Carboxylic Acid Amide Fungicides Research Articles

Triphenylphosphonium-Driven Targeting of Pyrimorph Fragment Derivatives Greatly Improved Its Action on Phytopathogen Mitochondria.

Pyrimorph is a carboxylic acid amide (CAA) fungicide, which shows excellent activity against oomycetes such as pepper phytophthora blight, tomato late blight, and downy mildew of cucumber. It works mainly by inhibiting the biosynthesis of cell wall of oomycetes. However, pyrimorph also shows weak activity of inhibiting mitochondrial complex III, which is the first CAA fungicide found to act on mitochondria. To improve this effect on mitochondria and develop fungicides that may have a novel mechanism of action, in this paper, by disassembling pyrimorph and conjugating the fragments with the mitochondrial-targeted delivery system (triphenylphosphonium), three series of mitochondrial-targeting analogues of pyrimorph were designed and synthesized. The results show that the pyridine-containing 1,1-diaryl is the core module of inhibition mitochondrial function of pyrimorph. Among these conjugates, compound 3b with a short linker showed the highest and broad-spectrum fungicidal activity, strong respiratory inhibition activity, and adenosine 5'-triphosphate synthesis inhibition activity, suggesting its potential as a fungicide candidate. 3b exhibited greatly improved action on mitochondria, such as by destroying the mitochondrial function of pathogens, causing mitochondrial swelling, weakening its influence on cell wall morphology, and so on. More importantly, this study provides a method to strengthen the drugs or pesticides with weak mitochondrial action, which is of special significance for developing mitochondrial bioactive molecules with the novel action mechanism.

PcCesA1 is involved in the polar growth, cellulose synthesis, and glycosidic linkage crosslinking in the cell wall of Phytophthora capsici

Phytophthora capsici is a destructive plant pathogen that infects a wide range of hosts worldwide. The P. capsici cell wall, rich in cellulose, is vital for hyphal growth and host interactions. However, the enzymes involved in its synthesis remain largely unelucidated. In the current study, we functionally characterized the cellulose synthase gene PcCesA1, which is highly conserved in Phytophthora. By using CRISPR/Cas9-mediated gene replacement and in situ complementation system, it was found PcCesA1 is essential for the mycelial growth, cystospore germination, and pathogenicity of P. capsici. The normal deposition of newly synthesized cell wall components and the polar growth point formation were disrupted in PcCesA1 knockout mutants, suggesting that PcCesA1 plays an important role in the polar growth of P. capsici. Compared with the wild-type strains, PcCesA1 knockout mutants displayed a thicker inner layer cell wall and were more sensitive to carboxylic acid amide fungicides (CAAs). The contents of the cell wall polysaccharides 1,4-Glc, 1,4,6-Glc, and 1,3,4-Glc were reduced in PcCesA1 knockout mutants, suggesting that PcCesA1 affected cellulose content and glycosidic linkage crosslinking in the cell wall. Our findings demonstrate that PcCesA1 is required for cell wall biogenesis. Therefore, PcCesA1 may be a potential target for Phytophthora disease control.

Multiple mutations in the predicted transmembrane domains of the cellulose synthase 3 (CesA3) of Phytophthora capsici can confer semi-dominant resistance to carboxylic acid amide fungicides

The current study established a clearer understanding of the molecular basis for resistance to carboxylic acid amide (CAA) fungicides. Although four cellulose synthase (CesA) genes were investigated, only F1073L, G1105A, V1109L in CesA3 were found to link to CAA-resistance in Phytophthora capsici. Back-transformation experiments confirmed the role of the three mutations in CAA-resistance. Inheritance studies also confirmed the link and indicated the resistance was semi-dominant with the heterozygous F1 and F2 progeny exhibiting intermediate resistance levels compared to the homozygous parents, which was validated by the pyrosequencing results. The semi-dominant nature of CAA-resistance implies that it could be easy for resistance to spread once resistance emerged, being facilitated by both sexual and asexual reproduction. Bioinformatic analysis indicated all mutations occurred in either the first or second of the predicted transmembrane domains at C-terminus of CesA3. Resistant isolates bearing different combinations of mutations were found to exhibit different resistance levels to different CAAs, indicating that each mutation could make different contributions to resistance phenotype depending on structural differences in different CAAs. The current results highlight the complex combinations of mutations and resistance phenotype, and further reinforces the research necessity to completely characterize CAA-resistance to develop appropriate strategies to manage resistance development.

