DIVERSITY OF ASTER YELLOWS PHYTOPLASMAS IN LETTUCE Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jianhua Zhang, B. S. ***** The Ohio State University 2003 Dissertation Committee: Approved by Dr. Sally A. Miller (adviser) Dr. Saskia A. Hogenhout (co-adviser) Adviser, Department of Plant Pathology Dr. Lowell R. Nault Dr. Terrence L. Graham Co-Adviser, Department of Entomology Dr. Sophien Kamoun ABSTRACT Aster yellows is a potentially devastating disease of lettuce, caused by a leafhopper- transmitted phytoplasma. Disease incidence varies in the range of 0-100% from year to year in Ohio lettuce fields. An unknown number of aster yellows phytoplasma strains infect lettuce. Aster yellows phytoplasma strains may be distributed differently in lettuce plants, potentially influencing leafhopper transmission of the pathogen. In this study I used molecular methods and symptom type to characterize several aster yellows phytoplasma strains. At least five aster yellows phytoplasma strains were identified based on the symptoms they cause in China aster and lettuce. Strain AY-Witches’ broom (AY-WB) caused wilting in lettuce and witches’-broom in aster plants at the late stage of infection. Aster yellows-Severe (AY-S) caused stunting, clustering and phyllody in aster. AY- Semi-geotropism (AY-SG) differed from the others by causing semi-geotropism at the late stage of infection in aster and lettuce. Strain Bolt-White (AY-BW) infection resulted in chlorosis of newly emerging leaves and Strain Bolt-Distortion (AY-BD2) resulted in leaf and stem distortion in lettuce, mild symptoms compared to those caused by other strains. ii Phytoplasmas were detected by polymerase chain reaction seven days before aster yellows symptoms appeared at the growing points of lettuce plants, indicating that aster yellows symptoms are closely related to the presence of phytoplasmas in an organ. Differences in phytoplasma distribution were found between two aster yellows phytoplasma strains. The phytoplasmas of AY-S strain spread faster and more widely than the AY-BD2 strain in lettuce plants. At the early stage of infection, phytoplasmas were detected first from the midrib of inoculated leaves, then the stems and growing points, but not from the margins of inoculated leaves, suggesting that movement of aster yellows phytoplasmas in the inoculated leaves is mainly unidirectional. RFLP analysis of PCR products amplified using the 16S rDNA primers F2/R2 indicates that all of the aster yellows phytoplasma strains belong to 16SrI phytoplasma group. AY-WB belongs to the 16SrI-A subgroup whereas the others belong to the 16SrI- B subgroup. AY-WB can be distinguished easily from the other strains by PCR with strain specific primers BF/BR. AY-WB, AY-S, AY-BW and AY-SS can be distinguished from one another either by PCR-RFLP analysis of AY19P/AY19m-amplified products or by multiplex PCR. Primer typing can be used to differentiate most strains except AY- BW and AY-SG. Phylogenetic analysis of partial sequences of 16S rRNA gene and 16S- 23S gene spacer regions showed that all aster yellows phytoplasma strains were clustered with most aster yellows phytoplasma sequences obtained from the Genbank database. Five µg aster yellows phytoplasma chromosomal DNA was isolated from AY-WB using pulse field gel electrophoresis (PFGE). A shotgun library was constructed by Integrated Genomics (Chicago, IL USA) and the 800 kb genome of AY-WB was sequenced with sequence saturation of 8.6-fold coverage. Sixteen contigs were iii constructed that cover approximately 87.5% of the sequence of the genome. The genome contains approximately 26.9% GC and 67% coding sequences. One contig contains the complete lettuce chloroplast genome with 128,839 bp and 36.5% GC content. I identified 11 putative transposase-coding regions were found in the near-complete aster yellows phytoplasma AY-WB genome. The length of the putative transposase coding regions ranged from 237 to 963 bp. Inverted repeats (IR) were found for seven of the transposase coding regions and direct repeats (DR) were found in three of the transposase coding regions with IRs. No IR was found overhanging singular ORF coding regions. The putative transposase genes contain 23.4 to 27.4% GC similar to the rest of the phytoplasma genome (26.9%). Phylogenetic analysis suggests that the putative transposase genes in the aster yellows phytoplasma genome are clustered into one clade. PCR with primers flanking transposase-coding regions amplified DNA fragments only from DNA extracts of aster yellows-symptomatic lettuce and aster yellows phytoplasma- infected leafhoppers, not from the extracts of healthy lettuce and leafhoppers. This suggests that the putative transposase coding sequences were in aster yellows phytoplasma DNA rather than contaminant DNA from lettuce. Amplification of putative transposase coding regions by external primers excludes the possibility of their existence in extrachromosomal elements. PCR analyses show that insertion sequences can be used to detect and separate strains of the aster yellows phytoplasma