Low frequency of resistance to thiophanate-methyl in Monilinia fructicola populations from southeastern United States peach orchards.

Methyl benzimidazole carbamate (MBC) fungicides were once widely used for brown rot (Monilinia fructicola) control of peach (Prunus persica (L.) Batsch) in the southeastern US, but their use was substantially reduced and often eliminated due to widespread resistance. In this study, 233 M. fructicola isolates were collected from major peach production areas in Alabama, Georgia, and South Carolina, and sensitivity to thiophanate-methyl was examined. Isolates were also collected from one organic and two experimental peach orchards. A discriminatory dose of 1 µg/ml was used to distinguish sensitive (S) and moderately sensitive (S-LR) isolates from low resistant phenotypes, while 50 and 500 µg/ml thiophanate-methyl concentrations were used to determine high resistant (HR) phenotypes. Sequence analyses were performed to identify mutations in the ß-tubulin target gene and detached fruit assays were performed to determine the efficacy of a commercial product against isolates representing each phenotype. Results indicated 55.7%, 63.5%, and 75.9% of isolates from Alabama, Georgia, and South Carolina, respectively, were S to thiophanate-methyl; 44.3%, 36.5%, and 21.4% were S-LR; no isolates were LR; and only 3 isolates (1.3%) from South Carolina were HR. No mutations in S or S-LR isolates were found, but HR isolates revealed the E198A mutation, an amino acid change of glutamic acid to alanine conferring high resistance. The high label rate of a commercial product containing thiophanate-methyl controlled brown rot caused by S and S-LR isolates in detached fruit studies but was ineffective against HR isolates. The combinations of thiophanate-methyl with azoxystrobin or isofetamid, when mixed together and applied in an experimental orchard 14 days preharvest, significantly reduced brown rot incidence on pre and postharvest commercially ripe fruit and efficacy was comparable to that of a grower standard fungicide. These results indicate that thiophanate-methyl may again be useful to peach growers in the southeastern US for brown rot and fungicide resistance management.

Unveiling the Impact of Thiophanate-Methyl on Arthrospira platensis: Growth, Photosynthetic Pigments, Biomolecules, and Detoxification Enzyme Activities.

BACKGROUND: The intensive and injudicious use of pesticides in agriculture has emerged as a major concern due to its detrimental impact on aquatic ecosystems. However, the specific impact of broad-spectrum fungicides, such as Thiophanate-methyl (TM), on cyanobacteria remains a subject of ongoing research and debate. METHODS: In order to fill this knowledge gap, The present study aimed to comprehensively investigate the toxicological effects of TM (10-30 µg/L) on the growth, photosynthetic pigments, oxidative stress, and biochemical composition of the non-nitrogen-fixing cyanobacterium Arthrospira platensis. RESULTS: Our findings unequivocally demonstrated that TM exposure significantly inhibited the growth of A. platensis. Moreover, the decrease in chlorophyll content indicated a pronounced negative impact on the photosynthetic system of A. platensis caused by TM exposure. Notably, TM induced oxidative stress in A. platensis, as substantiated by a significant increase in lipid peroxidation (MDA) within the culture. Furthermore, the intracellular generation of hydrogen peroxide (H2O2) exhibited a positive correlation with higher TM dosages, while the levels of vital antioxidant enzymes, such as catalase (CAT) and ascorbate peroxidase (APX), exhibited a discernible decrease. This suggests that TM compromises the antioxidant defense mechanisms of A. platensis. Additionally, TM was found to enhance the activity of a plethora of enzymes involved in the detoxification of pesticides, including peroxidase (POD) and glutathione-S-transferase (GST), thereby indicating a robust detoxification response by A. platensis. Interestingly, exposure to TM resulted in a general suppression of biocomponent production, such as total proteins and total carbohydrates, which exhibited a diminishing trend with increasing TM concentration. Conversely, the lipid content witnessed a significant increase, possibly as an adaptive response to TM-induced stress. CONCLUSIONS: These findings contribute to a deeper understanding of the ecological implications of pesticide usage and emphasize the urgent need for the adoption of sustainable and environmentally-friendly agricultural practices to safeguard aquatic ecosystems.

