Detection and molecular characterisation of Pyrenopeziza brassicae isolates resistant to methyl benzimidazole carbamates

BACKGROUND Methyl benzimidazole carbamate (MBC) fungicides are used to control the oilseed rape pathogen Pyrenopeziza brassicae. Resistance to MBCs has been reported in P. brassicae, but the molecular mechanism(s) associated with reductions in sensitivity have not been verified in this species. Elucidation of the genetic changes responsible for resistance, hypothesised to be target-site mutations in β-tubulin, will enable resistance diagnostics and thereby inform resistance management strategies. RESULTS P. brassicae isolates were classified as sensitive, moderately resistant or resistant to MBCs. Crossing P. brassicae isolates of different MBC sensitivities indicated that resistance was conferred by a single gene. The MBC-target encoding gene β-tubulin was cloned and sequenced. Reduced MBC sensitivity of field isolates correlated with β-tubulin amino acid substitutions L240F and E198A. The highest level of MBC resistance was measured for isolates carrying E198A. Negative cross-resistance between MBCs and the fungicides diethofencarb and zoxamide was only measured in E198A isolates. PCR-RFLP was used to screen isolates for the presence of L240F and E198A. The substitutions E198G and F200Y were also detected in DNA samples from P. brassicae populations after cloning and sequencing of PCR products. The frequencies of L240F and E198A in different P. brassicae populations were quantified by pyrosequencing. There were no differences in the frequencies of these alleles between P. brassicae populations sampled from different locations or after fungicide treatment regimes. CONCLUSIONS The molecular mechanisms affecting sensitivity to MBCs in P. brassicae have been identified. Pyrosequencing assays are a powerful tool for quantifying fungicide-resistant alleles in pathogen populations.

Alterations in predicted regulatory and coding regions of the sterol 14α-demethylase gene (CYP51) confer decreased azole sensitivity in the oilseed rape pathogen Pyrenopeziza brassicae

The incidence and severity of light leaf spot epidemics caused by the ascomycete fungus Pyrenopeziza brassicae on UK oilseed rape crops is increasing. The disease is currently controlled by a combination of host resistance, cultural practices and fungicide applications. We report decreases in sensitivities of modern UK P. brassicae isolates to the azole (imidazole and triazole) class of fungicides. By cloning and sequencing the P. brassicae CYP51 (PbCYP51) gene, encoding the azole target sterol 14α-demethylase, we identified two non-synonymous mutations encoding substitutions G460S and S508T associated with reduced azole sensitivity. We confirmed the impact of the encoded PbCYP51 changes on azole sensitivity and protein activity by heterologous expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a controllable promoter of native CYP51 expression. In addition, we identified insertions in the predicted regulatory regions of PbCYP51 in isolates with reduced azole sensitivity. The presence of these insertions was associated with enhanced transcription of PbCYP51 in response to sub-inhibitory concentrations of the azole fungicide tebuconazole. Genetic analysis of in vitro crosses of sensitive and resistant isolates confirmed the impact of PbCYP51 alterations in coding and regulatory sequences on a reduced sensitivity phenotype, as well as identifying a second major gene at another locus contributing to resistance in some isolates. The least sensitive field isolates carry combinations of upstream insertions and non-synonymous mutations, suggesting PbCYP51 evolution is on-going and the progressive decline in azole sensitivity of UK P. brassicae populations will continue. The implications for the future control of light leaf spot are discussed.

Effect of azole fungicide mixtures, alternations and dose on azole sensitivity in the wheat pathogen Zymoseptoria tritici

