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A Potential Energy-Saving Heat Treatment for Re-Circulated Irrigation Water and Its Biological Mechanisms

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A Potential Energy-Saving Heat Treatment for Re-Circulated Irrigation Water and Its Biological Mechanisms A potential energy-saving heat treatment for re-circulated irrigation water and its biological mechanisms Wei Hao Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Plant Pathology, Physiology, and Weed Science Chuanxue Hong, Chair Boris A. Vinatzer Antonius B. Baudoin Erik L. Stromberg D. Michael Benson October 29, 2012 Blacksburg, VA Keywords: 16S rRNA, α-proteobacteria, γ-proteobacteria, bacteria, biocontrol, chlamydospore, colony PCR-SSCP, culture-dependent plating, Firmicutes, greenhouse crops, oospore, pathogen-suppressing, PCR-DGGE, re-circulated irrigation water, zoospore Copyright 2012, Wei Hao A potential energy-saving heat treatment for re-circulated irrigation water and its biological mechanisms Wei Hao Abstract Heat pasteurization is an effective water treatment to address the emerging plant pathogen issue associated with increased water recycling practices in the ornamental horticulture industry. The current protocol that recommends treating water at 95°C for 30 s, however, faces two major challenges: its energy cost and environmental footprint. We hypothesized that temperature required to inactivate major pathogens in re-circulated water may be substantially lowered from 95°C with extended exposure time. The goal of this study was to test this hypothesis and make this water decontamination technology economically more attractive while reducing its environmental impact. Specific objectives were to (1) examine the effect of water temperature on the survival of Phytophthora and bacterial species, two major groups of plant pathogens in water recycling systems, and (2) elucidate the underlying biological mechanisms by which plant pathogens are killed at those temperatures. Lab assays were performed to determine the survival of zoospores and chlamydospores of P. nicotianae, and oospores of P. pini as well as seven bacterial species after heat treatments at given periods of time. Greenhouse experiments were conducted to determine the applicability of the lab assay data to the real world using annual vinca (Catharanthus roseus) and P. nicotianae as a model system. The results of these studies indicated that the water temperature required to eliminate Phytophthora and bacterial species can be lowered to 48°C from 95°C if treatment time extends to 24 h. Two major steps were taken to elucidate the underlying biological mechanisms. Firstly, a scheme based on the DNA fingerprint and sequence analysis was developed for characterizing bacterial species in irrigation water, after comparing two typing strategies, three sample concentration methods, and evaluating conditions in denaturing gradient gel electrophoresis (DGGE) profiling. Bacterial species detected by culture-dependent and -independent strategies were rather different. The greater bacterial diversity was detected when water samples were concentrated by using both methods than centrifugation or filtration alone. As for DGGE profiling, 40 to 60% denaturant concentrations at 70 V for 16 h revealed the highest bacterial diversity. Secondly, water samples were taken from an irrigation reservoir in a local nursery and analyzed for bacterial diversity following heat treatments at 42 and 48°C. After these heat treatments α-proteobacteria, γ-proteobacteria, and Firmicutes became dominant which presents a substantial shift of bacterial community structure compared to those in the control water at 25°C. Among the dominant in treated water were Bacillus, Pseudomonas, Paenibacillus, Brevibacillus, and Lysobacter species, which may have potential biocontrol activities against plant pathogens. This study provided the scientific basis for developing a more energy-efficient and environmentally sound heat pasteurization protocol for water decontamination. iii Acknowledgements I would like to express my sincere gratitude to my advisor Dr. Chuan Hong for his guidance, advice, and encouragement throughout the four years of my research projects. I also would like to thank the members of my graduate committee, Drs. Boris Vinatzer, Anton Baudoin, Erik Stromberg, and Michael Benson for their support and valuable advice throughout my doctoral program. Without their help, this work would not have been possible. I would like to thank the financial support provided by the Fred C. Gloeckner Foundation, Inc and a grant from the USDA National Institute of Food and Agriculture - Specialty Crop Research Initiative (Agreement #: 2010-51181-21140) for my research work and other expenses. I greatly appreciate all my colleagues and friends in the Department of Plant Pathology, Physiology, and Weed Science, and Hampton Roads Agricultural Research and Extension Center at Virginia Tech, whose advice, support, assistance, and encouragement have contributed to the completion of my research. My special thanks go to Dr. Ping Kong, Dr. Giovanni Cafa, Patricia Richardson, Xiao Yang, Dr. Venkatesan Parkunan, Dr. Zhihan Xu, Dr. Heather Olson, Lynn Rallos, Lauren Achtemeier, Michael Pistininzi, Andrew Rotzin, and Haijie Liu. I am also very thankful to the PPWS and HRAREC staff members including Donna Ford, Patsy Niece, Judy Fielder, Cris Thompson, and Ava Borden for all their help during my time in graduate school. I am forever grateful to my parents for their love, encouragement, and support. iv Attributions Several colleagues have contributed to this dissertation. Chuanxue Hong, Ph.D. is a professor in Department of Plant Pathology, Physiology and Weed Science and Hampton Roads Agricultural Research and Extension Center at Virginia Tech. He is the primary advisor and committee chair for this project. He advised each and every major step of this project from research planning to execution, data analyses and interpretation, and edited this dissertation. Chapter 2. Inactivation of Phytophthora and bacterial species in water by a potential energy-saving heat treatment Boris A. Vinatzer, Ph.D., an associate professor in Department of Plant Pathology, Physiology and Weed Science at Virginia Tech, serves on my graduate committee. He supported and guided the bacterial assays. He is listed as a co-author in a publication of work described in Chapter 2. Monday O. Ahonsi, Ph.D., a former post-doctoral research associate in Dr. Hong's lab, performed the assays investigating zoosporic survival of Phytophthora nicotianae (Figure 2-1). He is listed as a co-author in a publication of the work described in Chapter 2. v Table of Contents Abstract .............................................................................................................................. ii Acknowledgements .......................................................................................................... iv Attributions ........................................................................................................................v List of Tables .................................................................................................................. viii List of Figures .....................................................................................................................x Chapter 1. Introduction ...................................................................................................1 Literature review ..............................................................................................................1 Research objectives ........................................................................................................24 References ......................................................................................................................25 Chapter 2. Inactivation of Phytophthora and bacterial species in water by a potential energy-saving heat treatment .........................................................................................43 Abstract ..........................................................................................................................44 Introduction ....................................................................................................................45 Materials and methods ...................................................................................................47 Results ............................................................................................................................53 Discussion ......................................................................................................................55 Acknowledgements ........................................................................................................57 References ......................................................................................................................57 Tables .............................................................................................................................61 Figures ............................................................................................................................63 Chapter 3. Temperature effect on Phytophthora zoospore survival in irrigation water ..................................................................................................................................67 Materials and methods ...................................................................................................67 Results and discussion ....................................................................................................68
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