Prevalence of H6Y mutation in ß-tubulin causing thiophanate-methyl resistant in Monilinia fructicola from Fujian, China.

Brown rot disease broke out in stone fruit orchards of Fujian, China in 2019, despite pre-harvest application of methyl benzimidazole carbamate (MBC). To determine the reason, a total of 44 Monilinia fructicola strains were collected from nectarine, plum and peach fruits in this study, among which 79.5% strains were resistant to thiophanate-methyl, indicated by discriminatory dose of 5 µg/mL. The resistance of these strains was confirmed by treating detached peach fruit with label rates of formulated thiophanate-methyl which only completely inhibit infection of the sensitive strains, but not the resistant strains. Further analysis of the mechanism of MBC resistance revealed that all resistant strains carry a H6Y mutation in ß-tubulin protein Tub2, which was only reported previously in the M. fructicola strains from California, USA, and do not display obvious fitness penalties, as no significant defects in mycelial growth rate, sporulation, conidia germination, aggressiveness on detached peach fruit and temperature sensitivity was detected. In addition, we found that diethofencarb, the agent for managing MBC-resistance strains, was unable to inhibit growth of the H6Y strains. Taken together, our study, for the first time, identified a mutation form of H6Y in the ß-tubulin protein of M. fructicola in China, rendering the strains wide resistance to thiophanate-methyl. This mechanism of M. fructicola gaining resistance to MBC fungicides needs to be fully considered, when designing management strategies to control brown rot disease in stone fruit orchards.

Effect of fungicides on spike characteristics in winter wheat inoculated with Fusarium culmorum.

The impact of fungicides on the head blight (FHB) development disease index, percent spike harvest index (SHI), and deoxynivalenol (DON) accumulation in wheat kernels under field conditions was evaluated after artificial spike inoculation with Fusarium culmorum (S-14). The trial was carried out using commercially available fungicides and a sensitive cultivar of bread wheat (Flamura-85) in a field of a wheat producer located in the Trakya region of Turkey. Fungicides were applied at the beginning of anthesis (ZGS 61), 48 hours after the inoculation with the pathogen. Disease index was determined 10 days and 14 days post-inoculation. The application of fungicides containing tebuconazole, thiophanate methyl plus tetraconazole and prothioconazole plus trifloxystrobin reduced the FHB disease and increased kernel number, spike weight, and kernel weight, as compared to the inoculated/non-fungicide control. The efficacy of tebuconazole and of prothioconazole plus trifloxystrobin was higher than that of thiophanate methyl plus tetraconazole on FHB disease severity, percent spike harvest index (SHI), and DON accumulation in wheat kernels.

Chemical seed treatment and mycorrhizal inoculation provide better development and nutrition of common bean plants.

BACKGROUND: Chemical seed treatment is an established practice in agriculture to protect crops from soil-borne pathogens and pests. Arbuscular mycorrhizal fungi (AMF) benefit plants by extending soil exploration as well as water and nutrient uptake. The objective of this work was to analyze the effects of combinations of seed treatments with doses of inoculant containing Rhizoglomus intraradices on vegetative development, root colonization and nutrition of Phaseolus vulgaris plants and soil microbiota. RESULTS: Seed treatment benefited the vegetative development and nutrition of beans, with the treatments metalaxyl + fludioxonil + tiabendazole and pyraclostrobin + thiophanate methyl + fipronil standing out regarding the contents of nitrogen (N), phosphorus (P), iron (Fe) and zinc (Zn) of the aerial parts. Mycorrhizal inoculation linearly increased dehydrogenase activity, root biomass and total plant biomass, with increments reaching 27%. There was an interaction between seed treatment and inoculation dose for aboveground biomass and the contents of potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), manganese (Mn) and root colonization, with expressive results for the combination of the two highest doses of inoculant with metalaxyl + fludioxonil + tiabendazole or pyraclostrobin + methylthiophanate + fipronil in the seeds. CONCLUSION: Chemical seed treatment and mycorrhizal inoculation benefited bean plants and their nutritional status. The best combinations for the bean crop were metalaxyl + fludioxonil + tiabendazole with 41.4 mg of the inoculant per 100 seeds and pyraclostrobin + thiophanate methyl + fipronil with 62.1 mg of the inoculant per 100 seeds. © 2022 Society of Chemical Industry.

