Chemical Classes Of Fungicides Research Articles

Fungicide sensitivity of grapevine bacteria with plant growth-promoting traits and antagonistic activity as non-target microorganisms

This study evaluates the capacity of commercial formulations of synthetic fungicides to inhibit grapevine bacterial growth when sprayed on vineyards to control diseases, such as downy mildew, powdery mildew and secondary rots. Fungicide sensitivity plate assays were carried out on bacteria isolated from vineyards that were also identified and characterized for their plant growth-promoting (PGP) traits and antifungal activity. The high taxonomic variability of bacteria screened with different chemical classes of fungicides is one new finding of this study. Seven out of 11 fungicides were able to inhibit the growth of bacteria at a concentration corresponding to the maximum dose allowed by law in spray treatments of vineyards. Bacterial sensitivity to each fungicide varied greatly. Many sensitive isolates displayed PGP traits and/or antagonistic activity. This study shows the potential impact of fungicidal treatments on grapevine bacterial microbiota. The involvement of bacteria beneficial to the growth and health of plants underlines the importance of this investigation. Our data reveal that the control of a certain disease may be possible using fungicides that have no or low impact on natural non-target microbiota. Understanding the action mechanisms of the active ingredients in these products is a priority for the development of new eco-friendly pesticides.

Pyridachlometyl has a novel anti-tubulin mode of action which could be useful in anti-resistance management.

BACKGROUNDFungicide resistance is a growing problem affecting many crop pathogens owing to the low success rate in finding novel chemical classes of fungicides. Pyridachlometyl is a new fungicide that seems to belong to a new chemical class of tubulin polymerization promoters.RESULTSPyridachlometyl exhibited potent antifungal activity against a broad range of fungal species belonging to the phyla Ascomycota and Basidiomycota. No cross‐resistance was observed with other fungicide classes, such as ergosterol biosynthesis inhibitors, respiratory inhibitors, or tubulin polymerization inhibitors in Zymoseptoria tritici. Pyridachlometyl‐resistant strains were obtainable by UV mutagenesis in Z. tritici and Penicillium digitatum. Mutations in tubulin‐coding genes were found in all laboratory mutants but the patterns of mutation were distinct from that of tubulin polymerization inhibitors, such as benzimidazole fungicides.CONCLUSIONPyridachlometyl is a promising new tool for disease control. However, strict resistance management strategies should be recommended for the practical use of pyridachlometyl. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Competitive ability of multi-fungicide resistant Botrytis cinerea in a blackberry planting over three years

Botrytis cinerea isolates with multi-fungicide resistance have frequently been isolated from small fruit fields such as strawberries and blackberries. Individual B. cinerea isolates have been found resistant to up to seven chemical classes of fungicides. Fitness costs and less competitiveness have been observed in multi-fungicide resistant isolates, but this has not been examined under field conditions. In the spring of 2016, flowers of field-grown blackberries were either not inoculated or inoculated with B. cinerea isolates sensitive (0CCR), resistant to five or six chemical classes excluding phenylpyrroles (5CCR), or resistant to six or seven chemical classes including phenylpyrroles (6CCR/MDR1h). The experimental field was left unsprayed for the duration of this study and isolates of B. cinerea were collected from flowers and/or fruit in each of the three experimental years. Isolates collected in summer of 2016 revealed resistance phenotypes in each plot closely matching those of the respective inoculum, with 95% 0CCR, 55% 5CCR, and 91% 6CCR/MDR1h isolates recovered from 0CCR, 5CCR, and 6CCR/MDR1h inoculation plots, respectively. In the 2017 and 2018 isolate collections, 6CCR/MDR1h resistance phenotypes were found in plots inoculated and non-inoculated with this phenotype, indicating their persistence and movement between plots. Resistance phenotypes different from the inoculum were also recovered each year, indicating that the inoculum was successfully competing with a native Botrytis population. Despite the competition, 6CCR/MDR1h isolates were recovered in high frequency from all inoculated plots in 2018. G3pdh and mrr1 sequences of 6CCR/MDR1h isolates collected in 2018 were identical to the sequences of the inoculum, indicating that these isolates likely descended from the inoculum. This study demonstrates that isolates carrying multi-fungicide resistant phenotypes, specifically 6CCR/MDR1h, are competitive in the absence of fungicide selection pressure.

