ISOLATION, IDENTIFICATION, PATHOGENICITY

AND SENSITIVITY OF SPP. TO

PHENAZINE-1-CARBOXYLIC ACID (PCA)-

PRODUCING PSEUDOMONAS SPP.

By

AHMAD KAMIL MOHD JAAFFAR

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Department of

DECEMBER 2012

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of AHMAD

KAMIL MOHD JAAFFAR find it satisfactory and recommend that it be accepted.

Linda Thomashow, Ph.D., Chair

David Weller, Ph.D.

Timothy Paulitz, Ph.D.

Lori Carris, Ph.D.

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ACKNOWLEDGEMENTS

First of all, I thank God for giving me strength and ability to complete this study.

I would like to express my sincere gratitude to Drs. Linda Thomashow and David Weller for their guidance, understands, inspired and invaluable advices throughout the duration of this study and the preparation of this thesis and also for taking care my family life in

Pullman, Washington. I would like to thank Drs. Timorthy Paulitz and Lori Carris for serving in my committee and their valuable suggestions and encouragement for my project. Thanks also go to Drs. Pat Okubara, Kurt Schroeder, Olga Mavrodi, Dima

Mavrodi, Ming-Ming Yang, Grant Poole, Dipak Sharma-Poudyal, USDA-ARS Root

Disease and Biological Control Unit, Department of Plant Pathology, for their kind help through out the experiments. I am thankful to Jim Parejko for providing bacteria samples for biological control study and Shyam Kandel for help in collecting soil samples throughout the study.

I also wish to thank the Malaysian Cocoa Board for awarding me a scholarship during my

PhD program at Washington State University. Special thanks and gratitude to former

Director-General, Malaysian Cocoa Board, Dr. Azhar Ismail, for his understanding, invaluable advice, support, and inspired and constant encouragement during my graduate training and for his role in strengthening the Malaysian Cocoa Board.

iii

My sincere appreciation also goes to Aaron Prescott, Esmeralda Fernandez, Julia

Krauser, Zach Day, Karen Hansen, Karen Adams, Nathalie Walter, Kathleen Parker,

Jennifer , Bob Bonsall, Ira Mavrodi and Yahya Mohd Nor for their help in many ways throughout the study.

I am greatly indebted and appreciate very much my beloved wife, Siti Fatimah Jusoh for her encouragement, support and sacrifices throughout the study. Thanks also to my son,

Ahmad Faiz and daughters, Anis Mariam and Anis Maisarah, for they have inspired me in their own ways to finish my study. To my parents in law, thanks for your prayers, patience and sacrifices for my successful life in the United States of America. To all my dearest brothers and sisters, a big thank you for their prayers, support and encouragement and for putting colors in my life. May God bless you all.

Last but not least, I wish to express my sincere thanks to all those who have in one way or another helped me in making this study a success.

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ISOLATION, IDENTIFICATION, PATHOGENICITY

AND SENSITIVITY OF RHIZOCTONIA SPP. TO

PHENAZINE-1-CARBOXYLIC ACID (PCA)-

PRODUCING PSEUDOMONAS SPP.

Abstract

by Ahmad Kamil Mohd Jaaffar, Ph.D. Washington State University December 2012

Chair: Linda S. Thomashow

Rhizoctonia root rot and bare patch is the most important disease of direct-seeded and barley in the Inland Pacific Northwest. Major gaps remain in understanding the epidemiology of this disease and the biology and ecology of AG-8 and R. oryzae, the causal agents. In these studies, a collection of 498 isolates of R. solani,

AG-I-like binucleate Rhizoctonia sp., and R. oryzae groups was assembled from fields throughout the Inland Pacific Northwest. Isolates were identified by PCR with primers specific to internal transcribed spacers of the nuclear ribosomal DNA and/or by DNA sequence analysis of the ITS regions. The results revealed the geographic distribution of

Rhizoctonia isolates in cereal-based production systems. R. solani AG-8 and R. oryzae groups II and III (but not group I) caused severe root rot on wheat. R. solani AG-2-1

v caused only mild root rot and the other groups showed trace discoloration of the roots. In contrast, R. solani AG-2-1 caused severe damping-off of canola and killed seedlings in greenhouse assays. Distinctive morphological characteristics were described for isolates of R. solani AG-8, AG-2-1, and AG-10, AG-I-like binucleate Rhizoctonia, and R. oryzae groups I, II, III. These results demonstrated for the first time that colony morphology and amplification by specific PCR primers are predictive of the identity of an isolate of

Rhizoctonia on wheat and canola.

