In vitro selection for resistance to net blotch of barley and molecular characterization of the pathogen
Pyrenophora teres (anamorph: Drechslera teres), the causal agent of net blotch of barley, has been shown to produce toxins involved in pathogenicity. The objective of the first part of this study was to develop an in vitro screening procedure using concentrated culture filtrates of the pathogen to screen immature embryo-derived callus from resistant and susceptible barley genotypes. Three resistant (Fr926-77, Kao32-12, Kao22-3) and three susceptible (TR306, AC Oxbow and Deuce) genotypes were used for in vitro culture and Kao22-3 and TR306 were identified as potential culture-responsive parental combinations. However, the in vitro screening could not be carried out due to the inability to detect the toxins in culture filtrates by chromatographic separations or to delineate resistant and susceptible genotypes based on bioassays with excised leaves. In the second part of this study, molecular techniques were used to study the pathogen. Two forms of the net blotch pathogen have previously been identified. One form (Pyrenophora teres f. maculata) causes spot-type lesions and the other form (Pyrenophora teres f. teres) causes net-type lesions. These two forms are morphologically identical and differ only in the symptoms induced on barley. The RAPD methodology was used to distinguish between the spot and net forms and a closely related species, Pyrenophora graminea, which causes barley leaf stripe. Cochliobolus sativus, which causes spot blotch of barley, and Pyrenophora tritici-repentis, which causes tan spot of wheat, were included as outgroups. One hundred 10-mer primers were used for RAPD analysis and after agarose gel electrophoresis, the bands were scored as present or absent. Results were compiled in a binary matrix and cluster analysis performed. The net forms and the spot forms clustered close to each other with about 80% similarity. P. graminea clustered close to the net blotch cluster with about 10-25% similarity, followed by P. tritici-repentis. It was also found that the algorithm employed for cluster analysis affected the topology of the dendrograms in one investigation. However, upon deriving a consensus tree from all the dendrograms, the tree reverted back to the original topology. PCR primers were also designed based on the terminal sequences of a RAPD band unique to the net form. An approach, termed Pooled Isolates for Taxon Differentiation using RAPD (PITD-RAPD), was used to analyze pooled DNA from isolates of the net form, the spot form and P. graminea. The polymorphisms detected were similar to those generated when RAPD analysis was performed on individual DNA samples. This approach might be useful for the efficient development of taxon-specific probes or primers. The internal transcribed spacer (ITS) of the rRNA unit of the three pathogens was PCR amplified using universal primers. No size variation was observed. Amplification of the intergenic spacer region between two rRNA units was also attempted. Size variation was observed, but was not characteristic of any species or sub-species.