Fungicide Resistance in Botrytis fragariae and Species Prevalence in the Mid-Atlantic United States.

Botrytis fragariae was recently described causing gray mold of strawberry in Germany and the United States. The goal of the present study was to determine its prevalence, distribution, and sensitivity to fungicides in strawberry fields of five states. In total, 188 Botrytis isolates were obtained from flowers and fruit collected from the states of Maryland (n = 35), Virginia (n = 38), North Carolina (n = 46), South Carolina (n = 41), and Georgia (n = 28). Only 13 of these were fruit samples and came from South Carolina (n = 5) and Georgia (n = 8). B. fragariae made up 35.1% of the entire collection, and composed close to half of the Botrytis population in North Carolina (43.4%), South Carolina (61.0%), and Georgia (42.9%). One isolate of B. mali was also found, and the rest of the isolates were B. cinerea (sensu lato). B. fragariae and B. cinerea were found coexisting in 11 fields, while other field samples consisted of only B. fragariae (n = 3) or only B. cinerea (n = 10) isolates. B. fragariae isolates with resistance to one or more fungicides were found, and resistance profiles differed from those of B. cinerea, in that no resistance to cyprodinil (FRAC 8) or boscalid and other FRAC 7 botryticides was detected. We detected B. fragariae resistance to the active ingredients thiophanate-methyl, iprodione, fludioxonil, and fenhexamid. We also detected B. fragariae isolates with resistance to up to four chemical classes of fungicides, though most isolates were resistant to one or two chemical classes. In conclusion, isolates of the newly detected species B. fragariae were commonly found on strawberry flowers in the Mid-Atlantic United States, and have developed resistance to many of the most commonly used botryticides. Though the relevance of this species to pre- and postharvest fruit infections is unknown, fludioxonil applications may give this species a competitive advantage over B. cinerea. Controlling this fungus with FRAC 7 fungicides may be an effective way of limiting its spread in strawberry fields.

Genotypic and Phenotypic Variations in Botrytis spp. Isolates from Single Strawberry Flowers.

Gray mold, caused by Botrytis spp., is among the most devastating diseases affecting strawberry worldwide. The great diversity present in the pathogen enhances its ability to survive and adapt in the field. In this study, we explored the genotypic and phenotypic diversity present in single strawberry flowers. In total, 192 isolates were collected from 19 flowers and four farms, and 9 to 12 isolates were collected from each flower. Forty-two haplotypes were found using microsatellite fragment analysis. Multiple haplotypes of two different Botrytis spp. (Botrytis cinerea and B. fragariae) were found in 12 flowers. In the remaining seven flowers, the single-spore isolates examined were of identical haplotypes. In three flowers, the two Botrytis spp. were found to coexist. Isolates were either sensitive (zero chemical class resistance) or resistant to one, two, three, four, or five chemical classes of fungicides. Resistance to multiple fungicides was commonly observed in both species but resistance to boscalid and penthiopyrad was only found in B. cinerea isolates. Resistance to cyprodinil was found in B. fragariae for the first time in the United States. Each haplotype was generally linked to a single resistance profile; however, a single resistance profile often was represented by multiple haplotypes. Isolates from the same flower of multiple haplotypes were largely identical in resistance profiles. This study is a first detailed investigation of genotypic diversity combined with phenotypic analysis of Botrytis spp. at the single-tissue level. It demonstrates that high genotypic and phenotypic diversity is present not only within fields but also in individual blossoms as well. This information is important for understanding the epidemiology of Botrytis and also has implications for fungicide resistance management, particularly related to resistance monitoring practices.

Parasitic Fitness of Fungicide-Resistant and -Sensitive Isolates of Alternaria solani.