Stepwise accumulation of mutations in CesA3 in Phytophthora sojae results in increasing resistance to CAA fungicides.

Flumorph is a carboxylic acid amide (CAA) fungicide with high activity against oomycetes. However, evolution to CAAs from low resistance to high resistance has never been reported. This study investigated the basis of resistance evolution of flumorph in Phytophthora sojae. Total of 120 P. sojae isolates were collected and their sensitivity to flumorph was evaluated. Although no spontaneous resistance was found among the field isolates, adaptation on flumorph‐amended media resulted in the selection of five stable mutant types exhibiting varying degrees of resistance to CAAs. Type I, which exhibited the lowest resistance level, was obtained when the wild‐type isolate was exposed to a low concentration of flumorph, but no resistant mutants were obtained by direct exposure to higher concentrations. However, the more resistant types (Type II, III, IV and V) were obtained when Type I were exposed to higher concentrations of flumorph. Similar results were obtained when the entire screening process was repeated, which implied that evolution of resistance to flumorph in P. sojae could be a two‐step process, where high resistance phenotypes could develop gradually from low resistance ones. Further investigation into molecular mechanism strongly confirmed that evolution of isolates highly resistant to flumorph occurs in a stepwise process with Type I as intermediary, through accumulation of mutations in their target protein of CAAs (CesA3). Together, our findings indicate that application of low rates of flumorph in field could result in selection of low resistance Type I isolates, but that raising dosage to maintain comparable levels of control could elicit rapid evolution of more resistant Type II, III, IV and V isolates with stepwise accumulation of mutations in CesA3, which would render flumorph ineffective as a control method. Precautionary resistance management strategy should be implemented. The phenomenon described in the study could have broader biological significance.

Quarternary α-cyanobenzylsulfonamides: A new subclass of CAA fungicides with excellent anti-Oomycetes activity

A bioisosteric carboxamide - sulfonamide replacement explored during the optimization of an insecticide lead compound led to the surprising discovery of a formerly unknown subclass of the Carboxylic Acid Amide (CAA) fungicides, which is the very first CAA fungicide group without a carboxamide function. In this paper we present invention pathway, racemic and stereoselective synthesis routes, structure–activity relationship studies as well as resistance profile of this novel family of fungicides.

Efficacy of fungicide treatments for Plasmopara viticola control and occurrence of strobilurin field resistance in vineyards in Georgia, USA

Grapevine downy mildew, caused by Plasmopara viticola, is a major disease of cultivated wine grapes in Georgia (USA), where the climate is favorable for rapid epidemic development. Although loss of fungicide sensitivity has been documented for P. viticola populations in other viticultural regions, it had not been confirmed previously in Georgia. Downy mildew samples from three vineyards, two managed commercially and one located at a research station, were subjected to polymerase chain reaction (PCR) assays to identify the presence of known mutations conferring quinone outside inhibitor (QoI) and carboxylic acid amide (CAA) fungicide resistance. PCR assays detected the presence of the G143A mutation which confers resistance to the QoI fungicides in 100% of samples (n = 39) and the absence of the G1105S mutation that confers resistance to CAA fungicides at all three locations. To determine fungicide efficacy, field trials were conducted in these same vineyards with 10 oomycete-active fungicides or combinations. A significant reduction in efficacy of QoI fungicides (azoxystrobin and pyraclostrobin), with disease levels statistically equivalent to those of untreated vines, was observed. Based on the presence of the G143A mutation that confers resistance to QoIs and the associated field disease control failures observed where QoIs were applied, this fungicide class may no longer be effective for downy mildew management in many Georgia vineyards.

