots inoculated

with rifampicin-resistant Bacillus subtilis strains MBI600, B subtilis strain GBO3 and B amyloliquefaciens strain IN937 Colonies were isolated from roots on

rifampicin-amended medium Identification was based on colony morphology and rep-PCR with ERIC primers carried out on one colony per sample (Experiment I and II) 207

Table 5.3 Analysis of variance of rifampicin-resistant Bacillus subtilis strains MBI600 and GBO3 and B amyloliquefaciens strain IN937 population densities

(log CFU g-1 root +1) on 60 day-old tomato roots Colonies were isolated from roots of on rifampicin-amended medium Identification was based on colony morphology and rep-PCR with ERIC primers carried out on one colony per

sample Levene’s p-values were 0.635 and 0.280 for Experiment I and II,

respectively Normality was assumed 207

Table 5.4 Analysis of variance of the effect of rifampicin-resistant Bacillus subtilis strains MBI600 and GBO3 and B amyloliquefaciens strain IN937 on tomato

biomass and bacterial stem rot severity Area under the curve of plant height and nitrogen concentration was used for statistical analysis Averages are shown Levene’s p-values for plant height, root length, nitrogen in sap and lesion size were 0.667, 0.388, 0.775, 0.023 (Experiment I) and 0.648, 0.471, 0.838, 0.045 (Experiment II), respectively Normality was assumed 209

Table 5.5 Analysis of variance of the effect of irrigation rate on tomato biomass and bacterial stem rot severity Area under the curve of plant height and nitrogen concentration was used for statistical analysis Averages are shown Levene’s p-values for plant height, root length, nitrogen in sap and lesion size were 0.667, 0.388, 0.775, 0.023 (Experiment I) and 0.648, 0.471, 0.838, 0.045 (Experiment II), respectively Normality was assumed 210

LIST OF FIGURES

Figure 1.1 Colony morphology of Bacillus spp on nutrient agar medium after 24

hours incubation at 28oC A B subtilis MBI600 wild-type; B B subtilis MBI600 rifampicin-resistant mutant; C B subtilis GBO3 wild-type; D B subtilis GBO3 rifampicin-resistant mutant; E B amyloliquefaciens IN937 wild-type; F B

amyloliquefaciens IN937 rifampicin-resistant mutant 40 Figure 1.2 (*) Vegetative cells, (**) endospores within cell, and (***) free

endospores of Bacillus subtilis MBI600 rif on endospore-forming medium

incubated for 8 days at room temperature (945 x) 41 Figure 1.3 Production of endospores by wild-type and rifampicin-resistant strains

of Bacillus subtilis (MBI600 and GBO3) and B amyloliquefaciens (IN937) on

endospore-forming medium incubated at room temperature under white light for

14 days Experiment I: (+) Endospores; (∆) Rate of production of endospores; Experiment II: (x) Endospores; (◊) Rate of production of endospores 44 Figure 1.4 Enterobacterial repetitive intergenic consensus - polymerase chain

reaction (ERIC-PCR) products obtained from genomic DNA from Bacillus subtilis strains MBI600 and GBO3 and B amyloliquefaciens strain IN937 Arrows

indicate polymorphisms 48

Figure 2.1 Chromelosporium sp A Conidiophore; B Detail of conidiophores and conidia; C Antagonistic effect of IN937rif on a Chromelosporium spp

colony 73

Figure 3.1 Categories for tomato fruit assessment: A red marketable; B green marketable; C physiological disorders; D anthracnose; E bacterial diseases; F other diseases; G insect or bird damage and H other diseases on green fruit.

