RESUMO
Asian grapevine leaf rust, caused by Neophysopella meliosmae-myrianthae and N. tropicalis, is often controlled by quinone outside inhibitor (QoI) and demethylation inhibitor (DMI) fungicides in Brazil. Here, we evaluated the sensitivity of 55 Neophysopella spp. isolates to pyraclostrobin (QoI) and tebuconazole (DMI). To elucidate the resistance mechanisms, we analyzed the sequences of the cytochrome b (CYTB) and cytochrome P450 sterol 14α-demethylase (CYP51) target proteins of QoI and DMI fungicides, respectively. The CYP51 expression levels were also determined in a selection of isolates. In leaf disc assays, the mean 50% effective concentration (EC50) value for pyraclostrobin was about 0.040 µg/ml for both species. CYTB sequences were identical among all 55 isolates, which did not contain an intron immediately after codon 143. No amino acid substitution was identified at codons 129, 137, and 143. The mean EC50 value for tebuconazole was 0.62 µg/ml for N. tropicalis and 0.46 µg/ml for N. meliosmae-myrianthae, and no CYP51 sequence variation was identified among isolates of the same species. However, five N. meliosmae-myrianthae isolates grew on leaf discs treated at 10 µg/ml tebuconazole, and these were further exposed to tebuconazole selection pressure. Tebuconazole-adapted laboratory isolates of N. meliosmae-myrianthae showed an eight- to 25-fold increase in resistance after four rounds of selection that was not associated with CYP51 target alterations. In comparison with sensitive isolates, CYP51 expression was induced in the presence of tebuconazole in three out of four tebuconazole-adapted isolates tested. These results suggest a potential risk for QoI and DMI resistance development in Neophysopella spp.
Assuntos
Vitis , Citocromos b/genética , Íntrons/genética , Doenças das Plantas , Quinonas , EsteróisRESUMO
Downy mildew, caused by Plasmopara viticola, is the main disease affecting vineyards in subtropical Brazil. Here, we collected 94 P. viticola isolates from four organic and conventional vineyards in the two main grape-growing states of Brazil to evaluate the sensitivity to the quinone outside inhibitor (QoI) azoxystrobin by pheno- and genotyping assays. The impact of location, production system and sensitivity to QoI fungicides on the population genetics and structure of P. viticola was determined using 10 microsatellite markers. Cytochrome b sequencing revealed that 28 and 100% of the isolates from vineyards under organic and conventional management carried the G143A mutation, respectively. The G143A mutation was associated with high levels of azoxystrobin resistance. Three out of the 94 isolates analyzed carried the M125I alteration, not previously described in P. viticola, which was associated with a five-fold reduction in azoxystrobin sensitivity compared to wild-type isolates. Haplotype network analysis based on cytochrome b gene sequences suggested that the Brazilian populations are more closely related to the European than the North American population. A total of six haplotypes were identified, with two of them carrying the G143A mutation. Microsatellite analysis revealed high allelic and genotypic variation among the four populations. Population differentiation analyses indicated that state of origin directly influences the population biology of P. viticola, while production system and QoI sensitivity have little effect. Great genetic diversity, sexual reproduction and high levels of admixture were observed in Rio Grande do Sul State. In contrast, populations in São Paulo State were dominated by a few clonal genotypes, and no admixed genotype was detected between the two genetic pools identified in the state. This study raises the hypothesis that winter weather conditions influence the overwinter survival strategy with profound effects in the population biology of P. viticola.