Multiple resistance of Plasmopara viticola to QoI and CAA fungicides in Brazil

AbstractGrapevine downy mildew, caused by Plasmopara viticola, is commonly controlled by quinone outside inhibitor (QoI) and carboxylic acid amide (CAA) fungicides. QoI resistance is conferred by a mutation resulting in a G143A alteration in cytochrome b, while CAA resistance is associated with a G1105S mutation in cellulose synthase 3. The aims of this study were to (a) verify the presence of QoI and CAA resistance in P. viticola in Brazil, (b) develop a single nucleotide polymorphism (SNP) detection pyrosequencing assay for quantitative detection of G143A, and (c) determine the geographic prevalence and distribution of G143A and G1105S in Brazilian field populations. P. viticola isolates were sampled from two vineyards reporting failures of QoI and CAA control. Their growth was unaffected by azoxystrobin and mandipropamid, and resistance was associated with the G143A and G1105S mutations, respectively. The SNP detection pyrosequencing assay developed for G143A detection was precise and accurate. Pyrosequencing assays revealed that G143A and G1105S frequencies varied among the 66 field populations and were positively correlated with the number of QoI and CAA applications, respectively. The G143A mutation was observed in 60 populations, 39 of them with frequencies over 90%. G1105S was detected in 20 populations with frequencies ranging from 18.3% to 100%, and which also carried the G143A mutation. Multiple resistance to QoI and CAA in P. viticola was confirmed for the first time in South America. Future resistance monitoring surveys and management strategies must be adopted to prevent further increase in QoI and CAA resistance in Brazil.

Detection and Characterization of Carboxylic Acid Amide-Resistant Plasmopara viticola in China Using a TaqMan-MGB Real-Time PCR.

Grape production is increasing globally and so are problems with downy mildew, one of the main constraints in grape production. Downy mildew on grape is caused by Plasmopara viticola, an obligate biotrophic pathogen belonging to the oomycetes. Control of the disease is usually performed by fungicide applications, of which carboxylic acid amide (CAA) fungicides represent one of the most widely used groups of fungicides. Our previous research showed that the extensive application of CAA fungicides can result in fungicide resistance and in China, CAA-resistant isolates of P. viticola were collected from the field in 2014. To monitor the distribution and spread of CAA fungicide resistance, we developed a TaqMan-minor groove binder (MGB) real-time PCR-based method designed on a functional mutation in the PvCesA3 gene that allows efficient identification of CAA fungicide resistant and sensitive genotypes. The assay was validated on 50 isolates using Sanger sequencing and fungicide bioassays and exploited in a comprehensive survey comprising 2,227 single-sporangiophore isolates from eight major grapevine regions in China. We demonstrate that CAA fungicide resistance in P. viticola is widespread in China. On average, 53.3% of the isolates were found to be resistant, but marked differences were found between locations with percentages of resistant isolates varying from 0.3 to 96.6%. Furthermore, the frequency of CAA-resistant isolates was found to be significantly correlated with the exposure to CAA fungicides (P < 0.05). We further discussed the possibilities to apply the TaqMan-MGB real-time PCR assay to assess the frequency of fungicide-resistant P. viticola isolates in each region or vineyard, which would facilitate the correct choice of fungicide for grape downy mildew and resistance management strategies.

Fungicide Sensitivity Survey of Plasmopara viticola Populations in Georgia Vineyards

Grapevine downy mildew, caused by Plasmopara viticola, is among the most damaging diseases of grapes globally and in the viticultural regions of Georgia (U.S.A.). Although management of this disease typically involves fungicide applications, resistance development in P. viticola can render chemical management ineffective. The objective of this study was to survey fungicide sensitivities of P. viticola populations in vineyards across three regions in Georgia. Samples were collected from eight vineyards in 2017 and four in 2018 and tested phenotypically for sensitivity to quinone outside inhibitor (QoI), carboxylic acid amide (CAA), and phenylamide (PA) fungicides using leaf disc bioassays. In addition, DNA was extracted from P. viticola samples collected from 2015 through 2018 in these 12 vineyards and six others for a total of 18 vineyards sampled. All available DNA samples were tested for the presence of the known resistance-causing mutations G143A (QoI) and G1105S (CAA) using polymerase chain reaction (PCR). This study documented widespread occurrence of QoI resistance in P. viticola in Georgia, with 83.0% of isolates collected in 2017 and 2018 testing positive for QoI resistance based on bioassay and 82.9% of isolates collected from 2015 to 2018 testing positive for QoI resistance through PCR testing. In total, 94.4% (17/18) of surveyed vineyards had confirmed QoI resistance by the conclusion of the survey period. No reduced sensitivity to CAA or PA fungicides was identified. Due to widespread QoI resistance, these fungicides should not be relied upon for downy mildew management in Georgia vineyards with a long history of their use.