Molecular Diagnosis of Thiophanate-Methyl-Resistant Strains of Fusarium fujikuroi in Japan.

Fusarium fujikuroi is the pathogen of rice bakanae disease and is subclassified into gibberellin and fumonisin groups (G and F groups). Thiophanate-methyl (TM), a benzimidazole fungicide, has been used extensively to control F. fujikuroi. Previous investigation showed that F-group strains are TM sensitive (TMS), whereas most G-group strains are TM resistant (TMR) in Japan. The minimum inhibitory concentration in TMS strains was 1 to 10 µg ml-1, whereas that in TMR strains was >100 µg ml-1. E198K and F200Y mutations in ß2-tubulin were detected in TMR strains. A loop-mediated isothermal amplification-fluorescent loop primer method was developed for diagnosis of these mutations and applied to 37 TMR strains and 56 TMS strains. The results indicated that 100% of TMR strains were identified as having either the E198K mutation (41%) or the F200Y mutation (59%), whereas none of the TMS strains tested showed either mutation. We found one remarkable TMR strain in the F group that had an F200Y mutation. These results suggest that E198K and F200Y mutations in ß2-tubulin contribute to TM resistance in F. fujikuroi.

Resistance to Azoxystrobin and Thiophanate-Methyl Is Widespread in Colletotrichum spp. Isolates From the Mid-Atlantic Strawberry Fields.

Multiple Colletotrichum species have been found to be responsible for strawberry anthracnose, and prevalence of each species seems to vary by regions and/or host tissues. In this study, a total of 200 Colletotrichum isolates were obtained from different strawberry cultivars displaying anthracnose symptoms in the mid-Atlantic fields. Analysis of g3pdh, tub2, and/or internal transcribed spacer sequences revealed four Colletotrichum species, including C. nymphaeae, C. fioriniae, C. siamense, and C. lineola. C. nymphaeae was the predominant species, representing 90% of all isolates collected. This species was found from all strawberry organs/tissues examined, whereas C. siamense and C. fioriniae were limited to the crown and fruit, respectively. Further, all Colletotrichum isolates were screened for resistance to azoxystrobin in vitro, and all C. siamense isolates were additionally screened for resistance to thiophanate-methyl. The overall frequency of resistance to azoxystrobin and thiophanate-methyl was 48 and 67%, respectively. G143A in the cytochrome b gene was found in all C. nymphaeae and C. siamense isolates with high level of resistance, with EC50 >100 µg/ml, while F129L was found in two of the five C. nymphaeae isolates with moderate resistance, with EC50 values ranging from 2.6 to 7.8 µg/ml. All C. fioriniae isolates tested were found to be less sensitive to azoxystrobin, with EC50 values ranging from 9.7 to 14.4 µg/ml, despite no mutations detected in the cytochrome b gene. Moreover, E198A in tub2 was linked with C. siamense isolates resistant to thiophanate-methyl (EC50 >100 µg/ml). These results revealed that resistance in Colletotrichum spp. to primary fungicides is widespread in the mid-Atlantic strawberry fields.

Diversity in species composition and fungicide resistance profiles in Colletotrichum isolates from apples.