117

Figure 3.2 Identification of Bacillus subtilis strains MBI600 and GBO3 and B

amyloliquefaciens strain IN937 isolated from 29-day old tomato roots during seedling stage, using enterobacterial repetitive intergenic consensus -

polymerase chain reaction (ERIC-PCR) fingerprints Wild-type and resistant strain fingerprints were used as a reference The banding patterns of three colonies isolated from treatments (control, MBI600 and GBO3+IN937) were compared with the reference patterns for identification (Experiment I) 125

rifampicin-Figure 3.3 Tomato root colonization by Bacillus spp 29 (seedling stage), 49

(seedling stage), 102 (flowering stage) and 132 (fruiting stage) days after

seeding, Experiment I Control plants were colonized by MBI600 (flowering and fruiting stages), GBO3 (vegetative, flowering and fruiting stages), and IN937 (seedling, vegetative, flowering and fruiting stages) Means followed by the same letter are not significantly different (p-value < 0.05) 126

Figure 3.4 Tomato root colonization by Bacillus spp 36 (seedling stage), 60

(seedling stage), 114 (flowering stage) and 143 (fruiting stage) days after

seeding, Experiment II Control plants were colonized by MBI600 (vegetative, flowering and fruiting stages), GBO3 (fruiting stage), and IN937 (seedling and fruiting stages) Means followed by the same letter are not significantly different 127

Figure 3.5 Effect of irrigation on Bacillus spp population density on 102-day-old

‘Mountain Spring’ tomato roots Means followed by the same letter are not

significantly different (p-value < 0.05) Experiment I 128 Figure 3.6 Effect of soil drenches with rifampicin-resistant mutants of plant

growth-promoting rhizobacteria (PGPR) (Bacillus subtilis MBI600 and GBO3, and

B amyloliquefaciens IN937) on height of ‘Mountain Spring’ tomato seedlings 49 (Experiment I) and 60 days (Experiment II) after planting Means followed by the same letter are not significantly different (p-value < 0.05) 135 Figure 3.7 Effect of soil drenches with rifampicin-resistant strains of plant

growth-promoting rhizobacteria (PGPR) (Bacillus subtilis MBI600 and GBO3, and

B amyloliquefaciens IN937) on height of ‘Mountain Spring’ tomato plants under field conditions Plant height was evaluated on 81, 95 and 116 (Experiment I) and

101, 118, and 141 day-old plants (Experiment II) Means followed by the same letter are not significantly different (p-value < 0.05) 136

Figure 3.8 Effect of plastic and rye residue mulches on height of ‘Mountain

Spring’ tomato plants under field conditions Plant height was evaluated on 81,

95 and 116 (Experiment I) and 101, 118, and 141 day-old plants (Experiment II) Means followed by the same letter are not significantly different (p-value < 0.05) 136 Figure 3.9 Diseases and pathogens observed on ‘Mountain Spring’ tomato

plants A early blight; B Alternaria solani conidia; C septoria leaf spot; D

pycnidia and conidia of Septoria lycopersici; E bacterial stem rot; and F pith

desintegration induced by Pseudomonas cichorii 138

Figure 3.10 Effect of plastic and plant residue mulches on severity of early blight and septoria leaf spot on ‘Mountain Spring’ tomato plants under field conditions Severity of foliar diseases was evaluated every 7 days Area under the disease curve was used for statistical analysis Means followed by the same letter are not significantly different (p-value < 0.05) 139

Figure 3.11 Effect of plant growth-promoting rhizobacteria (PGPR) (Bacillus

subtilis MBI600 and GBO3, and B amyloliquefaciens IN937) on bacterial stem

rot severity of ‘Mountain Spring’ tomato plants under field conditions Severity of bacterial stem rot was evaluated every 7 days Area under the disease curve was used for statistical analysis Means followed by the same letter are not

significantly different (p-value < 0.05) 139

Figure 3.12 Effect of plastic and plant residue mulches on severity of bacterial stem rot on ‘Mountain Spring’ tomato plants under field conditions Severity of bacterial stem rot was evaluated every 7 days Area under the disease curve was used for statistical analysis Means followed by the same letter are not

significantly different (p-value < 0.05) 140

Figure 3.13 Effect of plant growth-promoting rhizobacteria (PGPR) (Bacillus

subtilis MBI600 and GBO3, and B amyloliquefaciens IN937) on marketable yield

of ‘Mountain Spring’ tomato plants Means followed by the same letter are not significantly different (p-value < 0.05) 141 Figure 3.14 Effect of plastic and plant residue mulches on marketable yield of