Resistance to chemistries of the succinate dehydrogenase inhibiting (SDHI) and quinone outside inhibiting (QoI) fungicides has developed rapidly in populations of Alternaria solani, the cause of early blight of potato. Reduced sensitivity to the anilinopyrimidine (AP) fungicide pyrimethanil has also been identified recently, determining that resistance to three chemical classes of fungicides is present within the A. solani population. Although no mutations have been characterized to confer resistance to APs, in A. solani five point mutations on three AsSdh genes have been determined to convey resistance to SDHIs, and the substitution of phenylalanine with leucine at position 129 (F129L) in the cytb gene confers resistance to QoIs. The objective of this study was to investigate the parasitic fitness of A. solani isolates with resistance to one or more of these chemical classes. A total of 120 A. solani isolates collected from various geographical locations around the United States were chosen for in vitro assessment, and 60 of these isolates were further evaluated in vivo. Fitness parameters measured were (i) spore germination in vitro, (ii) mycelial expansion in vitro, and (iii) aggressiveness in vivo. No significant differences in spore germination or mycelial expansion (P = 0.44 and 0.51, respectively) were observed among wild-type and fungicide-resistant isolates in vitro. Only A. solani isolates possessing the D123E mutation were shown to be significantly more aggressive in vivo (P < 0.0001) compared with wild-type isolates. These results indicate that fungicide-resistant A. solani isolates have no significant fitness penalties compared with sensitive isolates under the parameters evaluated regardless of the presence or absence of reduced sensitivity to multiple chemical classes. Results of these studies suggest that A. solani isolates with multiple fungicide resistances may compete successfully with wild-type isolates under field conditions.

Botrytis fragariae, a New Species Causing Gray Mold on Strawberries, Shows High Frequencies of Specific and Efflux-Based Fungicide Resistance.

Botrytis cinerea causes pre- and postharvest decay of many fruit and vegetable crops. A survey of German strawberry fields revealed Botrytis strains that differed from B. cinerea in diagnostic PCR markers and growth appearance. Phylogenetic analyses showed that these strains belong to an undescribed species in Botrytis clade 2, named Botrytisfragariae sp. nov. Isolates of Bfragariae were detected in strawberry fields throughout Germany, sometimes at frequencies similar to those of B. cinerea, and in the southeastern United States. Bfragariae was isolated from overwintering strawberry tissue but not from freshly infected fruit. Bfragariae invaded strawberry tissues with an efficiency similar to or lower than that of B. cinerea but showed poor colonization of inoculated nonhost plant tissues. These data and the exclusive occurrence of this fungus on strawberry plants indicate that Bfragariae is host specific and has a tissue preference different from that of B. cinerea Various fungicide resistance patterns were observed in Bfragariae populations. Many Bfragariae strains showed resistance to one or several chemical classes of fungicides and an efflux-based multidrug resistance (MDR1) phenotype previously described in B. cinerea Resistance-related mutations in Bfragariae were identical or similar to those of B. cinerea for carbendazim (E198A mutation in tubA), azoxystrobin (G143A in cytB), iprodione (G367A+V368F in bos1), and MDR1 (gain-of-function mutations in the transcription factor mrr1 gene and overexpression of the drug efflux transporter gene atrB). The widespread occurrence of Bfragariae indicates that this species is adapted to fungicide-treated strawberry fields and may be of local importance as a gray mold pathogen alongside B. cinereaIMPORTANCE Gray mold is the most important fruit rot on strawberries worldwide and requires fungicide treatments for control. For a long time, it was believed to be caused only by Botrytis cinerea, a ubiquitous pathogen with a broad host range that quickly develops fungicide resistance. We report the discovery and description of a new species, named Botrytisfragariae, that is widely distributed in commercial strawberry fields in Germany and the southeastern United States. It was observed on overwintering tissue but not on freshly infected fruit and seems host specific on the basis of its occurrence and artificial infection tests. Bfragariae has also developed resistance to several fungicides that is caused by mutations similar to those known in B. cinerea, including an efflux-based multidrug resistance. Our data indicate that Bfragariae could be of practical importance as a strawberry pathogen in some regions where its abundance is similar to that of B. cinerea.

Sedaxane, Isopyrazam and Solatenol™: Novel Broad-spectrum Fungicides Inhibiting Succinate Dehydrogenase (SDH) - Synthesis Challenges and Biological Aspects.

