Carboxylic Acid Amide Fungicides Research Articles

The cellulose synthase 3 (CesA3) gene of oomycetes: structure, phylogeny and influence on sensitivity to carboxylic acid amide (CAA) fungicides

Proper disease control is very important to minimize yield losses caused by oomycetes in many crops. Today, oomycete control is partially achieved by breeding for resistance, but mainly by application of single-site mode of action fungicides including the carboxylic acid amides (CAAs). Despite having mostly specific targets, fungicidal activity can differ even in species belonging to the same phylum but the underlying mechanisms are often poorly understood.In an attempt to elucidate the phylogenetic basis and underlying molecular mechanism of sensitivity and tolerance to CAAs, the cellulose synthase 3 (CesA3) gene was isolated and characterized, encoding the target site of this fungicide class. The CesA3 gene was present in all 25 species included in this study representing the orders Albuginales, Leptomitales, Peronosporales, Pythiales, Rhipidiales and Saprolegniales, and based on phylogenetic analyses, enabled good resolution of all the different taxonomic orders. Sensitivity assays using the CAA fungicide mandipropamid (MPD) demonstrated that only species belonging to the Peronosporales were inhibited by the fungicide. Molecular data provided evidence, that the observed difference in sensitivity to CAAs between Peronosporales and CAA tolerant species is most likely caused by an inherent amino acid configuration at position 1109 in CesA3 possibly affecting fungicide binding. The present study not only succeeded in linking CAA sensitivity of various oomycetes to the inherent CesA3 target site configuration, but could also relate it to the broader phylogenetic context.

Method for rapid detection of the PvCesA3 gene allele conferring resistance to mandipropamid, a carboxylic acid amide fungicide, in Plasmopara viticola populations

The occurrence of carboxylic acid amide (CAA)-fungicide-resistant Plasmopara viticola populations is becoming a serious problem in the control of grapevine downy mildew worldwide. The authors have developed a method, which utilises PCR-RFLP, for the rapid detection of resistance to the CAA fungicide mandipropamid in P. viticola populations. With this method, a glycine-to-serine substitution at codon 1105 of the cellulose synthase gene PvCesA3 of CAA-fungicide-resistant P. viticola was easily detected, although no resistant P. viticola was detected from 398 isolates in Japan. It is proposed that the PCR-RFLP method is a reliable tool for the rapid detection of CAA-fungicide-resistant P. viticola isolates. Only 4 h was required from the sampling of symptoms to the phenotyping of fungicide resistance.

Biological and genetic characterization of Phytophthora capsici mutants resistant to flumorph

Baseline sensitivity to flumorph, a carboxylic acid amide (CAA) fungicide used to control some oomycetes, was examined using 83 Phytophthora capsici isolates, resulting in a unimodal distribution of effective concentration for 50% inhibition of mycelial growth ranging from 0·716 to 1·363, with a mean of 1·033 ± 0·129 μg mL−1. To assess the potential risk of developing flumorph resistance, 13 flumorph‐resistant mutants of P. capsici were obtained using ultraviolet irradiation. Most of these mutants and their progeny had high levels of fitness, including mycelial growth, sporulation and virulence. The resistance to flumorph changed slightly, either increasing or decreasing, after 10 transfers on agar media. Cross‐resistance was found between flumorph and other CAA fungicides (dimethomorph and iprovalicarb), but not between flumorph and non‐CAA fungicides (cymoxanil, metalaxyl, azoxystrobin and cyazofamid). To investigate the genetics of the flumorph resistance, 619 progeny were obtained by self‐crossing and sexual hybridization. Segregation of sensitivity to fungicide was measured as a ratio of sensitive (S) to resistant (R) isolates. Segregation of the progeny, from self‐crossed isolate PCAS1 (flumorph resistant), was 1:15 in the first generation; and 0:1 or 1:15 in the second generation. In sexual hybridization, segregation of progeny was 0:1 and 1:7 for R × R hybridization; and 1:3 for R × S hybridization. Therefore, the resistance of P. capsici against flumorph was controlled by two dominant genes.