Outbreaks of bitter rot were observed in three commercial apple orchards in Illinois despite best management efforts during the 2018 production season. Three isolates from symptomatic fruit from these orchards and two isolates from an orchard in South Carolina were identified to the species level using morphological tools and calmodulin, glyceraldehyde-3-phosphate dehydrogenase, and beta-tubulin gene sequences. The isolates from Illinois were identified as Colletotrichum siamense of the Colletotrichum gloeosporioides species complex and the ones from South Carolina as Colletotrichum fioriniae and Colletotrichum fructicola of the Colletotrichum acutatum and the C. gloeosporioides species complex, respectively. Two of the three C. siamense isolates from Illinois were resistant to azoxystrobin and thiophanate-methyl as determined in mycelial growth tests in vitro. EC50 values were >100 µg/ml for both fungicides. One isolate was only resistant to azoxystrobin. None of the isolates from South Carolina was resistant to either of the two compounds. All five isolates were sensitive to fludioxonil (EC50 values <0.1 µg/ml), propiconazole (EC50 values ranged from 0.15 to 0.36 µg/ml), and benzovindiflupyr (EC50 values ranged from <0.1 to 0.33 µg/ml). Resistance in C. siamense to azoxystrobin and thiophanate-methyl was confirmed in detached fruit studies using apples treated with label rates of registered product. Resistance to thiophanate-methyl in C. siamense was based on E198A mutation in b-tubulin gene, whereas resistance to azoxystrobin was based on G143A in cytochrome b (CYTB). One isolate resistant to azoxystrobin possessed no amino acid variation in CYTB. This study shows that quinone outside inhibitor fungicide resistance in Colletotrichum from apple has emerged and is being selected for in Illinois apple orchards by current spray strategies. Resistance monitoring may alert growers to potential threats, but the employment of molecular tools based on current knowledge of resistance mechanisms will provide incomplete results.

Resistance risk assessment for fludioxonil in Sclerotinia homoeocarpa in China.

Sclerotinia homoeocarpa causes dollar spot disease on turfgrass and is a serious problem on many species worldwide. Fludioxonil, a phenylpyrrole fungicide, is not currently registered for dollar spot control in China. In this study, the baseline sensitivity to fludioxonil was established using an in vitro assay for 105 isolates of S. homoeocarpa collected from 10 locations in different regions of China. Results indicate that the frequency distribution of effective concentration for 50% inhibition of mycelial growth (EC50) values of the S. homoeocarpa isolates was unimodal (W = 0.9847, P = .2730). The mean EC50 value was 0.0020 ±â€¯0.0006 µg/ml with a range from 0.0003 to 0.0035 µg/ml. A total of 7 fludioxonil-resistant mutants were obtained in laboratory, the mutants were stable in fludioxonil sensitivity after the 10th transfer, with resistance factor (RF) ranging from 4.320 to >13,901.4. The mutants showed a positive cross-resistance between fludioxonil and the dicarboximide fungicide iprodione, but not propiconazole, fluazinam, and thiophanate-methyl. When mycelial growth rate, pathogenicity and osmotic sensitivity were assessed, the mutants decreased in the fitness compared with their parental isolates. Sequence alignment of the histidine kinase gene Shos1 revealed a 13-bp fragment deletion only in one mutant, no mutations were observed on Shos1 in the rest resistant mutants.

Genetic Differentiation Associated with Fumonisin and Gibberellin Production in Japanese Fusarium fujikuroi.

