Molecular and host specificity studies of Puccinia Striiformis in Australia

Abstract

ABSTRACT 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 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 spp. were frequently observed and these may assume a supporting role in Pst inoculum production and dispersal.