Baseline and differential sensitivity to mandipropamid among isolates of Peronophythora litchii, the causal agent of downy blight on litchi

Litchi downy blight caused by Peronophythora litchii is a devastating disease of litchi plants in China. Control of litchi downy blight requires numerous fungicide applications. A new carboxylic acid amide (CAA) fungicide, mandipropamid, was examined for its in vitro effects on multiple asexual stages of four single-sporangium P. litchii isolates and protective activity against downy blight in detached fruit assays. Though mandipropamid did not affect discharge of zoospores from sporangia, it strongly inhibited mycelial growth (mean EC50=0.0048μgml−1), sporangia production (mean EC50=0.0032μgml−1), germination of encysted zoospores (mean EC50=0.0023μgml−1), and germination of sporangia (mean EC50=0.0061μgml−1). On detached fruit, 0.39, 1.56 and 6.25μgml−1 of mandipropamid were superior in reducing downy blight compared to metalaxyl and flumorph, however, the 25μgml−1 application rate was necessary for all three CAA fungicides to completely inhibit the disease. In 2007, 100 isolates from Fujian, Guangdong, and Guangxi Provinces of China were characterized for the baseline sensitivity to mandipropamid. The isolates obtained from different provinces showed similar baseline sensitivities to mandipropamid. Baseline sensitivities formed a unimodal curve with mean EC50 values of 0.0055±0.0012μgml−1 for inhibition of mycelial growth. The described baseline sensitivities of P. litchii populations will be useful for monitoring possible shifts in sensitivity to mandipropamid.

EMS and UV irradiation induce unstable resistance against CAA fungicides in Bremia lactucae

Wild type (WT) field isolates of Bremia lactucae failed to germinate in vitro or infect lettuce leaves in the presence of CAA (carboxylic acid amide) fungicides. Minimal inhibitory concentrations (MIC) for mandipropamid, dimethomorph, benthiavalicarb and iprovalicarb were 0.005, 0.5, 0.5 and 5 μg ml−1, respectively. Mutagenesis experiments showed that spores exposed to EMS (ethyl methane sulphonate) or UV irradiation (254 nm) could infect lettuce leaves in the presence of up to 100 μg ml−1 CAA. The proportion of infected leaves relative to the number of spores inoculated (infection frequency) was inversely related to the concentration of CAA used, ranging between 0 and 160 per 1 × 106 spores. Resistant mutants (RM) lost their resistance within 1–14 reproduction cycles on CAA-treated plants. Crosses were made between RMxWT isolates and RMxRM isolates with an attempt to obtain stable homozygous resistant off-springs. Such crosses yielded few resistant but unstable progeny isolates. Mutagenic treatments given to hybrid isolates also failed to produce stable resistance. Previous gene sequencing data showed that stable resistance to CAAs is based on a single SNP in the cellulose synthase 3 (CesA3) gene of Plasmopara viticola. Therefore, we sequenced a 582 bp DNA fragment of Ces3A of WT, RM and hybrid isolates of B.lactucae. No mutation in this gene fragment was found. We conclude that mutagenic agents like EMS or UV may induce resistance to CAA in Bremia lactucae but this resistance is not stable and not linked to mutations in CesA3 gene.

A single point mutation in the novel PvCesA3 gene confers resistance to the carboxylic acid amide fungicide mandipropamid in Plasmopara viticola

