Positive Cross-resistance Research Articles

Resistance risk assessment of Rhizoctonia solani to four fungicides.

Hexaconazole, thifluzamide, difenoconazole and azoxystrobin are widely used fungicides for the control of Rhizoctonia solani in China. However, few studies have assessed the sensitivity and resistance risk of R. solani to these four fungicides. The sensitivities of 126 R. solani isolates to hexaconazole, thifluzamide, difenoconazole and azoxystrobin were determined, with average half maximal effective concentration (EC50) values of 0.0386, 0.0659, 0.663 and 1.508 μg mL-1, respectively. Field resistance monitoring of the four fungicides showed that the three isolates had moderate resistance to difenoconazole. Resistant mutants to the four fungicides were obtained by fungicide adaptation, and resistance could be stably inherited by most mutants. Compared with those of the parent isolates, the biological characteristics of hexaconazole-resistant mutants exhibited enhanced or similar compound fitness index (CFI), whereas most of the other mutants displayed reduced or comparable CFI. There was evidence of positive cross-resistance between hexaconazole and difenoconazole. In the presence of fungicides, the expression of the CYP51 genes in hexaconazole- and difenoconazole-resistant mutants significantly increased, the expression of SDH genes in thifluzamide-resistant mutants significantly decreased, and the expression of the Cyt b gene in azoxystrobin-resistant mutants did not significantly change. Based on these data, we speculated that R. solani had a low-to-medium resistance risk to four fungicides. The change of target gene expression may be one of the reasons for fungicide resistance in R. solani. This study provides a theoretical basis for monitoring resistance emergence and developing resistance management strategies to control R. solani. © 2024 Society of Chemical Industry.

Potential Mechanisms of Hexaconazole Resistance in Fusarium graminearum.

Fusarium head blight (FHB) caused by Fusarium graminearum is a serious fungal disease that can dramatically impact wheat production. At present, disease control is mainly achieved by the use of chemical fungicides. Hexaconazole (IUPAC name: 2(2,4-dichlorophenyl)-1-(1,2,4-triazol-1-yl)hexan-2-ol) is a widely used triazole fungicide, but the sensitivity of F. graminearum to this compound has yet to be established. The current study found that the EC50 values of 83 field isolates of F. graminearum ranged between 0.06 and 4.33 μg/ml, with an average EC50 value of 0.78 μg/ml. Assessment of four hexaconazole-resistant laboratory mutants of F. graminearum revealed that their mycelial growth and pathogenicity were reduced compared with their parental isolates and that asexual reproduction was reduced by resistance to hexaconazole. Meanwhile, the mutants appeared to be more sensitive to abiotic stress associated with SDS and H2O2, while their tolerance to high concentrations of Congo red, and Na+ and K+ increased. Molecular analysis revealed numerous point mutations in the FgCYP51 target genes that resulted in amino acid substitutions, including L92P and N123S in FgCYP51A, as well as M331V, F62L, Q252R, A412V, and V488A in FgCYP51B, and S28L, S256A, V307A, D287G, and R515I in FgCYP51C, three of which (S28L, S256A, and V307A) were conserved in all of the resistant mutants. Furthermore, the expression of the FgCYP51 genes in resistant strains was found to be significantly (P < 0.05) reduced compared with their sensitive parental isolates. Positive cross-resistance was found between hexaconazole and metconazole and flutriafol, as well as with the diarylamine fungicide fluazinam, but not with propiconazole, and the phenylpyrrole fungicide fludioxonil, or with tebuconazole, which actually exhibited negative cross-resistance. These results provide valuable insight into resistant mechanisms to triazole fungicides in F. graminearum, as well as the appropriate selection of fungicide combinations for the control of FHB to ensure optimal wheat production.

Amino acid substitutions in succinate dehydrogenase complex conferring resistance to the SDHI fungicide pydiflumetofen in Cochlibolus heterostrophus causing southern corn leaf blight1

Southern corn leaf blight (SCLB) caused by Cochlibolus heterostrophus, is a widespread foliar disease that has a substantial impact on maize yield in the Huanghuaihai region of China. Pydiflumetofen (Pyd), a new succinate dehydrogenase inhibitor (SDHI), has been found as a promising fungicide for the efficient control of SCLB, however, resistance of C. heterostrophus to Pyd has not been studied well. Here, five Pyd-resistant mutants were generated through fungicide adaptation. Sequence alignment analysis revealed that these mutants primarily mutated in ChSdhB and ChSdhD, with 3 genotypes: ChSdhBH277Y, ChSdhBI279T and ChSdhDH133Y, exhibiting two distinct categories of resistance: high resistance (HR) and moderate resistance (MR), which resistance factors (RF) is 214.22 and 44.33-53.67, respectively. These mutants were more pathogenic than the wild-type parental strains, but there was a significant reduction in mycelial growth rate and sporulation in the resistant mutants, indicating a significant fitness cost associated with resistance to Pyd. In addition, this study revealed a positive cross-resistance between Pyd and another SDHI fungicide cyclobutrifluram. However, no cross-resistance was found between Pyd and other classes of fungicide, including prochloraz, fludioxonil, iprodioneand pyraclostrobin. Homology modeling and molecular docking further confirmed that point mutation of ChSdhBH277Y, ChSdhBI279T, and ChSdhDH133Y could reduce binding affinity between Pyd and its target subunits from −74.07, −74.07, −152.52 kcal mol-1 to −3.90, −4.95, −9.93 kcal/mol, respectively. These findings not only provided valuable insights for managing SCLB caused by C. heterostrophus, but also enhanced our understanding of molecular mechanism underlying plant pathogen resistance to Pyd.

