In order to determine whether a given isolate of Pseudocercosporella herpotrichoides. the cause of eyespot disease of cereals, belongs to the W‐type or the R‐type, we have developed a test based on the polymerase chain reaction (PCR). Sequences of the internal transcribed spacer (ITS) region of the ribosomal genes were determined for three different isolates of each pathotype. Primers complementary to variable sequences were specifically chosen to amplify differentially DNA from W‐ and R‐types. Specific reaction conditions of the PCR assay were experimentally determined by testing four isolates of each type that exhibit a wide range of sensitivity to fungicides.
Using the ELISA method, the development of Pseudocercosporella herpotrtchoides var. herpotrichoides and var. acuformis in a susceptible cultivar of winter wheat was compared under controlled and held conditions. In the greenhouse, var. acufornis grew less vigorously, was slower in penetrating the coleoptile and the successive leaf sheaths and in colonizing the stem tissue than var. herpotrichoides. In the field, these differences were confirmed on the last leaf sheaths and the stem. At ripening stage, however, identical ELISA values were measured for both varieties. Moreover, a significant variation was observed between the individual isolates of each variety.
Comparison of the effect of both varieties of P. herpotrichoides on 20 wheat cultivars characterized by different resistance levels showed significant interactions. The cultivars carrying the Pch‐1 gene always remained the less diseased genotypes. In general, var. acuformis developed less antigen in the cultivars than var. herpotrichoides. It is concluded, that in tests for resistance to P. herpotrichoides mixtures of many highly pathogenic isolates of both fungus varieties should be used. Less complex mixtures or single isolates may result in wrong estimates of resistance.
Quantitative PCR and visual monitoring of Mycosphaerella graminicola epidemics were performed to investigate the effect of curative and preventative applications of azoxystrobin in wheat field crops. A non-systemic protectant and a systemic curative fungicide, chlorothalonil and epoxiconazole, respectively, were used as references. PCR diagnosis detected leaf infection by M graminicola 3 weeks before symptom appearance, thereby allowing a clear distinction between curative and preventative treatments. When applied 1 week after the beginning of infection, azoxystrobin curative activity was intermediate between chlorothalonil (low effect) and epoxiconazole. When applied preventatively, none of the fungicides completely prevented leaf infection. There was some indication that azoxystrobin preventative treatments may delay fungal DNA increase more than epoxiconazole at the beginning of leaf infection. Both curative and preventative treatments increased the time lapse between the earliest PCR detection and the measurement of a 10% necrotic leaf area. Azoxystrobin only slightly decreased the speed of necrotic area increase compared with epoxiconazole. Hence, azoxystrobin activity toward M graminicola mainly resides in lengthening the time lapse between the earliest PCR detection and the measurement of a 10% necrotic leaf area. Information generated in this way is useful for optimal positioning of azoxystrobin treatments on M graminicola.
Nirenberg's classification system and the polymerase chain reaction (PCR) combined with restriction enzyme digestion of an amplified ribosomal DNA fragment, were compared for the characterization of sixty isolates of Pseudocercosporella herpotrichoides, from various geographical areas and with differing fungicide sensitivity. With Nirenberg's system, it was possible to identify most isolates as P. herpotrichoides var. herpotrichoides or P. herpotrichoides var. acuformis. However, identification was slow and sometimes inconclusive as overlap occurred between the two varieties for all criteria examined. Molecular markers identified two distinct types among the isolates tested and generally good correlation was found between the PCR-based assay and Nirenberg's system, but the molecular assay was more accurate and faster.
Green fluorescent protein (GFP)-expressing transformants were used to investigate the effects of strobilurin fungicide azoxystrobin on Mycosphaerella graminicola infection. Azoxystrobin treatments (125 or 250 g AI ha-1) were applied at various stages of the infection process under controlled conditions. GFP transformants showed conserved in vitro sensitivity to azoxystrobin and pathogenicity. Azoxystrobin controlled over 90% of M graminicola infections when applied before or during penetration of the pathogen (15% of the incubation phase). Azoxystrobin also impaired the growth of intercellular hyphae in M graminicola post-penetration infection stages when applied at up to 50% of the incubation phase. Incubating infections observed in treated leaves were viable, but their growth was impaired and they did not induce necrosis under controlled conditions. Reduction by half of azoxystrobin dosage had little or no effect on azoxystrobin efficiency in controlling M graminicola. The contribution of post-penetration fungistatic effect to azoxystrobin curative properties toward M graminicola in a field situation is discussed.
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