‘Mountain Spring’ tomato plants under field conditions Means followed by the same letter are not significantly different (p-value < 0.05) 141

Figure 4.1 Irrigation system A General view B The single line serving each pot was situated so that solution dripped freely into the vermiculite C A piece of

transparent plastic covered the surface of the growing medium to reduce

evapotranspiration 160

Figure 5.1 Bacterial stem rot (Pseudomonas cichorii) A Brown lesions on

tomato variety ‘Mountain Spring’; B Lesion on tomato variety ‘Big Beef’ 212

LIST OF ABBREVIATIONS

g = gram Unit of weight

h = hour Unit of time

ha = hectare; acre = 0.404686 hectares

k =prefix kilo (103) Kilogram

l = unit of volume Liter

M = Mol Molecular mass of a particular molecule expressed in grams per liter Metric ton = 1000 kg

x g = gram-force 1 gram of mass subject to standard gravity = 980.66 cm s-2 = 9.806 mN

μ = prefix micro (10-6)

m = prefix milli (10-3) Milliliter, milligram

rpm = revolutions per minute

Ton = unit of weight equal to 1000 kg

u = unit Amount of enzyme that incorporates 10 nanomoles of dNTPs into acid insoluble form within 30 minutes at 74 oC

INTRODUCTION

Tomato (Lycopersicon esculentum Mill.) is a very important vegetable crop

worldwide In 2004, 120 million metric tons of tomatoes (fresh market and processing) were produced worldwide (FAO, 2004) In The United States, 52,892

ha of fresh market tomatoes were planted in 2004 producing 35,903 kg/ha and a value of $ 1,342,478,000 Ohio is third among the states in tomato production for fresh market, with 2,832 planted hectares, yielding 20,713 kg/ha and a value of

$49,549,000 Ohio is also third among the states in tomatoes for processing, with 2,670 planted hectares, 70,672 kg/ha and a value of $ 13,902,000 (USDA, 2005)

‘Mountain Fresh’, ‘Mountain Spring’, ‘Florida 47’, and ‘BHN543’ are high value varieties commonly used for fresh market tomato production in Ohio ‘BHN543’ matures at mid-season, producing large or extra large, globe-shaped fruit The plant has determinate growth and is reported to be resistant to Fusarium wilt

races 1 and 2, Meloidogyne incognita, M javanica, M arenaria, and Verticillium

dahliae race 1 ‘Florida 47’ is a mid-season variety that produces large fruit that are flattened at the poles The variety has determinate growth and shows

resistance to Fusarium wilt races 1 and 2, Stemphylium sp., and Verticillium

race 1 ‘Mountain Spring’ is a mid-season variety that produces extra

large, crack resistant, oblate fruit The variety has determinate growth and

resistance to Fusarium wilt races 1 and 2, Stemphylium, and Verticillium dahliae

race 1 ‘Mountain Fresh’ is a mid-season variety that produces large fruit, from oblate to globose in shape The plant has determinate growth (Anonymous, 2000)

A few elements have been determined to be essential for plant growth According

to their relative concentration in plant tissue, essential nutrients are classified as macronutrients (nitrogen, potassium, calcium, magnesium, phosphorus, sulfur and silicon) and micronutrients (chlorine, iron, boron, manganese, sodium, zinc, copper, nickel and molybdenum) Absence or inadequate supply of essential nutrients prevents a plant from completing its life cycle or causes nutritional disorders manifested by characteristic symtoms (Taiz and Zeiger, 2002)