The grapevine downy mildew, Plasmopara viticola, is one of the most devastating pathogens in viticulture. Effective control is mainly based on fungicide treatments, although resistance development in this pathogen is reported for a number of fungicides. In this study we describe for the first time the molecular mechanism of resistance to a carboxylic acid amide (CAA) fungicide. We identified a family of four cellulose synthase ( CesA) genes containing conserved domains that are found in all processive glycosyltransferases. Phylogenetic analysis revealed their close relationship to the cellulose synthases of Phytophthora sp. Sequencing of the CesA genes in a CAA- resistant and -sensitive field isolate revealed five single nucleotide polymorphisms (SNPs) affecting the amino acid structure of the proteins. SNP inheritance in F 1-, F 2- and F 3-progeny confirmed resistance to be correlated with one single SNP located in PvCesA3. Only if present in both alleles, this SNP led to the substitution of a glycine for a serine residue at position 1105 (G1105S) in the deduced amino acid sequence, thus conferring CAA- resistance. Our data demonstrate that the identified genes are putative cellulose synthases and that one recessive mutation in PvCesA3 causes inheritable resistance to the CAA fungicide mandipropamid.

Baseline Sensitivity to Mandipropamid Among Isolates of Phytophthora capsici Causing Phytophthora Blight on Pepper

Sensitivity to the new carboxylic acid amide fungicide, mandipropamid, of Phytophthora capsici causing pepper Phytophthora blight was determined on 187 isolates collected in Korea over 3 years, from 2005 to 2007. All isolates were sensitive to mandipropamid, with EC50 values for growth of mycelia ranging from 0.001 to 0.037 μg/ml. Among the isolates, 147 (79.0%) isolates were sensitive to metalaxyl, whereas others were resistant to this fungicide. Mandipropamid had the same effect on mycelium growth of both metalaxyl-sensitive and metalaxyl-resistant isolates, indicating an absence of cross-resistance between these two fungicides. Comparison of the sensitivities of P. capsici isolates showed a positive correlation between sensitivity to mandipropamid and dimethomorph (r2=0.8533). The results of this study indicate that there is no evidence for development of resistance to mandipropamid in this population of P. capsici isolates collected in Korea.

Sensitivity of Chinese Isolates of Plasmopara viticola to Metalaxyl and Dimethomorph

During 2007 and 2008, 392 isolates of Plasmopara viticola were collected from 11 regions in seven provinces in China, and their sensitivities to metalaxyl and dimethomorph were determined by the floating leaf disk technique. Among all isolates, 13% were classified as sensitive, 26% as low-level resistant, and 61% as resistant to metalaxyl. Of the 392, 85 were from vineyards never treated with carboxylic acid amide fungicides; these isolates were used to determine the baseline sensitivity to dimethomorph, and their EC50 values ranged from 0.01 to 0.21 (mean ± SD, 0.11 ± 0.04) μg/ml. The other 307 isolates were completely inhibited by a single discriminatory dose of 1.6 μg/ml of dimethomorph.

Baseline and differential sensitivity of Peronophythora litchii (lychee downy blight) to three carboxylic acid amide fungicides

Downy blight, caused by Peronophythora litchii, is an important disease of lychee (litchi) plants in China. The in vitro sensitivities of various asexual stages of P. litchii to the three carboxylic acid amide (CAA) fungicides dimethomorph, flumorph and pyrimorph were studied with four single‐sporangium isolates. None of the three fungicides affected zoospore discharge from sporangia, but they strongly inhibited mycelial growth (mean EC50 values of 0·075, 0·258 and 0·115 mg L−1, respectively); sporangial production (mean EC50 values of 0·085, 0·315 and 0·150 mg L−1, respectively); germination of cystospores (mean EC50 values of 0·140, 0·150 and 0·645 mg L−1, respectively); and germination of sporangia (mean EC50 values of 0·203, 0·5 and 0·743 mg L−1, respectively). As mycelial growth was the most sensitive stage to dimethomorph and pyrimorph, it was chosen to test baseline sensitivities to the three fungicides. In 2007, from 131 isolates collected in Fujian, Guangdong and Guangxi provinces, 127, 116 and 113 isolates were used to establish baseline sensitivity for dimethomorph, flumorph and pyrimorph respectively. Isolates from different provinces exhibited similar baseline sensitivity to the same fungicide. Baseline sensitivities to dimethomorph, flumorph and pyrimorph were distributed as unimodal curves, with mean EC50 values of 0·082 (± 0·01), 0·282 (± 0·047), and 0·115 (± 0·032) mg L−1, respectively. This information will serve as a baseline for tracking future changes in sensitivities of P. litchii populations to these three CAA fungicides.

