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

ROLE OF PLANT GROWTH-PROMOTING RHIZOBACTERIA IN INTEGRATED DISEASE MANAGEMENT AND PRODUCTIVITY OF TOMATO DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Cristian Nava Diaz, M.S. * * * The Ohio State University 2006 Dissertation Committee: Ph.D. Sally A. Miller, Adviser _______________________ Ph.D. Michael A. Ellis Ph.D. Matthew D. Kleinhenz Ph.D. Douglas J. Doohan UMI Number: 3197883 3197883 2006 UMI Microform Copyright All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 by ProQuest Information and Learning Company. ii 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 10 4 - 10 6 CFU g -1 during the iii seedling stage and dropped to less than 10 3 CFU g -1 during the flowering and fruiting stages. Significant increases in plant height were observed in all Bacillus- inoculated tomato plants, however, foliar diseases incited by A. solani and S. 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 10 1 -10 5 CFU 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. iv DEDICATION To my parents Maria Cristina Diaz Lozano Jose Guadalupe Nava Bernal reri v 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. vi 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 vii 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 viii 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 ix 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 rifampicin- amended medium. Identification was based on colony morphology and rep-PCR with ERIC primers carried out on three colonies per sample (Experiment I). 74 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 [...]... ‘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 IN9 37) on height of ‘Mountain Spring’ tomato. .. 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... GBO3 and B amyloliquefaciens strain IN9 37 isolated from 29-day old tomato roots during seedling stage, using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) fingerprints Wild-type and rifampicinresistant strain fingerprints were used as a reference The banding patterns of three colonies isolated from treatments (control, MBI600 and GBO3 +IN9 37) were compared with the... (Experiment I) 125 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 IN9 37 (seedling, vegetative, flowering and fruiting stages) Means followed by the... 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,... 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 (Wilcox,... of variance of the effect of rifampicin-resistant Bacillus subtilis strains MBI600 and GBO3 and B amyloliquefaciens strain IN9 37 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. .. or about 12 g /plant 3 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... promotion of ‘Mountain Spring’ tomato inoculated with rifampicin-resistant mutants of Bacillus subtilis strain MBI600, B subtilis strain GBO3 and B amyloliquefaciens strain IN9 37 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... 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 . ROLE OF PLANT GROWTH-PROMOTING RHIZOBACTERIA IN INTEGRATED DISEASE MANAGEMENT AND PRODUCTIVITY OF TOMATO DISSERTATION Presented in Partial Fulfillment of the Requirements. during the iii seedling stage and dropped to less than 10 3 CFU g -1 during the flowering and fruiting stages. Significant increases in plant height were observed in all Bacillus- inoculated. strains MBI600, and GBO3 +IN9 37 did not increase nitrogen concentration in tomato leaves. Lesions induced by Pseudomonas cichorii on tomato stems tended to be longer in GBO3 +IN9 37 inoculated plants