group. iv Dedicated to my mother’s hopes v ACKNOWLEDGMENTS As I begin to write this section, I am deeply touched by my recollection of the time that I have been studying and working under guidance of my adviser, and working with the professors in the Department of Plant Pathology and Entomology. First of all, I like to take the opportunity to thank my adviser, Dr. Sally A. Miller for giving me the opportunity to study under her guidance. I thank my co-adviser Dr. Saskia Hogenhout for her patience in teaching me molecular techniques. They not only taught me plant pathology, but also English writing skills; their continuous input and advice made it possible for me to complete this research project. The diversity of my SAC members broadened my knowledge in plant pathology; especially in vector-transmitted plant diseases. I should thank Dr. Lowell R. Nault for his genuine ideas on the aspects of entomology, Dr. Terrence L. Graham for his input from the aspect of plant-microbe interactions and Dr. Sophien Kamoun for his suggestions on bioinformatics. I shall never forget that it was Dr. Terrence L. Graham who signed my extension documents while I was waiting for the decision from graduate schools. Dr. Michael J. Boehm wrote a strong recommendation letter on behalf of my application. Without their full support and encouragement, I would never have had the chance to be accepted as a graduate student, no to mention to reach my academic goal. vi My sincere appreciation is expressed to Mr. Ian Holford, Mr. Xiaodong Bai, Dr. Laurence V. Madden, Dr. Brian McSpadden-Gardener, Dr. Trudy Torto and Dr. Walid Hamada for their help in lab techniques. I should thank Ms. Melanie Ivey, Ms. Shujing Dong, Ms. Kristen Willie, Ms. Joanne Hershberger, Ms. Helga Beke, Ms. Lynn West, Ms. Leedy Laurel, Ms. Angie Strock, Mr. Jhony Mera, Mr. Bill Styer, Mr. Bob James and the greenhouse crew for their technical support. Without their help, it would have been impossible for me to finish my research in time. My appreciation is also extended to Dr. Randy Rowe for his leadership of this department towards academic excellence. Finally, I thank my wife Qiuzhen Tong for her endurance and love. vii VITA 1979 ................................................. B. S. in agronomy, Shandong Agriculture University, P. R. China 1979 – 1982 .................................... Assistant researcher, Corn Research Institute, Shandong Academy of Agriculture Sciences 1983 –1985 ..................................... Associate researcher in agronomy Shandong Academy of Agriculture Sciences. 1986 –1988 .................................... Visiting scientist in The University of Sydney, Sydney, Australia. 1992 – 1995 .................................... Associate professor in agronomy Shandong Academy of Agriculture Sciences. 1995 – 1996 ..................................... Visiting scientist in The Ohio State University 1997 –1998 ..................................... Research assistant in The Ohio State University 1998—2003 ……………………… Ph D. student in plant pathology in The Ohio State University viii PUBLICATIONS Selected research publication after 1991 1 J. Zhang et al. “Effect of mineral ions on seedling growth before the third leaf stage in maize.” J. Soil & Fertilizer, 1,32-35 (1992). 2 J. Zhang, et al. “Effect of plant regulator C150B on malt.” Barley Science 4. (1993). 3 J. Zhang et al. Variety identification in maize and sweet potato by electrophoresis. J. Plant Biology 3(1993) 4 J. Zhang, et al. “Peroxidase electrophoretogram coding and variety identification in maize”. Acta Agronomica Sinica (1995). 5 J. Zhang et al. “Light, dry matter accumulation and distribution in the population of different plant type varieties” J. Plant Biology 3(1). (1993). 6 J. Zhang et al. “Quantitative selection for compact high-yielding maize hybrids.” J. Agric. Sci. 125, 39- (1995). 7 J. Zhang, M. B. McDonald and P. M. Sweeney, "Soybean cultivar identification using RAPD." Seed Sci. & Technol., 24, 589-592 (1996). 8 J. Zhang, M. B. McDonald and P. M. Sweeney, "Random amplified polymorphic DNA (RAPDs) from seed of differing soybean and maize genotypes." Seed Sci. & Technol., 24, 513-522 (1996). 9 J. Marcos-Filho, M. B. McDonald, D. M. TeKrony and J, Zhang, " RAPD fragment profiles from deteriorating soybean seeds." Seed Technology, 19, 34-44 (1997). 10 J. Zhang, M. B. McDonald P. M. Sweeney, " Testing for genetic purity in petunia and cyclamen seed using random amplified polymorphic DNA markers." HortScience 32(2), 246-247 (1997). 11 J. Zhang, M. B. McDonald, " The saturated salt accelerated aging test for small- seeded crops." Seed Sci. & Technol. 25, 123-131 (1997). ix 12 J. Zhang, S.A. Hogenhout and S.A. Miller, “Isolation of aster yellows phytoplasma genomic DNA from lettuce.” Phytopathology 91, S99 (2001). 13 S. A. Miller, J. Zhang, C.W. Hoy and L.R. Nault, “Monitoring aster yellows infectivity in leafhoppers during the vegetable growing season by the polymerase chain reaction” Phytopathology 87, S66 (1997). 14 J. Zhang, S.A. Miller, C. Hoy, X. Zhou, and L.