Fusarium fujikuroi is a pathogenic fungus that infects rice. It produces several important mycotoxins, such as fumonisins. Fumonisin production has been detected in strains of maize, strawberry, and wheat, whereas it has not been detected in strains from rice seedlings infested with bakanae disease in Japan. We investigated the genetic relationships, pathogenicity, and resistance to a fungicide, thiophanate-methyl (TM), in 51 fumonisin-producing strains and 44 nonproducing strains. Phylogenetic analyses based on amplified fragment length polymorphism (AFLP) markers and two specific genes (a combined sequence of translation elongation factor 1α [TEF1α] and RNA polymerase II second-largest subunit [RPB2]) indicated differential clustering between the fumonisin-producing and -nonproducing strains. One of the AFLP markers, EATMCAY107, was specifically present in the fumonisin-producing strains. A specific single nucleotide polymorphism (SNP) between the fumonisin-producing and nonproducing strains was also detected in RPB2, in addition to an SNP previously found in TEF1α. Gibberellin production was higher in the nonproducing than in the producing strains according to an in vitro assay, and the nonproducing strains had the strongest pathogenicity with regard to rice seedlings. TM resistance was closely correlated with the cluster of fumonisin-nonproducing strains. The results indicate that intraspecific evolution in Japanese F. fujikuroi is associated with fumonisin production and pathogenicity. Two subgroups of Japanese F. fujikuroi, designated G group and F group, were distinguished based on phylogenetic differences and the high production of gibberellin and fumonisin, respectively.IMPORTANCEFusarium fujikuroi is a pathogenic fungus that causes rice bakanae disease. Historically, this pathogen has been known as Fusarium moniliforme, along with many other species based on a broad species concept. Gibberellin, which is currently known as a plant hormone, is a virulence factor of F. fujikuroi Fumonisin is a carcinogenic mycotoxin posing a serious threat to food and feed safety. Although it has been confirmed that F. fujikuroi produces gibberellin and fumonisin, production varies among strains, and individual production has been obscured by the traditional appellation of F. moniliforme, difficulties in species identification, and variation in the assays used to determine the production of these secondary metabolites. In this study, we discovered two phylogenetic subgroups associated with fumonisin and gibberellin production in Japanese F. fujikuroi.

Thiophanate-methyl resistance in Sclerotinia homoeocarpa from golf courses in China.

Sclerotinia homoeocarpa causes dollar spot disease on many turfgrass species and is a significant problem worldwide. Thiophanate-methyl (TM), a methyl benzimidazole carbamate (MBC) fungicide, has been used for over forty years to manage dollar spot. Here we describe genetic mutations linked to three distinct TM fungicide resistance phenotypes: sensitive (S), moderately resistant (MR) and highly resistant (HR). These were established using multiple doses of TM, compared to previous studies using single discriminatory doses. In total, 19 S, 3 MR and 22 HR isolates were detected. Analysis of the ß-tubulin gene revealed the MR isolates had a point mutation from T to A at codon 200 changing phenylalanine (TTC) to tyrosine (TAC). Twenty HR isolates had a mutation at codon 198 changing glutamic acid (GAG) to alanine (GCG) and two HR isolates had a mutation at codon 198 changing glutamic acid (GAG) to lysine (AAG). Allele-specific PCR assays were developed for rapid detection of these mutations in isolates of S. homoeocarpa. In addition, our results suggest a two-dose system for in vitro screening provides useful information for monitoring the development of resistance.

Potential Impact of Populations Drift on Botrytis Occurrence and Resistance to Multi- and Single-Site Fungicides in Florida Southern Highbush Blueberry Fields.

Incidence of blossom blight and Botrytis fruit rot (BFR), caused by Botrytis cinerea, on two southern highbush blueberry cultivars was evaluated in several blueberry fields grown in the vicinity (BB-Str(+)) or not (BB-Str(-)) of strawberry fields in central Florida. Blossom blight and BFR incidence were higher in BB-Str(+) fields in 2014 and significantly higher in 2015 compared to BB-Str(-) fields. In total, 613 B. cinerea isolates (i.e., 181 and 432 isolates from BB-Str(-) and BB-Str(+) fields, respectively) were collected. The isolates were evaluated for sensitivity to eight single-site and one multisite fungicides using a spore germination and a germ tube elongation assay. Overall, 5, 15, 24, 28, 54, and 93% of isolates collected from BB-Str(-) were resistant to penthiopyrad, cyprodinil, boscalid, fenhexamid, pyraclostrobin, and thiophanate-methyl, respectively. Respective resistance frequencies in BB-Str(+) isolates were 10, 30, 65, 66, 89, and 99%. Resistance frequencies for all fungicides were always higher in BB-Str(+) fields compared to BB-Str(-) fields. Isolates exhibiting resistance to six or five fungicides simultaneously were predominant (50 to 70%) in blueberry fields regardless if they were grown in the vicinity of strawberry fields or not. Among 308 and 305 B. cinerea isolates tested in 2014 and 2015, 41.8 and 47.1%, respectively, showed reduced sensitivity to the multisite fungicide captan. The lower label rate of captan applied preventively did not control isolates with reduced sensitivity on detached blueberry fruit. These findings suggest a potential population flow between strawberry and blueberry fields that may impact blossom blight and gray mold development in blueberry fields. The relatively lower fungicide input applied to blueberry fields compared with strawberry fields seems to be sufficient to select for resistance and multiple-resistant phenotypes in B. cinerea populations in blueberry.