Nitrogen-deficient plants grow slowly Leaves are small, thick, and light green Stems are thick and hard Flower buds generally drop off, fruits are small, and

yield is reduced (Scholberg et al 2000; Wilcox, 1993) Phosphorous-deficient

plants grow slowly, and leaves become dark, with purple interveinal tissue on the underside Stems become slender and hard (Wilcox, 1993) Potassium-deficient tomato plants have small stems and shortened internodes Young leaves are

dark green, crinkled and curled Old leaves are chlorotic and bronzed Fruits are slender and surfaces are blotchy They ripen unevenly and drop off the plant after ripening (Wilcox, 1993) Calcium deficiency results in reduced growth of tomato Root tips die and seedling leaves show upward cupping and have necrotic margins Limited calcium availability during the fruiting stage results in the disorder blossom end rot, since translocation from vegetative portions and root uptake cannot compensate for the deficiency in the fruit during development (Wilcox, 1993)

Total nitrogen uptake by tomato averages 197 kg/ha (Wilcox, 1993) The variety

‘Mountain Spring’ requires a nitrogen rate of only 67 kg/ha to produce 48 ton/ha (Taber, 1998) Nitrogen accumulation is highest during the vegetative stage (185 mg/plant/day) Average nitrogen concentration in plant tissue is relatively high during seedling (4.38% dry weight) and vegetative stages (4.30% dry weight) and decreases at flowering (3.99% dry weight) and fruiting (3.34% dry weight) stages Nitrogen concentrations in petiole sap of the tomato variety ‘Mountain Spring’ at seedling, flowering, and fruiting stages was reported to be 180, 203, and 90 ppm respectively (Taber, 1998) Average total phosphorous uptake by tomatoes is 26 kg/ha or about 1.052 g/plant, of which 75% is in the fruit and the rest in leaves and stems Phosphorus uptake is particularly rapid during the fruiting stage

Nearly 70% of potassium is in the fruit Potassium uptake is rapid during vegetative and fruiting stages (Wilcox, 1993)

During vegetative growth, the root system, stems and leaves are developed (first

42 days) then, flowers appear and develop Fruits begin to develop about 55 days after emergence During flowering and fruiting, a rapid increase in dry weight of stems and leaves occurs up to 70 days after emergence, with little increase after 77 days Accumulation of dry weight in the fruits is almost linear from day 70 to 105 Fruit ripening begins at 84 days and progresses to ripeness

at 112 days (Wilcox, 1993)

Water management influences uptake and utilization of nutrients, development of plants, disease development, and quality and quantity of tomato production Irrigation is particularly important in tomato in late summer when there is a reduction in rainfall Soil moisture deficiencies during vegetative, flowering or fruiting stages resulted in yield reductions of about 25, 52 and 43%, respectively

(Rutledge et al 1999) Irrigation delivery system (sprinklers vs furrow vs buried

drip) and irrigation frequency have a significant impact not only on quality and quantity of tomato production, but also on the development of diseases (Strange 2000) Drip irrigation is one of the most common delivery systems in which

water under pressure flows through a pipe This system utilizes less water than over-the-top irrigation Drip irrigation places water directly where it is needed and reduces diseases since it avoids wetting the foliage One disadvantage of the system is that it requires clean water since soil and mineral deposits result in line

blockage and non-uniform water application (Rutledge et al 1999)

The use of black plastic is widely accepted for growing tomatos Plastic mulch controls weeds and certain diseases, conserves moisture, and increases quality and quantity of marketable fruit Plastic mulch is generally installed over a 100

cm wide x 12 cm high bed The irrigation system is set up ahead of laying the plastic Transplant holes are punched through the plastic The main disadvantage of plastic mulch is the expense associated with installation,

removal, and disposal of the black plastic (Rutledge et al 1999)

Nearly 200 biotic diseases have been reported on tomato throughout the USA

(Jones et al 1997) Worldwide, one of the most important foliar bacterial diseases is bacterial spot caused by Xanthomonas euvesicatoria (formerly X

campestris pv vesicatoria, X axonopodis pv vesicatoria, race T1, phenotypic group A), X vesicatoria (formerly X vesicatoria, race T2, phenotypic group B), X