Low evolutionary risk of iprovalicarb resistance in Phytophthora infestans

Iprovalicarb is a carboxylic acid amide (CAA) fungicide, highly effective in the control of potato late blight, causing by Phytophthora infestans. Due to cross-resistance with other CAA fungicides and moderate resistance risk of P. capsici to iprovalicarb, the evolutionary risk of P. infestans resistance to this fungicide and the contribution of inherited genes and environmental effect was evaluated using a common garden experiment. The results showed that the ratio of heritability and plasticity of iprovalicarb in the seven populations equaled 1.0, indicating both inherited genes and environmental factors were essential to iprovalicarb sensitivity in P. infestans. The pairwise population differentiation determined by SSR loci (FST) between populations ranged from 0.007 to 0.133 and the overall FST was significantly higher than population differentiation in RGR (QST), suggesting constraining selection acting on iprovalicarb sensitivity. We also found a new indicator of growth rate inhibition (GRI) for fungicide sensitivity, which was negatively correlated to growth rate in the absence of iprovalicarb, indicating a trade-off between iprovalicarb resistance and pathogen's growth. The constraining selection plus a trade-off between GRI and growth rate revealed low risk of P. infestans evolving resistance to iprovalicarb.

First Report of Carboxylic Acid Amide Fungicide Resistance in Plasmopara viticola (Grapevine Downy Mildew) in North America

This report documents the first known occurrence in North America of resistance in grape downy mildew (Plasmopara viticola) to the carboxylic acid amide (CAA) fungicides mandipropamid and dimethomorph. These fungicides (FRAC group 40) have been an important component of downy mildew management programs for the past decade. Resistant isolates were obtained at three locations in Virginia and one in North Carolina, at considerable distances from each other. Resistance was documented by bioassay and the presence of the G1105S mutation, which has been associated with CAA resistance of P. viticola in other areas. Further survey is needed to determine the geographic extent of this resistance.

Tetra-primer ARMS PCR for rapid detection and characterisation of Plasmopara viticola phenotypes resistant to carboxylic acid amide fungicides.

The occurrence of Plasmopara viticola populations resistant to carboxylic acid amide (CAA) fungicides is becoming a serious problem in the control of grapevine downy mildew worldwide. The resistance is caused by point mutations in the PvCesA3 gene. These isolates with this mutation have been detected mainly by determining the minimum inhibitory concentration of fungicides, which is always time consuming and inefficient. To establish a suitable method for rapid detection of the G1105S mutation in P. viticola, an efficient and simple molecular method was developed, based on tetra-primer ARMS PCR. A set of four primers were designed and optimised to distinguish the different genotypes within one step. Only 2 h was required from the sampling of symptoms to the phenotyping of fungicide resistance. Using this method, CAA-resistant P. viticola were identified for the first time in China. Also, the finding of sensitive heterozygotes indicated that the resistant allele is spreading in the population in Ziyuan. This new method proved to be useful as an early warning system for resistance outbreaks of P. viticola to CAA fungicides in the field and may be helpful in decisions concerning rotation of different fungicide groups. © 2016 Society of Chemical Industry.

Insights into the adaptive response of the plant-pathogenic oomycete Phytophthora capsici to the fungicide flumorph.

Phytophthora capsici is an important oomycete plant pathogen that causes significant losses worldwide. The carboxylic acid amide fungicide flumorph has shown excellent activity against oomycete plant pathogens. Despite its potential, there remains concern that the sexual reproduction of oomycete pathogens, which results in genetic recombination, could result in the rapid development of resistance to flumorph. The current study utilized an iTRAQ (isobaric tags for relative and absolute quantitation) based method to compare differences between the proteome of the parental P. capsici isolate PCAS1 and its sexual progeny S2-838, which exhibits significant resistance to flumorph. A total of 2396 individual proteins were identified, of these, 181 were considered to be associated with the adaptive response of P. capsici to flumorph. The subsequent bioinformatic analysis revealed that the adaptive response of P. capsici to flumorph was complex and regulated by multiple mechanisms, including utilising carbohydrate from the host environment to compensate for the cell wall stress induced by flumorph, a shift in energy generation, decreased amino acids biosynthesis, and elevated levels of proteins associated with the pathogen’s response to stimulus and transmembrane transport. Moreover, the results of the study provided crucial data that could provide the basis for early monitoring of flumorph resistance in field populations of P. capsici.

