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Effect of Heterotrophic Bacterial Communities on Pythium Spp. in Recycled Irrigation Water

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Effect of Heterotrophic Bacterial Communities on Pythium Spp. in Recycled Irrigation Water The Pennsylvania State University The Graduate School Department of Plant Pathology and Environmental Microbiology EFFECT OF HETEROTROPHIC BACTERIAL COMMUNITIES ON PYTHIUM SPP. IN RECYCLED IRRIGATION WATER A Dissertation in Plant Pathology by María L. Burgos-Garay © 2013 María L. Burgos-Garay Submitted in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy May 2013 :by the following ٭The dissertation of María L. Burgos-Garay was reviewed and approved Gary W. Moorman Professor of Plant Pathology Dissertation Advisor Chair of Committee Edward G. Dudley Associate Professor of Food Science Beth Gugino Assistant Professor of Plant Pathology María del Mar Jiménez-Gasco Assistant Professor of Plant Pathology Timothy W. McNellis Associate Professor of Plant Pathology Frederick E. Gildow Professor of Plant Pathology Head of the Department of Plant Pathology Signatures are on file in the Graduate School٭ iii Abstract A main concern in commercial greenhouses is the harboring of Pythium species in recycled irrigation water and their spread into susceptible crops. Pythium species are among the most damaging pathogens of horticultural crops causing damping-off of seedlings as well as root and stem rots in ornamental plants. Despite frequent attempts to recover Pythium from recycled water, success in isolation is sporadic. This raises the question of possible microbial suppression of Pythium present in recycled irrigation water. The present study focused on the deleterious effect of microbial communities in recycled irrigation water on the development of Pythium aphanidermatum, P. irregulare, and P. cryptoirregulare. Microscopic observations of the interaction between microorganisms in recycled irrigation water and Pythium species indicated a deleterious effect on Pythium development. The amount of sporangia produced in the presence of the microbial community was negatively affected when compared to water samples with no microorganisms. In addition, research was conducted to evaluate heterotrophic bacteria isolated from recycled irrigation water that reduced the growth in vitro of the three highly pathogenic Pythium species. We isolated a variety of bacteria some of which inhibited the three Pythium spp. growth, others bound to their hyphae, and still others seemed to enhance the growth of the Pythium species. Known biocontrol agents against Pythium (Pseudomonas and Bacillus) and some potentially new biocontrol agents (Acidovorax, Microbacterium, and Chryseobacterium) were identified as isolates inhibiting the growth of P. aphanidermatum, P. irregulare, and P. cryptoirregulare in vitro. The use of biological control agents to control Pythium spp. is not new. However, in most cases, commercially available beneficial organisms are from other environments. Utilization of naturally occurring suppressive bacterial communities in recycled irrigation water may provide an environmentally friendly ecologically sound biological control strategy. We observed that Pythium grew slower when bacterial isolates bound to their hyphae. This phenomenon was previously observed but is not fully understood. These isolates were identified as Acinetobacter, Enterobacter, Pantoea, and Pseudomonas species. Surprisingly, we also isolated iv bacteria that enhanced the growth of Pythium in vitro. More research is needed to understand this phenomenon. Further studies were conducted to determine if changes in bacterial community composition were associated with the presence of Pythium species in the water. We used automated ribosomal intergenic spacer analysis (ARISA) to examine the impact the presence of P. aphanidermatum, P. irregulare, or P. cryptoirregulare had on bacteria diversity in recycled irrigated water. ARISA profiles indicated that all three Pythium species had an impact on the bacterial community. To confirm ARISA results, we used quantitative PCR (q-PCR) to quantify the change in γ-Proteobacteria populations when P. aphanidermatum, P. irregulare, or P. cryptoirregulare were added to recycled irrigation water samples. All samples showed a significant increased in γ- Proteobacteria population when one of the three Pythium was present in the water sample. The results suggest that Pythium presence plays a role in structuring bacterial community composition. In an effort to gain knowledge of the aquatic ecology of Pythium spp., we isolated and identified Pythium species from greenhouse water reservoirs to elucidate temporal patterns of Pythium in that aquatic environment. This is an important aspect to increase farmer awareness of this genus and the species that are more prevalent in this ecosystem. The results suggest that some Pythium species survived for long periods (e.g. Pythium group F isolates) while other species are transient (e.g. P. catenulatum) in this environment. P. aphanidermatum, P. irregulare, and P. cryptoirregulare were rarely isolated (one time only) from greenhouse water reservoirs suggesting that microbial populations in recycled irrigation water suppress these three pathogenic Pythium species. v TABLE OF CONTENTS LIST OF FIGURES ...................................................................................................... viii LIST OF TABLES ........................................................................................................... xi ACKNOWLEDGEMENTS ............................................................................................. xii Chapter 1 Literature Review .......................................................................................... 1 Pythium species ............................................................................................................. 1 Plant Pathogenic Species .............................................................................................. 3 Pythium aphanidermatum (Edson) Fitzp. ............................................................ 6 Pythium irregulare Buisman ................................................................................ 6 Pythium cryptoirregulare Garzón, Yánez & Moorman ......................................... 7 Diseases Caused by Pythium ............................................................................. 8 Sources of Pythium Inoculum in the Greenhouse ............................................... 9 Survival of Pythium species in Recycled Irrigation Water .................................. 11 Recycled irrigation water .............................................................................................. 12 Water Environment............................................................................................ 13 Microbial communities in Recycled Irrigation Water System ............................. 15 Biological control .......................................................................................................... 16 Mode of Action .................................................................................................. 16 Biological control of Pythium diseases .............................................................. 17 Microbial Interaction in Recycled Irrigation Water ............................................. 19 Research Objectives .................................................................................................... 19 Literature Cited ............................................................................................................ 22 Chapter 2 Effect of microbial communities present in recycled irrigation water on the development of P. aphanidermatum, P. irregulare, and P. cryptoirregulare in vitro ............................................................................................................... 32 Abstract ........................................................................................................................ 32 Introduction .................................................................................................................. 32 Materials and Methods ................................................................................................. 34 Pythium species ................................................................................................ 34 Recycled irrigation water sampling .................................................................... 35 Microbial community fractionation ..................................................................... 35 Testing the effect of microbial communities on Pythium.................................... 37 Microscopic observations ............................................................................................. 38 Data analysis ..................................................................................................... 38 Results ......................................................................................................................... 39 Influence of microbial community on sporangia ................................................ 39 A. Pythium aphanidermatum ................................................................. 39 vi B. Pythium cryptoirregulare .................................................................... 40 C. Pythium irregulare .............................................................................. 42 Influence of microbial community in the production of zoospores ..................... 43 Discussion.................................................................................................................... 45 Acknowledgments .......................................................................................................
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