The distribution of fluorescent Pseudomonas spp. capable of producing the antibiotic phenazine-1-carboxylic acid (PCA) was shown to overlap closely with that of

R. solani AG-8 but not that of R. solani AG-2-1 and to exhibit a highly significant inverse correlation with annual precipitation. Sensitivity of R. solani AG-8 and AG-2-1 to PCA was not correlated with exposure in nature or with virulence, indicating that tolerance of these pathogens to the antibiotic does not develop in nature.

Representative isolates from each of the four major phylogenetic groups of Phz+ pseudomonads were shown to control root rot of wheat caused by R. solani as well or better than did the model PCA producer P. fluorescens 2-79.

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TABLE OF CONTENTS

Page ACKNOWLEDGEMENTS ...... iii

ABSTRACT ...... v

LIST OF TABLES ...... ix

LIST OF FIGURES ...... xi

GENERAL INTRODUCTION

ROOT DISEASES OF WHEAT AND BARLEY ...... 1

RHIZOCTONIA ROOT ROT OF WHEAT ...... 2

CHARACTERIZATION OF THE PATHOGENS R. SOLANI AND R.ORYZAE ...... 3

MOLECULAR IDENTIFICATION AND DETECTION OF RHIZOCTONIA SOLANI ...8

MANAGEMENT OF THE DISEASE ...... 9

BIOLOGICAL CONTROL OF PLANT DISEASE ...... 12

BIOLOGICAL CONTROL BY FLUORESCENT PSEUDOMONAS SPP...... 13

PHENAZINES IN BIOLOGICAL CONTROL ...... 17

REFERENCES ...... 21

CONTRIBUTION PAGE ...... 40

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CHAPTER 1. MOLECULAR CHARACTERISTICS, MORPHOLOGICAL

CHARACTERISTICS, VIRULENCE AND GEOGRAPHIC DISTRIBUTION OF

RHIZOCTONIA SPP. IN THE INLAND PACIFIC NORTHWEST

ABSTRACT ...... 42

INTRODUCTION ...... 44

MATERIALS AND METHODS ...... 48

RESULTS ...... 59

DISCUSSION ...... 83

REFERENCES ...... 91

CHAPTER 2. SENSITIVITY OF RHIZOCTONIA ISOLATES FROM THE INLAND

PACIFIC NORTHWEST OF THE UNITED STATES TO PHENAZINE-1-

CARBOXYLIC ACID AND BIOLOGICAL CONTROL BY PHENAZINE-

PRODUCING PSEUDOMONAS SPP.