(formerly X axonopodis pv vesicatoria, race T3, phenotypic group C),

and X gardneri (formerly X vesicatoria, race T4, phenotypic group D) (Jones et

al 2004a; Jones et al 2004c)

Xanthomonas euvesicatoria and X vesicatoria are widely distributed throughout the world (Bouzar et al 1994; Bouzar et al 2004) These species of

Xanthomonas can reduce tomato production under environmental conditions that

occur in Ohio (Abbasi et al 2002; Sahin, 1997), Alabama and the southeastern United States (Cambell et al 1998) In Ohio, three species have been reported

as causal agents of bacterial leaf spot: X euvesicatoria, X vesicatoria, and X

perforans (Sahin, 1997) X perforans has been reported in several tomato production areas in the United States and in Mexico X gardneri was isolated in Costa Rica, and reported from Michigan and Brazil (Bouzar et al 2004)

Xanthomonas euvesicatoria, X vesicatoria and X perforans are gram negative,

aerobic, oxidase negative, catalase positive, rod-shaped bacteria with a single

polar flagellum The yellow color of Xanthomonas spp colonies is caused by

brominated arylpolyene esters Colonies are mucoid on yeast CaCO3 medium (Schaad et al 2001) These species can be distinguished easily

extract-dextrose-by physiological tests, extract-dextrose-by distinctive SDS PAGE profile, and DNA similarities X

is weakly amylolytic and pectolytic, able to use cis-aconitic acid

and has a distinctive SDS PAGE profile X vesicatoria is strongly amylolytic and pectolytic, utilizes acetic acid, but does not utilize cis-aconitate X perforans is

strongly amylolytic and pectolytic, but does not utilize acetic acid and has a

distinctive SDS PAGE profile (Jones et al 2004c)

Xanthomonas euvesicatoria and X vesicatoria induce brown, roughly circular

spots on stems, leaves, and petioles of tomato and pepper plants The lesions are generally smaller than 3 mm in diameter, surrounded by a prominent halo, and become water-soaked during periods with high moisture When environmental conditions are optimal for disease development, the lesions coalesce On affected fruits symptoms begin as minute slightly raised blisters

that become brown and scab like (Jones et al 1997; Basim et al 2004)

The pathogens of bacterial spot survive on infected crop residue at least 6

months (Bashan et al 1982; Jones et al 1997), and or tomato volunteers and weeds (Jones et al 1986) The pathogen persists for 16 days in sandy loam soil, and at least 3 months in tomato seed (Bashan et al 1982) Development of

bacterial spot disease is favored by temperatures of 24-30 oC and abundant moisture Dissemination is mainly by wind-driven rain droplets and clipping of

transplants The bacteria penetrate plant tissue through stomata and wounds

(Jones et al 1997)

Partial control of bacterial leaf spot is achieved by crop rotation, disease free transplants, destruction of infected leaves, and eradication of weeds or volunteer plants Pesticides with efficacy against bacterial leaf spot include bactericides such as Cuprofix 40 DF (2.24 kg/ha sprayed every 7-10 days), and the plant

activator Actigard 50WG (35 g/ha sprayed every 7-10 days) (Lewis Ivey et al 2005; Briceno and Miller, 2004; Jones et al 2004b; Obradovic et al 2005; Vavrina et al 2004)

Stem rot of tomato is a bacterial disease that is characterized by breakdown of pith, vascular discoloration, external black lesions, eventual wilting and death of

the plant Bacteria that cause stem rot are Erwinia carotovora (Speights et al 1967), E carotovora ssp carotovora (Dhanvantari and Dirks, 1987),

Pseudomonas viridiflava (Lukezic et al 1983), P corrugata (Scarlett et al 1978) and P cichorii (Wilkie and Dye, 1974)

