New insights into the infection process of Rhynchosporium secalis in barley using GFP.

Through the use of a Rhynchosporium secalis isolate transformed with the green fluorescent protein gene and LASER scanning confocal microscopy (LSCM), fungal development during the R. secalis/barley interaction was analysed. Each infection stage was investigated from 0.5h to 14 days post-inoculation (p.i.) with extensive sampling within the first 48 h p.i. Early germination events were observed that had not been previously described. A specific time of germination was noted, with germ tube formation appearing as early as 1h p.i. Conidia were observed within anticlinal grooves of epidermal cells and the formation of bubbles within these pectin-rich regions was observed within 24h p.i. The study reports R. secalis pectinase production and suggests degradation of these pectin-rich regions. Reactive oxygen species were present during early penetration, 3h p.i. and co-localised with fungal development. LSCM allowed the visualisation of fungal growth deep within tissues at the later stage of the infection.

Genetic structure of South Australian Pyrenophora teres populations as revealed by microsatellite analyses.

The aim of this study was to determine the genetic structure of South Australian field populations of the barley net blotch pathogens, Pyrenophora teres f. sp. teres (PTT) and P. teres f. sp. maculata (PTM), using microsatellite DNA markers. Three PTT populations (76 isolates total) and two PTM populations (43 isolates total) were sampled from separate fields during a single growing season. The results showed that of the 20 microsatellite loci examined, 17 (85%) were polymorphic within the PTT and PTM populations. In total, 120 distinct alleles were identified of which only 11 (9%) were shared between the two population types. Nei's measure of gene diversity across the PTT and PTM populations was similar at 0.38 and 0.40, respectively, and also much higher than previously reported from studies in which other types of molecular markers were used. The coefficient of genetic differentiation among both populations was the same (G(ST)=0.03) and the low and insignificant estimates of F(ST), as indicated by θ, between populations of the same type (PTT: θ<0.008, PTM: θ=0.014) indicated that isolates sampled from different areas within the same field were genetically similar. In contrast, high and significant genetic differentiation was observed among and between populations of different type (G(ST)=0.42, θ>0.567). The high number of unique multilocus haplotypes observed within the PTT (84%) and PTM (100%) populations, combined with a 1:1 distribution of both mating types, suggested that sexual reproduction was predominant among these populations. However, tests for multilocus associations showed that both PTT and PTM populations were in significant linkage disequilibrium. Although the levels of disequilibrium were low, an asexual reproductive component could not be excluded.

Pathogenic and molecular variation support the presence of genetically distinct clonal lineages in Australian populations of Puccinia graminis f. sp. avenae.

Previous studies of the causal agent of stem rust of oats (Puccinia graminis f. sp. avenae; P. g. avenae) in Australia have demonstrated a high level of pathogenic variability. In this work, the pathotypic structure of the Australian P. g. avenae population in 1999 was investigated, as well as the pathotypic and genetic diversity of a collection of 26 Australian isolates representing a 25-year period (1971-1996). In the 1999 sample, 16 races belonging to six international standard races were identified from 97 isolates, with standard race 94 predominant in all regions. Race 94+Pg-13,Pg-Sa,Pg-a, detected in southern New South Wales (sNSW) and northern New South Wales (nNSW), was virulent on all of the differential genotypes used. Detailed analyses of pathogenicity and AFLP variability among 26 isolates collected from 1971-1996 revealed that isolates of standard race 94 collected in 1999 were genetically distinct from other Australian races of P. g. avenae. This evidence, along with data from annual pathogenicity surveys, suggests that the group to which standard race 94 belongs appeared during the late 1980s, and that it increased in frequency to dominate P. g. avenae pathogen populations throughout Australia from 1992 onward. The existence of groups of P. g. avenae isolates in Australia that differ in pathogenicity and AFLP phenotype suggests that current populations have evolved from a number of isolates of the fungus that differ in their genetic backgrounds, which may have originated from independent introductions or from asexual hybridisational events.

Molecular genetic variability of Australian isolates of five cereal rust pathogens.

Rust fungi cause economically important diseases of cereals, and their ability to rapidly evolve new virulent races has hindered attempts to control them by genetic resistance. PCR-based molecular tools may assist in understanding the genetic structure of pathogen populations. The high multiplex DNA fingerprinting techniques, amplified fragment length polymorphisms (AFLP), selectively amplified microsatellites (SAM) and sequence-specific amplification polymorphisms (S-SAP) were assessed for their potential in investigations of the genetic relationships among isolates of the wheat rust pathogens, Puccinia graminis f. sp. tritici (Pgt), Puccinia triticina (Pt), and P. striiformis f. sp. tritici (Pst), the oat stem rust pathogen P. graminis f. sp. avenae (Pga), and a putative new P. striiformis special form tentatively designated Barley grass yellow rust (Bgyr). Marker information content, as indicated by the number of species-specific fragments, polymorphic fragments among pathotypes, percentage of polymorphic loci, and the marker index, was highest for the SAM assay, followed by the AFLP and S-SAP assays. UPGMA analysis revealed that all marker types efficiently discriminated the five different taxa and Mantel tests revealed significant correlations between the marker types. Within pathogen groups, the marker types differed in the amount of variation detected among isolates; however, the major differences were consistent and polymorphism was generally low. This was reflected by the AMOVA analysis that significantly partitioned 90% of the genetic variation between taxa. Of the three marker types, SAMS were the most informative, and have the potential for the development of locus-specific microsatellites.