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On Phymatotrichopsis Omnivora (Cotton Root Rot) EFFECTS OF OILSEED MEALS AND ISOTHIOCYANATES (ITCS) ON PHYMATOTRICHOPSIS OMNIVORA (COTTON ROOT ROT) AND SOIL MICROBIAL COMMUNITIES A Dissertation by PING HU Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2012 Major Subject: Soil Science Effects of Oilseed Meals and Isothiocyanates (ITCS) on Phymatotrichopsis omnivora (Cotton Root Rot) and Soil Microbial Communities Copyright 2012 Ping Hu EFFECTS OF OILSEED MEALS AND ISOTHIOCYANATES (ITCS) ON PHYMATOTRICHOPSIS OMNIVORA (COTTON ROOT ROT) AND SOIL MICROBIAL COMMUNITIES A Dissertation by PING HU Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Co-chairs of Committee, Terry J. Gentry Frank M. Hons Committee Members, Hongbin Zhang Shuhua Yuan Emily B. Hollister Head of Department, David Baltensperger May 2012 Major Subject: Soil Science iii ABSTRACT Effects of Oilseed Meals and Isothiocyanates (ITCS) on Phymatotrichopsis omnivora (Cotton Root Rot) and Soil Microbial Communities. (May 2012) Ping Hu, B.S., Nankai University; M.S., Kansas State University Co-Chairs of Advisory Committee: Dr. Terry J. Gentry Dr. Frank M. Hons The meals from many oilseed crops contain biocidal chemicals that are known to inhibit the growth and activity of several soil pathogens, though little is known concerning impacts on whole soil microbial communities. We investigated the effect of oilseed meals (SMs) from both brassicaceous plants, including mustard and camelina, as well as non-brassicaceous plants, including jatropha and flax, on P. omnivora (the casual agent of cotton root rot) in Branyon clay soil (at 1 and 5% application rates). We also investigated the effect of SMs from camelina, jatropha, flax, and wheat straw on microbial communities in Weswood loam soil. We also used four types of isothiocyanates (ITCs) including allyl, butyl, phenyl, and benzyl ITC to test their effects on P. omnivora growth on potato dextrose agar (PDA), as well as on soil microbial communities in a microcosm study. Community qPCR assays were used to evaluate relative abundances of soil microbial populations. Soil microbial community composition was determined through tag-pyrosequencing using 454 GS FLX titanium technology, targeting ITS and 16S rRNA gene regions for fungal and bacterial iv communities, respectively. The results showed that all tested brassicaceous and jatropha SMs were able to inhibit P. omnivora sclerotial germination and hyphal growth, with mustard SM being the most effective. Flax didn’t show any inhibitory effects on sclerotial germination. All tested ITCs inhibited P. omnivora OKAlf8 hyphal growth, and the level of inhibition varied with concentration and ITC type. Total soil fungal populations were reduced by ITC addition, and microbial community compositions were changed following SM and ITC application. These changes varied according to the type of SM or ITC added. Our results indicated that SMs of several brassicaceous species as well as jatropha may have potential for reducing cotton root rot as well as some other pathogens. Different SMs releasing varied ITCs may result in differential impacts on soil microorganisms including some pathogens. v DEDICATION To my parents vi ACKNOWLEDGEMENTS I would like to thank my committee chairs, Dr. Gentry and Dr. Hons, and my committee members, Dr. Hollister, Dr. Zhang and Dr. Yuan, for their guidance and support throughout the course of this research. Thanks also to my friends and colleagues and the department faculty and staff for making my time at Texas A&M University a great experience. I also want to extend my gratitude to Chevron Corp. and Cotton Inc., which provided funding for this study. Finally, thanks to my mother and father for their encouragement and love. vii NOMENCLATURE ITC Isothiocyanate GLS Glucosinolates NMDS Non-metric multidimensional scaling PCA Principal component analysis SM Oilseed meal CLPP Community level physiological profiling viii TABLE OF CONTENTS Page ABSTRACT .............................................................................................................. iii DEDICATION .......................................................................................................... v ACKNOWLEDGEMENTS ...................................................................................... vi NOMENCLATURE .................................................................................................. vii TABLE OF CONTENTS .......................................................................................... viii LIST OF FIGURES ................................................................................................... xi LIST OF TABLES .................................................................................................... xiii CHAPTER I INTRODUCTION AND LITERATURE REVIEW ............................ 1 1.1. Introduction ............................................................................................ 1 1.2. Background ............................................................................................ 2 1.2.1. Biofuel oilseed crops .................................................................. 2 1.2.2. SMs as organic fertilizers ........................................................... 5 1.2.3. Chemicals in various SMs .......................................................... 6 1.2.4. Environmental fate and persistence of allelochemicals from SMs 8 1.3. Effects of SMs on soil microorganisms ................................................. 10 1.3.1. Soil fungal pathogens ................................................................. 10 1.3.2. Other soil microorganisms ......................................................... 12 1.4. Conclusions ............................................................................................ 17 II PHYMATOTRICHOPSIS OMNIVORA INHIBITION BY OILSEED MEALS AND ISOTHIOCYANATES ............................... 19 2.1. Introduction ............................................................................................ 19 2.2. Materials and methods ........................................................................... 22 2.2.1. Soil collection and characterization ........................................... 22 2.2.2. Phymatotrichopsis omnivora cultures and sclerotia ................... 23 2.2.3. SM analysis ................................................................................ 24 2.2.4. Experimental plan ...................................................................... 26 ix CHAPTER Page 2.2.5. Statistical analysis ...................................................................... 30 2.3. Results .................................................................................................... 31 2.3.1. SM chemical composition .......................................................... 31 2.3.2. Sclerotial germination and survival rate ..................................... 32 2.3.3. P. omnivora hyphal growth in soil ............................................. 34 2.3.4. P. omnivora OKAlf8 hyphal growth on PDA ............................ 38 2.4. Discussion .............................................................................................. 38 2.4.1. GLS in SMs and ITC concentrations in the soil ......................... 38 2.4.2. P. omnivora inhibition by SMs: sclerotial germination ............. 39 2.4.3. P. omnivora inhibition by SMs: hyphal growth ......................... 41 2.4.4. P. omnivora inhibition by ITCs .................................................. 41 2.5. Conclusions ............................................................................................ 43 III SOIL MICROBIAL COMMUNITY CHANGES DUE TO OILSEED MEAL APPLICATION ...................................................... 45 3.1. Introduction ............................................................................................ 45 3.2. Materials and methods ........................................................................... 48 3.2.1. Soil collection and characterization ........................................... 48 3.2.2. SM analysis ................................................................................ 48 3.2.3. Experimental plan ...................................................................... 49 3.2.4. Functional diversity using BIOLOG analysis ............................ 50 3.2.5. DNA extraction and quantification ............................................ 50 3.2.6. qPCR on general bacterial and fungal abundance ...................... 52 3.2.7. Fungal tag-encoded amplicon pyrosequencing and analysis ..... 52 3.2.8. Statistical analysis ...................................................................... 54 3.3. Results .................................................................................................... 55 3.3.1. SM and wheat straw chemical composition ............................... 55 3.3.2. Abundance of soil bacterial and fungal populations .................. 56 3.3.3. Soil fungal community composition .......................................... 58 3.3.4. Soil community level physiological profiles (CLPP) ................ 62 3.4. Discussion .............................................................................................. 67 3.4.1. Soil fungal and bacterial abundance enhanced by SMs ............. 67 3.4.2. Soil fungal community
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