Sensitivity of Botrytis cinerea Isolates from Conventional and Organic Strawberry Fields in Brazil to Azoxystrobin, Iprodione, Pyrimethanil, and Thiophanate-Methyl.

Botrytis fruit rot, caused by Botrytis cinerea, is one of the most important strawberry diseases worldwide, and fungicide applications are often used to manage the disease in commercial production. Isolates of B. cinerea were collected from conventional and organic strawberry fields in four Brazilian States from 2013 to 2015 and their sensitivity to the main single-site mode-of action fungicides used in Brazil was tested. Resistance to azoxystrobin, iprodione, pyrimethanil, and thiophanate-methyl was found and values for effective concentration that inhibited mycelial growth by 50% were higher than 71.9, 1.2, 5.0, and 688 µg/ml, respectively, regardless the production system. Resistance to these fungicides was observed in 87.5, 76.6, 23.4, and 92.2% of isolates from conventional fields and 31.4, 22.9, 14.3, and 51.4% of isolates from organic fields, respectively. Moreover, frequencies of isolates with multiple fungicide resistance to the four active ingredients were 20.6 and 2.8% whereas 6.3 and 27.8% were sensitive to the four fungicides for conventional and organic areas, respectively. Molecular analyses of the cytochrome b, ß-tubulin, and bos1 genes revealed the presence of G143A; E198A; and I365 N/S, Q369P, or N373S mutations, respectively, in resistant isolates of B. cinerea. Field rates of fungicides sprayed preventively to inoculated strawberry fruit failed to control disease caused by the respective resistant isolates.

Oxidative stress and histopathological changes induced by methylthiophanate, a systemic fungicide, in blood, liver and kidney of adult rats.

BACKGROUND: Methyl-thiophanate (MT), a fungicide largely used in agriculture throughout the world including Tunisia, protects many vegetables, fruits and field crops against a wide spectrum of fungal diseases. Oxidative stress has been proposed as a possible mechanism involved in MT toxicity on non-target organism. METHODS: In the present study, the effect of MT injected intraperitoneally to adult rats at 300 or 500 mg/kg of body weight was studied on blood, liver and kidney. RESULTS: Our results showed 3 days after MT injection, a significant decrease in hemoglobin and hematocrit values. A disruption in total white blood cells and platelets also occurred. Accordingly, an increased in malondialdehyde, H2O2 and advanced oxidation protein levels in liver and kidney were noted with the two doses. A significant change in plasma biomarkers and organ enzymatic and non-enzymatic activities were observed after MT treatment. The modifications in biochemical parameters were substantiated by histopathological data. CONCLUSION: These data confirmed the pro-oxidant effects of this fungicide. Accordingly, care must be taken to avoid mammalian and human exposure to MT.

Thiophanate-Methyl Resistance and Fitness Components of Colletotrichum musae Isolates from Banana in Brazil.

