COPYRIGHT AND USE OF THIS THESIS This thesis must be used in accordance with the provisions of the Copyright Act 1968. Reproduction of material protected by copyright may be an infringement of copyright and copyright owners may be entitled to take legal action against persons who infringe their copyright. Section 51 (2) of the Copyright Act permits an authorized officer of a university library or archives to provide a copy (by communication or otherwise) of an unpublished thesis kept in the library or archives, to a person who satisfies the authorized officer that he or she requires the reproduction for the purposes of research or study. The Copyright Act grants the creator of a work a number of moral rights, specifically the right of attribution, the right against false attribution and the right of integrity. You may infringe the author’s moral rights if you: - fail to acknowledge the author of this thesis if you quote sections from the work - attribute this thesis to another author - subject this thesis to derogatory treatment which may prejudice the author’s reputation For further information contact the University’s Director of Copyright Services sydney.edu.au/copyright Molecular and Host Specificity studies in Puccinia striiformis in Australia By Jordan Bailey A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy The University of Sydney Plant Breeding Institute May 2013 Statement of Authorship This thesis contains no material which has been accepted for the award of any other degree or diploma in any university, and to the best of my knowledge, it contains no material previously published by any other person, except where references are made in text Jordan Bailey i Acknowledgments I would like to thank the Cooperative Research Centre for National Plant Biosecurity for supporting this project and myself. I appreciate the support of the postgraduate team and the numerous conferences and workshops I was able to attend being a CRCNPB student. It goes without saying that without the continual guidance and advice of my supervisor Dr. Colin Wellings this thesis would not have made it to fruition. Thank you for allowing me to pursue to my own ideas and creativity, at times, and always being there when I had a question, even in retirement. I truly appreciate the dedication and insight of Dr. Haydar Karaoglu. I thank you for the time and effort you gave me and for treating me as an equal and a friend. Your training and high standards have afforded me expert skills in the field of molecular biology and I thank you for fuelling my passion in this area. Prof. Robert F. Park, thank you for your dedication in the final months, when I needed you the most. I know you are a busy man in high demand and I really appreciate the time you allocated to reviewing my work and your words of encouragement. A special thank you to technician Mr. Matthew Williams for helping whenever I needed it. Thank you to Mr. Keshab Kandel for constant technical assistance and advice and Dr. Neil Wilson for sharing your facilities and expertise with me. I would also like to thank the numerous staff at PBI who were always happy to take time out of their day to help. Last but not least, thank you to my Husband and family for their support. ii "There are ... rusts that attack grains and grasses, trees and shrubs, the most beautiful flowers of the gardens and the ugliest weeds of the fields" E. C. Stakman (Stakman, 1957) iii Summary The development of 26 SSR markers, specific for selective and sensitive amplification of Puccinia striiformis Westend forma specialis tritici Eriks (Pst), and related stripe rust pathogens, is documented. These markers were designed using genomic sequences from collaborators at the Australian National University and data published by Cantu et al. (2010). The allelic diversity observed varied from 2 to 8 alleles per locus and PIC values ranged from 0.5 to 0.76 with an average of 0.54. The marker set discriminated major Pst lineages in Australia, and separate host specific forms of the stripe rust pathogen on various graminaceous hosts. There was limited evidence for specific molecular phenotypes associating with Pst pathotypes. The SSR markers were able to identify a putative hybrid form of Pst, and were also used to develop a diagnostic test for application in biosecurity and incursion detection. The diagnostic protocol was based on simple and reliable visualisation using 3% agarose gel electrophoresis and was able to adequately amplify PCR products even with minute and degraded DNA samples from urediniospores and stripe rust infected leaf tissue. Fifteen of the SSR markers developed were used to genotype a set of 115 Australian isolates of Puccinia striiformis. The isolates were collected over the years 1979 – 2010 and represented 14 pathotypes from two major Pst pathotype lineages (pre-2002 and post-2002). Three isolates of Pst from the USA were also included. Genotyping was also performed for isolates of non-wheat infecting P. striiformis f. sp. pseudo-hordei and P. striiformis f. sp. hordei. Isolates of the stripe rust pathogens P. striiformoides (Psds) and P. pseudostriiformis (Pps), infecting cocksfoot grass (Dactylis glomerata) and Kentucky bluegrass (Poa pratensis), respectively, were also included. The results confirmed the clonal nature of the Pst iv population in Australia. Isolates from each of the two major Pst pathotype lineages were strongly clustered and pathotype demarcation beyond this point was limited. The USA isolates strongly resembled post-2002 isolates detected in Australia with limited additional variation between isolates from both continents. Distinct groupings, congruent with host preferences, were evident among the formae speciales Pst, Psp-h and Psh. However, many alleles were shared between the forms at various SSR loci, making intraspecific relationships difficult to resolve. The pathogens Psds and Pps were separated from Pst, Psp-h and Psh, largely due to extensive non-amplification of target product in isolates of Psds and Pps but also allelic polymorphism, when using the SSRs developed here from Pst genomic sequence data. This agrees with recent studies that have elevated both to species rank (Liu & Hambleton, 2010). The role of wild Hordeum Link spp. as an ancillary host of Pst in Australia was explored in this study. A differential set of Hordeum spp. was developed in order to examine avirulence/virulence characteristics of Pst isolates originating from cultivated cereals and weedy Hordeum communities. The differential set was used to screen isolates from a range of Pst pathotypes collected over a 30 year period and representing diverse geographical origins. Five distinct pathotypes were described with respect to the Hordeum differential set. The majority of Pst isolates derived from the original 1979 incursion and all isolates derived from the 2002 incursion were considered avirulent for Hordeum and were designated pathotype H1. Representative isolates collected between 1980 to 2001 showed evidence for increased virulence of Pst on Hordeum spp., classified as pathotypes H2 to H5. There was no association between the pathotypes determined using the Hordeum differential developed here and the pathotypes already established for these isolates using wheat differential lines. Therefore, weedy Hordeum spp, although widely distributed and frequently associated with commercial wheat production, were concluded to play a negligible selective role in the evolution of pathotype variability on wheat. However, susceptible genotypes of Hordeum v spp. were frequently observed and these may assume a supporting role in Pst inoculum production and dispersal. Contents Statement of Authorship .......................................................................................................... i Acknowledgments .................................................................................................................... ii Summary .................................................................................................................................. iv Contents ................................................................................................................................... vi List of Tables ........................................................................................................................... ix List of Figures .......................................................................................................................... xi 1. General Introduction ........................................................................................................... 1 2. Literature Review................................................................................................................. 5 Puccinia striiformis .............................................................................................................. 5 Impact and lifecycle ............................................................................................................. 7 Puccinia striiformis f. sp. tritici in Australia ...................................................................... 10 Molecular studies in P. striiformis ..................................................................................... 15 Interactions between Graminaceous hosts and P. striiformis with particular reference to Hordeum spp. ....................................................................................................................