Sedaxane (SDX) 1, isopyrazam (IZM) 2 and Solatenol™ (STL) 3 are broad-spectrum pyrazole carboxamides, which originate from novel chemical classes of fungicides. Their mode of action (MoA) is inhibition of succinate dehydrogenase (SDH), which was recognized for a long time to deliver only compounds with a narrow biological spectrum. This view changed with the market introduction of BASF's boscalid in 2003. All major agro-companies subsequently worked in parallel on this MoA successfully and recently introduced new compounds to the market. Syngenta entered the SDHI area in 1998 and was able to introduce three complementary compounds to the market between 2010 and 2012. In this short review some synthesis challenges and biological effects of SDX 1, IZM 2 and STL 3 will be covered. New cost-efficient synthesis strategies for the preparation of o-biscyclopropyl-aniline, new benzonorbornene intermediates and the key pyrazole carboxylic acid intermediate which is essential for all three Syngenta SDHIs, will be in the focus of this review.

Independent Emergence of Resistance to Seven Chemical Classes of Fungicides in Botrytis cinerea.

Gray mold, caused by the fungal pathogen Botrytis cinerea, is one of the most destructive diseases of small fruit crops and control is largely dependent on the application of fungicides. As part of a region-wide resistance-monitoring program that investigated 1,890 B. cinerea isolates from 189 fields in 10 states of the United States, we identified seven isolates (0.4%) from five locations in four different states with unprecedented resistance to all seven Fungicide Resistance Action Committee (FRAC) codes with single-site modes of action including FRAC 1, 2, 7, 9, 11, 12, and 17 registered in the United States for gray mold control. Resistance to thiophanate-methyl, iprodione, boscalid, pyraclostrobin, and fenhexamid was based on target gene mutations that conferred E198A and F200Y in β-tubulin, I365N/S in Bos1, H272R/Y in SdhB, G143A in Cytb, and T63I and F412S in Erg27. Isolates were grouped into MDR1 and MDR1h phenotypes based on sensitivity to fludioxonil and variations in transcription factor mrr1. MDR1h isolates had a previously described 3-bp deletion at position 497 in mrr1. Expression of ABC transporter atrB was increased in MDR1 isolates but highest in MDR1h isolates. None of the isolates with seven single resistances (SR) had identical nucleotide variations in target genes, indicating that they emerged independently. Multifungicide resistance phenotypes did not exhibit significant fitness penalties for the parameters used in this study, but MDR1h isolates produced more sclerotia at low temperatures and exhibited increased sensitivity to salt stress. In this study we show that current resistance management strategies have not been able to prevent the geographically independent development of resistance to all seven site-specific fungicides currently registered for gray mold control in the United States and document the presence of MDR1h in North America.

Location-Specific Fungicide Resistance Profiles and Evidence for Stepwise Accumulation of Resistance in Botrytis cinerea

The fungicide resistance profiles to seven chemical classes of fungicides were investigated in 198 Botrytis cinerea isolates from five blackberry fields and 214 B. cinerea isolates from 10 strawberry fields of North and South Carolina. Populations of B. cinerea tended to have a single dominant, location-specific resistance profile that consisted of resistance to multiple fungicides in fields sprayed weekly with site-specific fungicides. The most prevalent profile in blackberry fields consisted of resistance to thiophanate-methyl, pyraclostrobin, and boscalid. The most prevalent resistance profile found in conventional strawberry fields consisted of resistance to thiophanate-methyl, pyraclostrobin, boscalid, and cyprodinil. A statistical model revealed that multifungicide resistance patterns did not evolve randomly in populations from both crops. Instead, strains resistant to thiophanate-methyl were more likely to acquire resistance to pyraclostrobin, the resulting dual-resistant population was more likely to acquire resistance to boscalid, the resulting triple-resistant population was more likely to acquire resistance to cyprodinil, and the resulting quadruple-resistant population was more likely to acquire resistance to fenhexamid (strawberry population only) compared with random chance. Resistance to iprodione and fludioxonil evolved from a pool of strains with different fungicide resistance profiles. Resistance to thiophanate-methyl, pyraclostrobin, boscalid, and fenhexamid in blackberry isolates was, without exception, based on target gene mutations, including E198A and E198V in β-tubulin, G143A in cytochrome b, H272Y and H272R in SdhB, and F412I in Erg27, respectively. A new genotype associated with fenhexamid resistance was found in one strain (i.e., Y408H and deletion of P298). Fungicide-resistant strains were present but rare in an unsprayed blackberry field, where some unique phenotypes, including low and medium resistance to fludioxonil, had emerged in the absence of fungicide pressure. The isolates resistant to fludioxonil had effective dose that inhibited 50% of mycelial growth values of 0.16 μg/ml (low resistance) and 0.32 and 0.38 μg/ml (medium resistance) and were also resistant to the anilinopyrimidine fungicide cyprodinil, indicating that this and similar phenotypes will eventually be selected by continued applications of the fludioxonil + cyprodinil premixture Switch. This study shows that multifungicide-resistant phenotypes are common in conventionally maintained strawberry and blackberry fields and that resistance to multiple fungicides evolved from stepwise accumulation of single resistances.