Differences in the efficacy of carboxylic acid amide fungicides against less sensitive strains of Plasmopara viticola.

Plasmopara viticola is controlled by fungicides with different modes of action, including carboxylic acid amides (CAAs). The aim of this study was to evaluate differences in the response of CAA-resistant P. viticola strains towards CAAs. The G1105S mutation affects all four CAAs, but with different impacts. While this confirms that they have the same mode of action, it shows that differences between CAAs can occur. Further molecular modelling and docking studies are needed to gain a better understanding of the different behaviours reported here. © 2015 Society of Chemical Industry.

Monitoring of a Single Point Mutation in the PvCesA3 Allele Conferring Resistance to Carboxylic Acid Amide Fungicides in Plasmopara viticola Populations In Yamanashi Prefecture, Japan

The use of the carboxylic acid amide (CAA) fungicide mandipropamid to manage grapevine downy mildew in vineyards in Japan has been increasing since 2010, because of widespread quinone outside inhibitor fungicide resistance in the Plasmopara viticola population. However, CAA fungicide resistance in P. viticola is becoming a serious problem worldwide. In 2013, we monitored for the presence of a single point mutation at codon 1105 of the cellulose synthase gene PvCesA3, which confers resistance to mandipropamid in P. viticola samples collected from four vineyards in Yamanashi prefecture in Japan. Five out of 157 samples were found to be heterozygotes, carrying both the mutated and nonmutated PvCesA3 alleles. Although CAA fungicide-resistant P. viticola isolates have not been reported yet in Japan, the emergence of heterozygous P. viticola populations indicates the potential risk of emergence of resistant homozygotes. Accepted for publication 14 March 2015. Published 1 May 2015

Design, synthesis and antifungal activity of carboxylic acid amide fungicides: part 2: substituted 1-phenyl-2-phenoxyethylamino valinamide carbamates

Most of the “stretched” valinamide carbamate analogues had higher anti-oomycetic potency than dimethomorph. The field trial showed that 8b is a promising candidate for future development.

Competition Between Pyrimorph-Sensitive and Pyrimorph-Resistant Isolates of Phytophthora capsici

Phytophthora capsici causes significant losses to vegetable production worldwide. Pyrimorph, a new carboxylic acid amide fungicide, has been registered to control P. capsici in China. A mutation (Q1077K) in cellulose synthase 3 has been reported to confer resistance to pyrimorph. In this study, we measured the competition between pyrimorph-resistant and pyrimorph-sensitive isolates of P. capsici. Mixed zoospore suspensions of resistant (R) and sensitive (S) isolates at five ratios (1R:9S, 3R:7S, 5R:5S, 7R:3S, and 9R:1S) were applied to carrot agar in vitro test (with five successive transfers) and to the soil surface around pepper plants in planta test (with 10 successive disease cycles). The proportion of resistant isolates was measured by a conventional assay in which single zoospore isolates recovered after transfers or disease cycles were grown on agar medium with a discriminatory concentration of pyrimorph. The results were then compared with those of a real-time polymerase chain reaction (PCR)-based method developed here, the results were similar. Both assays showed that the competitive ability of the resistant isolates was similar to or less than that of the sensitive isolates. The real-time PCR assay developed will be useful for high-throughput analysis and monitoring the development of pyrimorph resistance in field populations of P. capsici.

Activity of Ten Fungicides against Phytophthora capsici Isolates Resistant to Metalaxyl