Anthracnose, caused by Colletotrichum musae, is the most important postharvest disease of banana and is widely distributed among the banana production regions in Brazil. Although thiophanate-methyl is a fungicide frequently used in Brazilian banana orchards to control Sigatoka leaf spot, Collettotrichum populations are also exposed, resulting in the evolution of fungicide resistance and the inability to manage banana anthracnose. We investigated 139 Brazilian isolates of C. musae for thiophanate-methyl sensitivity in vitro. The 50% mycelial growth inhibition (EC50) values varied between 0.003 and 48.73 µg/ml. One-hundred and thirty isolates were classified as sensitive, with EC50 values ranging from 0.003 to 4.84 µg/ml, while the remaining nine isolates were considered moderately resistant, with EC50 values ranging between 10.43 and 48.73 µg/ml. Resistant or highly resistant isolates (EC50 > 100 µg/ml) were not found. A substitution of TAC for TTC at codon 200 in a coding region of the ß-tubulin gene was associated with the moderately resistant phenotype. Applications of thiophanate-methyl formulation to detached banana fruit at the label rate (500 µg/ml) showed low efficacy in controlling the moderately resistant isolates on banana fruits. However, there is no indication of a reduction in fitness associated with fungicide resistance as sensitive and moderately resistant isolates do not differ with respect to mycelial growth rate (P = 0.098), spore production (P = 0.066), spore germination (P = 0.366), osmotic sensitivity (P = 0.051), and virulence (P = 0.057). Our results revealed absence of adaptability cost for the moderately resistant isolates, suggesting that they can be dominant in population if the fungicide continue to be applied.

Resistance to Increasing Chemical Classes of Fungicides by Virtue of "Selection by Association" in Botrytis cinerea.

Previous research has shown that Botrytis cinerea isolates with resistance to multiple chemical classes of fungicides exist in eastern strawberry fields. In this study, the fungicide resistance profiles of 2,130 isolates from flowers of commercial strawberry fields located in multiple states was determined over four consecutive strawberry production seasons. Producers were asked to alternate single-site fungicides that were considered low risk in their specific location based on resistance monitoring results in their fields. This recommendation led to an increase of chemical class diversity used in the spray programs. Results indicated that simultaneous resistance in individual isolates to two, three, four, five, six, and seven classes of fungicides increased over time. The increase in chemical class resistances within isolates was likely due to a process we termed "selection by association", where fungicide resistance traits were often linked to the trait being selected rather than the selectable trait itself. Data analysis also indicated that the odds were highest for isolates resistant to one chemical class (1CCR) to be resistant to thiophanate-methyl; for 2CCR isolates to be resistant to thiophanate-methyl and pyraclostrobin; and for 3CCR isolates to be resistant to thiophanate-methyl, pyraclostrobin, and either cyprodinil or fenhexamid. We hypothesize that the more chemical classes are used in a spray program, the faster isolates will be selected with increasing numbers of chemical class resistances by virtue of selection by association if such isolates preexist in the population.

Sensitivity of Podosphaera xanthii populations to anti-powdery-mildew fungicides in Spain.

BACKGROUND: Cucurbit powdery mildew caused by Podosphaera xanthii limits crop production in Spain, where disease control is largely dependent on fungicides. In previous studies, high levels of resistance to QoI and DMI fungicides were documented in south-central Spain. The aim of this study was to investigate the sensitivity of P. xanthii populations to other fungicides and to provide tools for improved disease management. RESULTS: Using a leaf-disc assay, sensitivity to thiophanate-methyl, bupirimate and quinoxyfen of 50 isolates of P. xanthii was analysed to determine discriminatory concentrations between sensitive and resistant isolates. With the exception of thiophanate-methyl, no clearly different groups of isolates could be identified, and as a result, discriminatory concentrations were established on the basis of the maximum fungicide field application rate. Subsequently, a survey of P. xanthii resistance to these fungicides was carried out by testing a collection of 237 isolates obtained during the 2002-2011 cucurbit growing seasons. This analysis revealed very high levels of resistance to thiophanate-methyl (95%). By contrast, no resistance to bupirimate and quinoxyfen was found. CONCLUSION: Results suggest that thiophanate-methyl has become completely ineffective for controlling cucurbit powdery mildew in Spain. By contrast, bupirimate and quinoxyfen remain as very effective tools for cucurbit powdery mildew management. © 2014 Society of Chemical Industry.