Pseudomonas cichorii is a gram negative, rod-shaped bacterium with one to several polar flagella The bacterium is aerobic, levan negative, oxidase positive,

non-pectolytic and arginine dihydrolase negative, inducing a positive hypersensitivity reaction on tobacco Colonies produce diffusable fluorescent pigments that are visible on iron-deficient medium such as King’s medium B, and

Pseudomonas agar F (Braun-Kiewnick and Sands, 2001)

Pseudomonas cichorii induces elongated dark lesions on the surface of tomato stems Lesions may extend along petioles and leaves, causing dark green water-soaked blotches without halos Internally, vascular tissues show dark brown discoloration Infected stem pith is brown and watery then disintegrates leaving a hollow stem Dark brown spots appear on fruits Eventually the plant wilts and dies (Wilkie and Dye, 1974)

Pseudomonas cichorii infection is favored by extended leaf wetness and temperatures around 20 oC Symptoms appear within 48 hours after inoculation

under these conditions (Wilkie and Dye, 1974) Pseudomonas cichorii severity

may be reduced by applying copper hydroxide and Actigard (Ustun, 2004)

Early blight (Alternaria solani, A alternata) has been reported from most tomato production areas Alternaria solani is present in England, India, Australia, and the

United States It is particularly damaging in the central states (Jones, 1997) This

fungus produces septate and branched mycelium Conidiophores are simple and dark Conidia are obclavate to elliptical, 150 - 300 x 15 – 19 μm, dark, with both transversal and longitudinal septa, and a very long beak (almost the size of the body), developing through pores in the outer wall at the apex of conidiophore (Barnett and Hunter, 1998; Evans and Howards, 1994; Menzies and Jarvis,

1994a) The pathogen affects leaves, stems, flowers, and fruits On leaves, A

solani induces small circular spots with concentric rings, becoming irregular in shape and generally surrounded by a yellow area Black elongated, slightly sunken lesions occur on stems and pedicels Black, leathery, sunken lesions appear on the fruit, generally around the calix, wounds or cracks Heavy fruit load, age, and inadequate nutrition increase tomato susceptibility to early blight (Jones, 1997; Menzies and Jarvis, 1994a; Pitblado and Howard, 1994)

Alternaria solani survives in plant residue, tomato seed or volunteer Solanaceous weeds Spores are spread by wind, rain, and infested plant debris Conidia germinate within 2 hours in water at 6 - 34 oC and penetrate directly through the cuticle Lesions usually appear on the older leaves in 2 - 3 days and spread to the upper leaves Extended leaf wetness and high temperature (24 – 29 oC) favor the disease, which can completely defoliate tomato plants and expose fruit to

sunscald and other diseases, and reduce yield (Jones, 1997; Pitblado and Howard, 1994)

Management of early blight may be achieved by crop rotation, use of free transplants, minimizing plant injury, balancing nutrition, destruction of infected leaves, eradication of weeds or volunteer plants, irrigation, and use of resistant varieties (e.g ‘HY9478’, ‘Malinta’ and ‘Medalist’) and fungicides (Jones, 1997; Menzies and Jarvis, 1994a; Pitblado and Howard, 1994)

disease-Septoria leaf spot (disease-Septoria lycopersici) is another common disease of tomato in

Ohio that can significantly reduce yield if not properly managed It is widely distributed throughout the world, having been reported from Europe, Asia, Africa,

Australia and North and South America (Stevenson, 1997) Septoria lycopersici

produces septate and immerse mycelium The pycnidia are globose, about 66

μm in width with a thin wall of textura angularis and a smaller-celled inner layer The ostiole is circular and central The conidiogenous cells are holoblastic, hyaline, ampuliform, doliform or short cylindrical that produce hyaline, multiseptate, filiform 67 x 3.2 μm conidia (Menzies and Jarvis, 1994b; Sutton, 1980)

