Genetics of pathogenicity in Pyrenophora leaf diseases of barley

[摘要] ENGLISH ABSTRACT:Net blotch of barley, caused by Pyrenophora teres, is one of the mostimportant diseases of this cereal in the south Western Cape Province ofSouth Africa. This fungus exists as two different types (forms), namely a nettypeand a spot-type that are distinguished by differential symptom expressionon barley leaves. Based on this specific plant pathological difference a seriesof studies of agricultural importance were executed to investigate the effectsof sexual recombination between these two types. In addition, studies weredone to determine the difference between local net- and spot-type populationswith regards to population structure and fungicide sensitivity. This dissertationtherefore, consists of a collection of separate publications and as a result acertain degree of redundancy has been unavoidable.Recombination is one of the most important evolutionary forcesinvolved with sexual reproduction. In plant-fungal agricultural ecosystems thismay result in pathogenic fungal populations adapting more rapidly to controlprograms such as fungicide applications. The first section of the review inpart 1 of this dissertation covers different aspects of sexual reproduction inascomycetes, specifically focussing on mating-type genes, vegetativeincompatibility and recombination. The major part of the review is thendedicated to various plant pathological aspects of P.teres, specificallyaddressing the differences between the two types, and in various caseshighlighting the significance of sexual recombination within and between thenet- and spot-type.Using morphological criteria for identification purposes there have beenmany conflicting reports concerning the identity of leaf spot isolates in theWestern Cape Province of South Africa. In part 2, the correct identity waseventually achieved employing mating studies and molecular markers .: Thiswas accomplished after single ascospores were obtained from pseudotheciaafter in vitro mating had occurred between a verified P. teres net-blotch isolatefrom Denmark and a representative Pyrenophora leaf spot isolate from SouthAfrica. Using amplified fragment length polymorphism (AFLP) and RAPDmarkers, recombination was demonstrated in the progeny that had DNAbanding patterns different from the two parental isolates. Pathogenicity trialsalso confirmed that recombination had taken place during mating.Inoculations were conducted on the differential cultivars susceptible to thenet-blotch and leaf spot forms. The two parents induced typical net-blotch orleaf spot symptoms whereas the progeny mostly induced a jagged spotsymptom on each cultivar. Fungicide sensitivity tests using the ergosterolbiosynthesis inhibitors showed that, due to recombination, some progenycould have increased resistance to these fungicides. Due to mating andsubsequent recombination between a net blotch isolate of P. teres and arepresentative leaf spot isolate, it was concluded that the latter was P. teres f.maculata.Fifteen of the net-spot hybrid progeny (F1) produced from the matingstudy in Part 2 were screened in Part 3 to assess their viability and geneticstability. Hybrid progeny (F1) inoculated onto barley seedlings consisting ofthe cultivars Stirling (differentially susceptible to net-type isolates), B87/14 andClipper (both differentially susceptible to spot-type isolates) producedintermediate symptoms on all cultivars. Axenic cultures (F1-1) isolated fromfoliar lesions, followed by repeated inoculation and isolation (F1-2) onto ahealthy set of seedlings produced similar intermediate symptoms. RAPDsconducted with two 1Q-mer primers on all isolates of F1-1and F1-2progenyrevealed profiles similar to those obtained for F1 isolates. RAPD moleculardata, therefore, indicated that hybrid progeny of this net x spot mating weregenetically stable after having been subjected to two repetitive inoculation andreisolation cycles. Phylogenetic analysis of DNA sequences of the internaltranscribed spacers (ITS1 and ITS2) flanking the 5.8S nuclear ribosomal RNAgene and the 5' end partial histone-3 gene confirmed the genetic stability ofthe hybrid progeny. These results also indicated that the hybrid progenyproduced consistent symptoms throughout the series of experiments, andmaintained their virulence to the differential cultivars screened.Both types of P. teres are prevalent in the south Western CapeProvince of South Africa, found on susceptible cultivars often grown withinclose proximity of each other. In Part 4, a net- and spot-type population werecharacterised in terms of their population structure using RAPD markers.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. A total of 65 loci were produced of which 54 werepolymorphic. Total gene diversities determined for all loci resulted in meanindices of 0.063 and 0.082 being obtained respectively for the net- and spottypepopulations. A coefficient of genetic differentiation (Gs) of 0.0149 wasobtained between sites within populations while a coefficient (GT) of 0.63 wasobtained between the two populations. Genotypic variation revealed 13distinct multilocus genotypes (haplotypes) in the net-type population whilethere were 12 in the spot-type population. UPGMA cluster analysis done onthe two populations together with six progeny from the mating between a netandspot-type isolate resulted in three main clusters being produced, one foreach population and one for the progeny. One isolate collected from the nettypepopulation also contained a unique spot-type RAPD fragment. Thissuggested that sexual recombination may be taking place between isolates ofthe net- and spot-type under field conditions.Fungicide application is the most important method used in the controlof net blotch in South Africa. In Part 5 the fungicide sensitivities (ICsD values)of 89 monoconidial isolates (46 net-type and 43 spot-type) of P. teres to steroldemethylation inhibiting fungicides were determined, based on the inhibitoryeffect on radial mycelial growth. The fungicides evaluated were triadimenol,bromuconazole, flusilazole, propiconazole and tebuconazole. Both net- andspot-type isolates revealed strong resistance to triadimenol while flusilazolewas shown to be the strongest inhibitor of fungal growth. Spot-type isolatesshowed a higher resistance than net-type isolates to all five fungicidesscreened. The ICsD values indicated significant differences between four of thefungicides (triadimenol, tebuconazole, flusilazole and propiconazole). TheICsD values between propiconazole and bromuconazole were not significant.This study suggested that spot-type isolates showed a higher degree ofresistance to commercially used fungicides than net-type isolates.The overall conclusion of this study is that the spot-type of P. teres isthe pathogen associated with leaf spots of barley in the south western Capeprovince of South Africa and not P. japonica as earlier reported. Togetherwith the net-type, both types exist as genetically variable populations in thisbarley production region. Mating between the two types results in sexual progeny that are genetically stable. This implies that barley fields adjacent toone another in which either net- or spot-type susceptible cultivars are beingcultivated may lead to sexual progeny being produced. This in turn may leadto an increased rate at which fungal populations may become resistant tocommercially used fungicides. It is furthermore suggested that an alternativefungicide seed treatment is used instead of triadimenol due to high resistanceof P. teres to this fungicide.

[发布机构] Stellenbosch University