Methyl-thiophanate increases reactive oxygen species production and induces genotoxicity in rat peripheral blood.

Methylthiophanate is one of the widely used fungicides to control important fungal diseases of crops. The aim of this study was to elucidate the short-term hematoxicity and genotoxicity effects of methylthiophanate administered by intraperitoneal way at three doses (300, 500 and 700 mg/kg of body weight) after 24, 48 and 72 h. Our results showed, 24 h after methylthiophanate injection, a hematological perturbation such as red blood cells (p < 0.05, p < 0.05 and p < 0.01) and hemoglobin content (p < 0.05), respectively, and a noticeable genotoxic effect in WBC evidenced by a significant increase in the frequency of the micronuclei and a decrease in cell viability. An increase in erythrocyte osmotic fragility was also noted after 24 and 48 h of methylthiophanate treatment at graded doses. A significant increase in hydrogen peroxide, advanced oxidation of protein products and malondialdehyde levels, in erythrocytes of methylthiophanate-treated rats with 300, 500 and 700 mg/kg of body weight, was also observed after 24 h of treatment (p < 0.05, p < 0.01 and p < 0.001, respectively), suggesting the implication of oxidative stress in its toxicity. Antioxidants activities of superoxide dismutase and glutathione peroxidase in erythrocytes significantly increased (p < 0.001) 24 h after the highest dose injected. While all these parameters were improved after 72 h of methylthiophanate injection (300, 500 and 700 mg/kg body weight). In conclusion, these data showed that the exposure of adult rats to methylthiophanate resulted in oxidative stress leading to hematotoxicity and the impairment of defence system, confirming the pro-oxidant and genotoxic effects of this fungicide.

Simultaneous quantification of alleles E198A and H6Y in the ß-tubulin gene conferring benzimidazole resistance in Monilinia fructicola using a duplex real-time (TaqMan) PCR.

BACKGROUND: The benzimidazole fungicide thiophanate-methyl is commonly used for the control of brown rot of stone fruits. Low and high levels of resistance to this fungicide have been found in field isolates of the causal pathogen Monilinia fructicola. RESULTS: The minor groove binding (MGB) TaqMan probes specific for alleles E198A and H6Y conferring the high and low levels of resistance in the ß-tubulin gene of M. fructicola were designed. A duplex real-time PCR assay based on these probes was developed for simultaneous quantification of both mutations in a pathogen population. The specificity tests for the primers and probes were conducted using different fungal species of stone and pome fruit pathogens. Similar results were obtained between the duplex real-time (TaqMan) PCR assay and the conventional method to quantify the frequencies of alleles E198A and H6Y of eight samples from different peach orchards. CONCLUSION: The MGB TaqMan probe based duplex real-time PCR provides a useful tool for simultaneous quantification of both alleles, E198A and H6Y, conferring high and low resistance, and has a potential in monitoring the benzimidazole-resistance in M. fructicola populations in stone and pome fruit orchards.

Unlikely remedy: fungicide clears infection from pathogenic fungus in larval southern leopard frogs (Lithobates sphenocephalus).

Amphibians are often exposed to a wide variety of perturbations. Two of these, pesticides and pathogens, are linked to declines in both amphibian health and population viability. Many studies have examined the separate effects of such perturbations; however, few have examined the effects of simultaneous exposure of both to amphibians. In this study, we exposed larval southern leopard frog tadpoles (Lithobates sphenocephalus) to the chytrid fungus Batrachochytrium dendrobatidis and the fungicide thiophanate-methyl (TM) at 0.6 mg/L under laboratory conditions. The experiment was continued until all larvae completed metamorphosis or died. Overall, TM facilitated increases in tadpole mass and length. Additionally, individuals exposed to both TM and Bd were heavier and larger, compared to all other treatments. TM also cleared Bd in infected larvae. We conclude that TM affects larval anurans to facilitate growth and development while clearing Bd infection. Our findings highlight the need for more research into multiple perturbations, specifically pesticides and disease, to further promote amphibian heath.