ABSTRACT ...... 100

INTRODUCTION ...... 102

MATERIALS AND METHODS ...... 107

RESULTS ...... 119

DISCUSSION ...... 145

REFERENCES ...... 153

GENERAL CONCLUSIONS ...... 161

viii

LIST OF TABLES

CHAPTER 1

1. Polymerase chain reaction (PCR) primers used for amplification of Rhizoctonia solani,

R. oryzae and AG-I-like binucleate Rhizoctonia (teleomorph ) ...... 52

2. Characteristics of the Inland Pacific Northwest agronomic zones that are highlighted in

maps (Figs. 9, 10 and 11) showing the distribution of Rhizoctonia spp...... 58

3. Morphological and molecular characteristics and virulence of Rhizoctonia solani AG-8

and AG-2-1 isolates from the Inland Pacific Northwest ...... 62

4. Virulence of Rhizoctonia solani AGs, AG-I-like binucleate Rhizoctonia spp. and R.

oryzae on wheat (cv. Louise) ...... 73

5. Pathogenicity and virulence of Rhizoctonia solani AGs, AG-I-like binucleate

Rhizoctonia sp., and R. oryzae on canola cv. TR8 ...... 74

6. Anastomosis groups and of Rhizoctonia isolates from cereal-based cropping

systems in the Inland Pacific Northwest ...... 79

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CHAPTER 2

1. Bacterial strains used in this study ...... 108

2. Population sizes of Phz+ and total culturable bacteria and plant colonization

frequencies on crops from fields in the Inland Pacific Northwest (PNW) collected in

2010...... 122

3. Growth rate (mm/day) among Rhizoctonia AG groups and species in 1/5 PDA buffered

to different pH values...... 130

4. Growth rate (mm/day) of isolates of Rhizoctonia in 1/5 PDA buffered to different pH

values ...... 131

5. Sensitivity of isolates from different anastomosis groups of Rhizoctonia solani,

binucleate Rhizoctonia AG-I, and Rhizoctonia oryzae to phenazine-1-carboxylic acid

(PCA) ...... 135

6. Sensitivity of selected isolates of R. solani AG-8 and AG-2-1 from fields with low or

high frequencies of Phz+ isolates ...... 137

7. Biological control of Rhizoctonia solani AG-8 C-1 in natural soil by PCA-producing

Pseudomonas spp...... 143

8. Biological control of Rhizoctonia solani AG-8 C-1 in pasteurized soil by PCA-

producing Pseudomonas spp...... 144

x

LIST OF FIGURES

CHAPTER 1

1. Tube assay for testing the virulence and pathogenicity of Rhizoctonia isolates ...... 57

2. Bands associated with amplification of DNA by primers specific for R. solani AG-8,

AG-2-1 and AG-10, AG-I-like binucleate Rhizoctonia sp., and R. oryzae group II

(RO-2) ...... 60

3. Optimization of PCR using different annealing temperatures to distinguish between

AG-2-1 and AG-8 ...... 61

4. Colony characteristics of Rhizoctonia solani AG-2-1 (A, C and E) and AG-8 (B, D,

and F) isolates on PDA at 7 days (A, B, C and D) and 30 days (E and F) after

inoculation...... 68

5. Colony characteristics of isolates of Rhizoctonia solani AG-10 (A and C) and AG-I-

like binucleate Rhizoctonia sp. (B and D) on PDA at 7 days after inoculation ...... 69

6. Colony characteristics of Rhizoctonia oryzae groups I (A and D), II (B and E) and III

(C and F) isolates on PDA at 7 days after inoculation ...... 70

7. Typical symptoms caused by isolates of R. solani AG-2-1 (A and C) and AG-8 (B, D

and E) on wheat roots ...... 75

8. Typical symptoms of Rhizoctonia root rot caused by R. oryzae groups II and III ...... 76

9. Geographic distribution of Rhizoctonia AGs and species in the Inland PNW collected

from cereal-based cropping systems by Dr. Timothy Paulitz from 2000-2009 ...... 80

10. Geographic distribution of Rhizoctonia AGs and species in the Inland PNW collected

from cereal-based cropping systems during this study in 2010 ...... 81

xi

11. Geographic distribution of Rhizoctonia AGs and species in the Inland PNW collected

from cereal-based cropping systems during this study in 2011 ...... 82

CHAPTER 2

1. Flow chart of the PCR-based terminal dilution endpoint assay to determine the

population sizes of Phz+ bacteria and total culturable aerobic bacteria in the wheat

rhizosphere ...... 111

2. Diagram of the tube assay used to test the biocontrol activity of bacteria against

Rhizoctonia root rot ...... 118

3. Distribution of Rhizoctonia solani AG-8 isolates collected in 2010 and 2011, and

frequency of Phz+ isolates on the roots of wheat recovered in 2010 in Eastern

Washington State ...... 121

4. Relationship between frequencies of plant colonization by indigenous Phz+

pseudomonads and annual precipitation