Septoria leaf spot is a common foliar disease late in the growing season Circular brown spots (2-5 mm) generally with a narrow halo appear on the lower leaves The center of old lesions turns light brown with dark margins and black pycnidia appear within the lesion Small circular, water-soaked spots occur in stems and petioles Under favorable conditions, the disease spreads from lower leaves to stems and upper leaves, inducing defoliation and causing exposure of fruit to sunscald and other pathogens, with potentially heavy yield losses (Menzies and

Jarvis, 1994b; Pitblado, 1994; Stevenson, 1997) Septoria lycopersici survives on

tomato seed, wood stakes and plant debris Spores are spread by water, workers, equipments, insects, wind and infested plant debris Conidia penetrate tomato plants through stomates Symptoms appear within 6 days and pycnidia are produced about 14 days after inoculation Temperatures between 20 - 25 oC and extended leaf wetness favor the disease At 100% relative humidity, 10 days are required to complete the life cycle (Menzies and Jarvis, 1994b; Pitblado, 1994; Stevenson, 1997) As for early blight, management of septoria leaf spot is achieved by crop rotation, use of disease-free transplants, elimination of weeds, crop debris removal, minimizing plant injury, balancing nutrition, timing of irrigation, and application of fungicides (Stevenson, 1997; Pitblado, 1994)

Most growers rely on fungicides/bactericides to manage foliar diseases and achieve acceptable yield of tomato However, new resistant strains of

Xanthomonas species causing bacterial spot (Cambell et al 1997; Jones et al 1991; Marco and Stall, 1983) and fungal and oomycetes pathogens (Day et al 2004; Grech, 1990; Yun et al 1999) appear, reducing the effectiveness of these

products Furthermore, alternative measures of disease control that require fewer

or low-risk pesticides are needed to prevent human health problems and damage

to the environment (Bell et al 2000; Cambell et al 1997) Under these

circumstances, the use of pathogen-free tomato seed, as well as cultural and biological control tactics are needed for integrated management of diseases caused by both bacteria and fungi The effectiveness of hot-water treatment of

tomato seed to reduce bacterial spot has been demonstrated (Miller et al 2004)

Optimizing the fertilizer program may be a practical approach to help manage bacterial diseases (Harkness and Marlatt, 1970) Nitrogen has been studied in relation to disease intensity in several crop-disease combinations (Huber and Watson, 1974) High rates of nitrogen induce a reduction (Chase and Poole, 1987; Harkness and Marlatt, 1970; Chase and Jones, 1986) or increase

(Bachelder et al 1956; Nayudu and Walker, 1961; Thomas, 1965; Haygood et al 1982; Jones et al 1985) in the susceptibility of the host to the pathogen

Promising results have been reported using biorational products such as

bacteriophages (Snowden, 2004; Jackson, 2004; Jones et al 2004b), Bacillus

amyloliquefaciens (Briceno and Miller, 2004), B subtilis (Briceno and Miller, 2004), Pseudomonas syringae (Briceno and Miller, 2004), P fluorescens (Briceno and Miller, 2004), Bacillus subtilis strains QRD131, 132, 137 and 141

(Highland, 2004) against bacterial spot and foliar diseases of tomato

The use of plant growth-promoting rhizobacteria (PGPR) is a potentially attractive approach to disease management and improved crop productivity in sustainable agriculture, since PGPR have been reported to increase yield and protect crops

from disease simultaneously (Ramamoorthy et al 2002; Raupach, 1998) PGPR

are strains of bacteria that live in the rhizosphere, stimulate plant growth, and

improve stand under stress conditions (van Loon et al 1998) Several classes of

PGPR have been reported to enhance plant growth and suppress pathogens:

biofertilizers, such as Rhizobium, Sinorhizobium, Mesorhizobium,

Bradyrhizobium, Azorhizobium and Allorhizobium fix nitrogen into a form that can

be used by the plant (Bloemberg and Lugtenberg, 2001) Phytostimulator PGPR enhance plant growth, usually by the production of phytohormones such as

auxins (Asghar et al 2002) Biocontrol agents protect plants (niche exclusion,

antibiotic synthesis, competition for nutrients, antagonism) and enhance defense