G.F. Campbell

Pyrenophora teres f. maculata, the cause of Pyrenophora leaf spot of barley in South Africa

Net blotch caused by Pyrenophora teres is a serious disease of barley in many cereal production areas world-wide, including the Western Cape province of South Africa. The pathogen occurs as two forms, P. teres f. teres, which produces net-blotch symptoms, and P. teres f. maculata which produces leaf spots. Pyrenophora japonica and P. hordei, which have also been reported in South Africa, also produce spots on susceptible barley cultivars. Using RAPD markers, spot-forming isolates from the South African population were found to be relatively uniform. Single ascospores were obtained from pseudothecia after in vitro mating had occurred between a verified P. teres net-blotch isolate from Denmark and a representative Pyrenophora leaf spot isolate from South Africa. Using amplified fragment length polymorphism (AFLP) and RAPD markers, recombination was demonstrated in the progeny which had DNA banding patterns different from the two parental isolates. Pathogenicity trials also confirmed that recombination had taken place during mating. Inoculations were conducted on differential cultivars susceptible to the net-blotch or leaf spot forms. The two parents induced typical net-blotch or leaf spot symptoms whereas the progeny mostly induced a jagged spot symptom on each cultivar. Fungicide sensitivity tests using the ergosterol biosynthesis inhibitors triademinol, bromuconazole and triticonazole showed that, due to recombination, some progeny could have increased resistance to these fungicides. Due to mating and subsequent recombination between a net blotch isolate of P. teres and a representative leaf spot isolate, it was concluded that the latter was P. teres f. maculata. These results contrast with the earlier belief that Pyrenophora leaf spot isolates in the Western Cape are P. japonica and P. hordei.

Evidence of recombination between net- and spot-type populations of Pyrenophora teres as determined by RAPD analysis

The genetic structure of Pyrenophora teres , the causal agent of net blotch of barley, was examined in two fields 30 km apart in the south-western Cape of South Africa. The two fields respectively represented a net- and spot-type population, the two types being distinguished on the basis of symptom expression on differentially susceptible cultivars. The number of isolates sampled from each field was 36 for the net-type population and 29 for the spot-type population. Samples were collected from infected barley leaves from two separate quadrants in each field, the two quadrants positioned in corners of the fields, diagonal to one another. Of the 40 10-mer random oligonucleotide primers screened, five produced scorable, reproducible DNA bands suitable for the determination of population structure. A total of 65 loci were produced of which 54 were polymorphic. Genetic analysis of bands produced by one of the primers has revealed single locus segregation in a mating between a net- and spot-type isolate, indicating that RAPD bands can be interpreted as alleles at genetic loci. Total gene diversities determined for all loci resulted in mean indices of 0.063 and 0.082 being obtained respectively for the net- and spot-type populations. Genetic diversity among the two populations was divided into within- (variation between sampling quadrants) and among population components using Neis G ST . A coefficient of genetic differentiation ( G S ) of 0.0149 was obtained between quadrants within populations while a coefficient ( G T ) of 0.63 was obtained between the two populations. Genotypic variation revealed 13 distinct multilocus genotypes (haplotypes) in the net-type population while there were 12 in the spot-type population. UPGMA cluster analysis of the two populations together with six progeny from a mating between a net- and spot-type isolate resulted in three main clusters being produced, one for each population and one for the progeny. One isolate collected from the net-type population that did not cluster with the other net-type isolates clustered directly next to the cluster containing the sexual progeny. This isolate also contained a unique spot-type DNA band. This suggested that sexual recombination may be occurring between net- and spot-type isolates under field conditions.

Genetic stability of net × spot hybrid progeny of the barley pathogen Pyrenophora teres

Hybrid progeny produced from a mating between a net- and a spot-type isolate of the barley net blotch pathogen Pyrenophora teres were screened to assess their viability and genetic stability. Progeny (F1) inoculated onto seedlings of the barley cultivars Stirling (highly susceptible to net-, and slightly susceptible to spot-type isolates), B87/14 and Clipper (only susceptible to spot-type isolates) produced jagged-type symptoms on all cultivars. When cultures (F1-1) isolated from leaves inoculated with Fj cultures were inoculated onto these cultivars, they produced similar symptoms. F1-2) cultures isolated from F1-1) infections also produced similar symptoms. RAPDs produced on all isolates of F1-1) and F1-2) progeny revealed identical profiles to those obtained for F1) isolates. Molecular and infection data indicated that jagged-type hybrid progeny of this net × spot mating retained their virulence and fertility overtime, and were genetically stable after two cycles of inoculation, mitosis and re-isolation. Hybridisation between net- and spot-type isolates of P. teres may, therefore, play a significant role in net blotch epidemiology in barley-growing areas.