Carboxylic acid amide (CAA) fungicide resistance prevention strategy

CAA fungicides are currently represented in New Zealand by the three active ingredients dimethomorph iprovalicarb and mandipropamid They are currently registered in New Zealand for use against oomycete fungi in grapes onions potatoes and lettuce These fungicides are considered to have low to medium risk of resistance development and resistance management is recommended by the Fungicide Resistance Action Committee (FRAC) in Europe Guidelines are presented for fungicide use that will avoid or delay the development of resistance to CAA fungicides

Activity of carboxylic acid amide (CAA) fungicides against Bremia lactucae

Four carboxylic acid amide (CAA) fungicides, mandipropamid (MPD), dimethomorph (DMM), benthiavalicarb (BENT) and iprovalicarb (IPRO) were examined for their effects on various developmental stages of Bremia lactucae, the causal agent of downy mildew in lettuce, in vitro and in planta. Spore germination in vitro or on leaf surfaces was inhibited by all CAA fungicides (technical or formulated). MPD was more effective in suppressing germination than DMM or BENT, whereas IPRO was least effective. CAA induced no disruption of F-actin microfilament organisation in germinating spores of B. lactucae. CAA applied to germinating spores in vitro prevented further extension of the germ tubes. When applied to germinated spores on the leaf surface they prevented penetration. Preventive application of CAA to intact plants inhibited infection. MPD was more effective in suppressing infection than DMM or BENT, whereas IPRO was least effective. Curative application was effective at ≤3 h post-inoculation (hpi) but not at ≥18 hpi. CAA (except IPRO) applied to upper leaf surfaces inhibited spore germination on the lower surface and hence reduced infection. CAA suppressed sporulation of B. lactucae on floating leaf discs and when sprayed onto infected plants two days before onset of sporulation. BENT and DMM were more effective in suppressing sporulation than MPD or IPRO. Epidemics of downy mildew in shade-house grown lettuce were suppressed by CAA. A single spray applied to five-leaf plants before transplanting controlled the disease for 50 days. The results suggest that CAA are effective inhibitors of spore germination and therefore should be used as preventive agents against downy mildew of lettuce caused by B. lactucae.

Inheritance of resistance to carboxylic acid amide (CAA) fungicides in Plasmopara viticola

Mandipropamid is a new mandelic acid amide fungicide expressing high activity against foliar infecting oomycetes, including the grapevine downy mildew, Plasmopara viticola. Because cross‐resistance with the valinamide fungicides iprovalicarb and benthiavalicarb and the cinnamic acid amide fungicides dimethomorph and flumorph was postulated, all five compounds are classified as carboxylic acid amide (CAA) fungicides. To support this classification, cross‐resistance among these compounds with field isolates and the segregation of resistance in F1 and F2 progeny of P. viticola were evaluated. A bimodal distribution of sensitivity in field isolates and cross‐resistance among all CAAs for the vast majority of isolates were detected. Crosses between sensitive (s) and CAA‐resistant (r) isolates of opposite mating types, P1 and P2, yielded abundant oospores. All F1‐progeny isolates were sensitive to CAAs (s:r segregation 1:0), whereas in F2 progeny segregation of about 9:1 (s:r) was observed suggesting that resistance to CAA fungicides is controlled by two recessive nuclear genes. Mating type segregated in a ratio P1:P2 of c. 2:1 in F1 and 1:1 in F2 progeny. In the same crosses, resistance to the phenylamide fungicide mefenoxam segregated in a ratio of c. 1:3:2 (sensitive:intermediate:resistant), reflecting the monogenic, semidominant nature of resistance. The risk of resistance in P. viticola was classified as high for phenylamide and moderate for CAA fungicides. This is the first report on the inheritance of phenotypic traits in P. viticola.