AbstractPepper Phytophthora blight (PPB), caused by Phytophthora capsici, is an important disease of pepper in China. The extensive application of metalaxyl has resulted in widespread resistance to this fungicide in field. This study has evaluated the activities of several fungicides against the mycelial growth and sporangium germination of metalaxyl‐sensitive and metalaxyl‐resistant P. capsici isolates by determination of EC50 values. The results showed that the novel carboxylic acid amide (CAA) fungicide mandipropamid exhibited excellent inhibitory activity against PPB both in vitro and in vivo, with averagely EC50 values of 0.075 and 0.004 μg/ml in mycelial growth and sporangium germination, respectively, and over 88% efficacy in controlling PPB. The other three CAA fungicides also provided over 70% efficacy in controlling PPB. The mycelial growth was less sensitive to quinone outside inhibitor (QoI) fungicides azoxystrobin and trifloxystrobin than that of sporangium germination in P. capsici isolates. However, azoxystrobin and trifloxystrobin provided over 80% efficacy in controlling PPB. It was noted that propamocarb and cymoxanil did not exhibit activity against the mycelial growth or sporangium germination of P. capsici isolates in the in vitro tests, with over 70% efficacy in controlling PPB. The new fungicide mixture 62.5 g/l fluopicolide + 625 g/l propamocarb (trade name infinito, 687.5 g/l suspension concentrate (SC)) produced over 88% efficacy in controlling PPB caused by both metalaxyl‐sensitive and metalaxyl‐resistant isolates. The data of this study also proved that there was obviously no cross‐resistance between metalaxyl and the other tested fungicides. Therefore, these fungicides should be good alternatives to metalaxyl for the control of PPB and management of metalaxyl resistance.

Assessing the risk that Phytophthora melonis can develop a point mutation (V1109L) in CesA3 conferring resistance to carboxylic acid amide fungicides.

The risk that the plant pathogen Phytophthora melonis develops resistance to carboxylic acid amide (CAA) fungicides was determined by measuring baseline sensitivities of field isolates, generating resistant mutants, and measuring the fitness of the resistant mutants. The baseline sensitivities of 80 isolates to flumorph, dimethomorph and iprovalicarb were described by unimodal curves, with mean EC50 values of 0.986 (±0.245), 0.284 (±0.060) and 0.327 (±0.068) µg/ml, respectively. Seven isolates with different genetic background (as indicated by RAPD markers) were selected to generate CAA-resistance. Fifty-five resistant mutants were obtained from three out of seven isolates by spontaneous selection and UV-mutagenesis with frequencies of 1×10−7 and 1×10−6, respectively. CAA-resistance was stable for all mutants. The resistance factors of these mutants ranged from 7 to 601. The compound fitness index (CFI = mycelial growth × zoospore production × pathogenicity) was often lower for the CAA-resistant isolates than for wild-type isolates, suggesting that the risk of P. melonis developing resistance to CAA fungicides is low to moderate. Among the CAA-resistant isolates, a negative correlation between EC50 values was found for iprovalicarb vs. flumorph and for iprovalicarb vs. dimethomorph. Comparison of the full-length cellulose synthase 3 (CesA3) between wild-type and CAA-resistant isolates revealed only one point mutation at codon position 1109: a valine residue (codon GTG in wild-type isolates) was converted to leucine (codon CTG in resistant mutants). This represents a novel point mutation with respect to mutations in CesA3 conferring resistance to CAA fungicides. Based on this mutation, an efficient allelic-specific PCR (AS-PCR) method was developed for rapid detection of CAA-resistance in P. melonis populations.

Insights into the molecular mechanism of tolerance to carboxylic acid amide (CAA) fungicides in Pythium aphanidermatum

Tolerance to the oomycete-specific carboxylic acid amide (CAA) fungicides is a poorly understood mechanism in Pythium species. The root-rot and damping-off causative agent Pythium aphanidermatum and the CAA fungicide mandipropamid (MPD) were used to investigate the molecular basis of CAA tolerance. Five genes putatively involved in carbohydrate synthesis were identified and characterised: one chitin synthase gene, PaChs, and four cellulose synthase genes PaCesA1 to PaCesA4, of which PaCesA3 encodes the MPD target enzyme. These genes were differentially expressed throughout the life cycle of P. aphanidermatum. Mycelium treated with MPD concentrations slightly affecting mycelial growth did not cause a change in PaCesA3 expression nor a strong upregulation of PaCesA homologues. The high tolerance level of P. aphanidermatum and the lack of PaCesA upregulation imply that MPD tolerance is the result of a specific amino acid configuration in the cellulose synthase 3 (CesA3) target enzyme. Indeed, P. aphanidermatum displays the amino acid L1109 which is also associated with MPD resistance in artificial mutants of Phytophthora species. It is concluded that MPD tolerance in P. aphanidermatum is not caused by compensatory mechanisms but most likely by an inherent target-site configuration in PaCesA3 that hinders MPD binding to the enzyme pocket.