Fungicide Resistance Profiles in Botrytis cinerea from Strawberry Fields of Seven Southern U.S. States.

The sensitivity to seven chemical classes of fungicides was investigated in 1,810 Botrytis cinerea isolates collected from strawberry blossoms and fruit in 181 strawberry fields from seven southern states in the United States across 2 years. Ten isolates were examined from each field. Fungicide sensitivity assays were carried out based on visual assessment of diametrical mycelial growth after 4 days of incubation on media amended with discriminatory doses of fungicides in microtiter plates. Results of visual assessments were verified with selected isolates using a previously published germination assay and by inoculating representative isolates with resistant phenotypes on fungicide-sprayed fruit. The overall resistance frequencies of 750 isolates collected in 2012 for thiophanate-methyl, pyraclostrobin, boscalid, cyprodinil, fenhexamid, iprodione, and fludioxonil were 76, 42, 29, 27, 25, 3, and 1%, respectively. Frequencies of 1,060 isolates collected in 2013 were 85, 59, 5, 17, 26, 2, and 1%, respectively. Resistance to thiophanate-methyl and pyraclostrobin was found in virtually every location in both years, whereas resistance to iprodione and fludioxonil was rarely found. Resistant isolates were resistant to either one (23%), two (18%), three (19%), four (14%), five (3%), or six (0.1%) chemical classes of fungicides in 2012. In 2013, this distribution was 24, 29, 26, 8, 2, and 0.3%, respectively. Multifungicide-resistant isolates of B. cinerea were widespread in southern states and evidence suggests that the frequency of isolates with multifungicide resistance increased from 2012 to 2013. The data also show that fungicide resistance in B. cinerea was already present in blossoms, indicating that resistance management needs to be implemented early in the season.

Fungicide modes of action and resistance in downy mildews

Among oomycetes, Plasmopara viticola on grape and Phytophthora infestans on potato are agronomically the most important pathogens requiring control measures to avoid crop losses. Several chemical classes of fungicides are available with different properties in systemicity, specificity, duration of activity and risk of resistance. The major site-specific fungicides are the Quinone outside inhibitors (QoIs; e.g. azoxystrobin), phenylamides (e.g. mefenoxam), carboxylic acid amides (CAAs; e.g. dimethomorph, mandipropamid) and cyano-acetamide oximes (cymoxanil). In addition, multi-site fungicides such as mancozeb, folpet, chlorothalonil and copper formulations are important for disease control especially in mixtures or in alternation with site-specific fungicides. QoIs inhibit mitochondrial respiration, phenylamides the polymerization of r-RNA, whereas the mode of action of the other two site-specific classes is unknown but not multi-site. The use of site-specific fungicides has in many cases selected for resistant pathogen populations. QoIs are known to follow maternal, largely monogenic inheritance of resistance; they bear a high resistance risk for many but not all oomycetes. For phenylamides, inheritance of resistance is based on nuclear, probably monogenic mechanisms involving one or two semi-dominant genes; resistance risk is high for all oomycetes. The molecular mechanism of resistance to QoIs is mostly based on the G143A mutation in the cytochrome b gene; for phenylamides it is largely unknown. Resistance risk for CAA fungicides is considered as low to moderate depending on the pathogen species. Resistance to CAAs is controlled by two nuclear, recessive genes; the molecular mechanism is unknown. For QoIs and CAAs, resistance in field populations of P. viticola may gradually decline when applications are stopped.