The evolution of fungicide resistance in the cereal pathogen Zymoseptoria tritici, is a serious threat to the sustainability and profitability of wheat production in Europe. Application of azole fungicides has been shown to affect fitness of Z. tritici variants differentially, so it has been hypothesised that combinations of azoles could slow the evolution of resistance. This work was initiated to assess the effects of dose, mixtures and alternations of two azoles on selection for isolates with reduced sensitivity and on disease control. Naturally infected field trials were carried out at six sites across Ireland and the sensitivity of Z. tritici isolates monitored pre- and post-treatment. The azoles epoxiconazole and metconazole were applied as solo products, in alternation with each other and as a pre-formulated mixture. Full and half label doses were tested. The two azoles were partially cross-resistant, with a common azole resistance principal component accounting for 75% of the variation between isolates. Selection for isolates with reduced azole sensitivity was correlated with disease control. Decreased doses were related to decreases in sensitivity but the effect was barely significant (P = 0.1) and control was reduced. Single applications of an active ingredient (a.i.) caused smaller decreases in sensitivity than double applications. Shifts in sensitivity to the a.i. applied to a plot were greater than to the a.i. not applied, and the decrease in sensitivity was greater to the a.i. applied at the second timing. These results confirm the need to mix a.i.s with different modes of action.

Farmers’ perceptions of the effectiveness of the Cocoa Disease and Pest Control Programme (CODAPEC) in Ghana and its effects on poverty reduction

The study examined the contribution of the Cocoa Disease and Pest Control Programme (CODAPEC), which is a cocoa production-enhancing government policy, to reducing poverty and raising the living standards of cocoa farmers in Ghana. One hundred and fifty (150) cocoa farmers were randomly selected from five communities in the Bibiani-Anhwiaso-Bekwai district of the Western Region of Ghana and interviewed using structured questionnaires. Just over half of the farmers (53%) perceived the CODAPEC programme as being effective in controlling pests and diseases, whilst 56.6% felt that their yields and hence livelihoods had improved. In some cases pesticides or fungicides were applied later in the season than recommended and this had a detrimental effect on yields. To determine the level of poverty amongst farmers, annual household consumption expenditure was used as a proxy indicator. The study found that 4.7% of cocoa farmers were extremely poor having a total annual household consumption expenditure of less than GH¢ 623.10 ($310.00) while 8.0% were poor with less than GH¢ 801.62 ($398.81). An amount of money ranging from GH¢ 20.00 ($9.95) to GH¢ 89.04 ($44.29) per annum was needed to lift the 4.7% of cocoa farmers out of extreme poverty, which could be achieved through modest increases in productivity. The study highlighted how agricultural intervention programmes, such as CODAPEC, have the potential to contribute to improved farmer livelihoods.

Detection of SDHI insensitivity in a Zymoseptoria tritici field population associated with the SdhC-H152R and SdhD-R47W substitutions

BACKGROUND: Succinate dehydrogenase inhibitor fungicides are important in the management of Zymoseptoria tritici in wheat. New active ingredients from this group of fungicides have been introduced recently and are widely used. Because the fungicides act at a single enzyme site, resistance development in Z. tritici is classified as medium-to-high risk. RESULTS: Isolates from Irish experimental plots in 2015 were tested against the SDHI penthiopyrad during routine monitoring. The median of the population was approximately 2 x less sensitive than the median of the baseline population. Two of the 93 isolates were much less sensitive to penthiopyrad than least sensitive of the baseline isolates. These isolates were also insensitive to most of commercially available SDHIs. Analysis of the succinate dehydrogenase coding genes confirmed the presence of the substitutions SdhC-H152R and SdhD-R47W in the very insensitive isolates. CONCLUSION: This is the first report showing that the SdhC-H152R mutation detected in laboratory mutagenesis studies also exists in the field. The function and relevance of this mutation, combined with SdhD-R47W, still needs to be determined.

Proposal for a unified nomenclature for target site mutations associated with resistance to fungicides

Evolved resistance to fungicides is a major problem limiting our ability to control agricultural, medical and veterinary pathogens and is frequently associated with substitutions in the amino acid sequence of the target protein. The convention for describing amino-acid substitutions is to cite the wild type amino acid, the codon number and the new amino acid, using the one letter amino acid code. It has frequently been observed that orthologous amino acid mutations have been selected in different species by fungicides from the same mode of action class, but the amino acids have different numbers. These differences in numbering arise from the different lengths of the proteins in each species. The purpose of the current paper is to propose a system for unifying the labelling of amino acids in fungicide target proteins. To do this we have produced alignments between fungicide target proteins of relevant species fitted to a well-studied “archetype” species. Orthologous amino acids in all species are then assigned numerical “labels” based on the position of the amino acid in the archetype protein.