Amino acid mutation of succinate dehydrogenase complex induced resistance to benzovindiflupyr in Magnaporthe oryzae

Magnaporthe oryzae is a rice blast pathogen that seriously threatens rice yield. Benzovindiflupyr is a succinate dehydrogenase inhibitor (SDHI) fungicide that effectively controls many crop diseases. Benzovindiflupyr has a strong inhibitory effect on M. oryzae; however, control of rice blast by benzovindiflupyr and risk of resistance to benzovindiflupyr are not well studied in this pathogen. In this study, six benzovindiflupyr-resistant strains were obtained by domestication induced in the laboratory. The MoSdhBH245D mutation was the cause of M. oryzae resistance to benzovindiflupyr, which was verified through succinate dehydrogenase (SDH) activity assays, molecular docking, and site-specific mutations. Survival fitness analysis showed no significant difference between the benzovindiflupyr-resistant and parent strains. Positive cross-resistance to benzovindiflupyr and other SDHIs and negative cross-resistance to azoxystrobin were observed. Therefore, the risk of benzovindiflupyr resistance in M. oryzae might be medium to high. It should be combined with other classes of fungicides (tebuconazole and azoxystrobin) to slow the development of resistance.

Various amino acid substitutions in succinate dehydrogenase complex regulating differential resistance to pydiflumetofen in Magnaporthe oryzae

Rice blast, caused by Magnaporthe oryzae, is a devastating fungal disease worldwide. Pydiflumetofen (Pyd) is a new succinate dehydrogenase inhibitor (SDHI) that exhibited anti-fungal activity against M. oryzae. However, control of rice blast by Pyd and risk of resistance to Pyd are not well studied in this pathogen. The baseline sensitivity of 109 M. oryzae strains to Pyd was determined using mycelial growth rate assay, with EC50 values ranging from 0.291 to 2.1313 μg/mL, and an average EC50 value of 1.1005 ± 0.3727 μg/mL. Totally 28 Pyd-resistant (PydR) mutants with 15 genotypes of point mutations in succinate dehydrogenase (SDH) complex were obtained, and the resistance level could be divided into three categories of very high resistance (VHR), high resistance (HR) and moderate resistance (MR) with the resistance factors (RFs) of >1000, 105.74–986.13 and 81.92–99.48, respectively. Molecular docking revealed that all 15 mutations decreased the binding-force score for the affinity between Pyd and target subunits, which further confirmed that these 15 genotypes of point mutations were responsible for the resistance to Pyd in M. oryzae. There was positive cross resistance between Pyd and other SDHIs, such as fluxapyroxad, penflufen or carboxin, while there was no cross-resistance between Pyd and carbendazim, prochloraz or azoxystrobin in M. oryzae, however, PydR mutants with SdhBP198Q, SdhCL66F or SdhCL66R genotype were still sensitive to the other 3 SDHIs, indicating lack of cross resistance. The results of fitness study revealed that the point mutations in MoSdhB/C/D genes might reduce the hyphae growth and sporulation, but could improve the pathogenicity in M. oryzae. Taken together, the risk of resistance to Pyd might be moderate to high, and it should be used as tank-mixtures with other classes of fungicides to delay resistance development when it is used for the control of rice blast in the field.

Resistance risk assessment for benzovindiflupyr in Sclerotium rolfsii and transmission of resistance genes among population.

Sclerotium rolfsii is a destructive soil-borne fungal pathogen which is distributed worldwide. In previous study, the succinate dehydrogenase inhibitor (SDHI) fungicide benzovindiflupyr has been identified for its great antifungal activity against Sclerotium rolfsii. This study is aimed to investigate the resistance risk and mechanism of benzovindiflupyr in Sclerotium rolfsii. Eight stable benzovindiflupyr-resistant isolates were generated by fungicide adaptation. Although the obtained eight resistant isolates have a stronger pathogenicity than the parental sensitive isolate, they have a fitness penalty in the mycelial growth and sclerotia formation compared to the parental isolate. A positive cross-resistance existed in the resistant isolates between benzovindiflupyr and thifluzamide, carboxin, boscalid and isopyrazam. Three-point mutations, including SdhBN180D, SdhCQ68E and SdhDH103Y, were identified in the benzovindiflupyr-resistant isolates. However, molecular docking analysis indicated that only SdhDH103Y could influence the sensitivity of Sclerotium rolfsii to benzovindiflupyr. After mycelial co-incubation of resistant isolates and the sensitive isolate, resistance genes may be transmitted to the sensitive isolate. The in vivo efficacy of benzovindiflupyr and thifluzamide against benzovindiflupyr-resistant isolates was a little lower than that against the sensitive isolate but with no significant difference. The results suggested a low to medium resistance risk of Sclerotium rolfsii to benzovindiflupyr. However, once resistance occurs, it is possible to spread in the population of Sclerotium rolfsii. This study is helpful to understanding the risk and mechanism of resistance to benzovindiflupyr in multinucleate pathogens such as Sclerotium rolfsii. © 2024 Society of Chemical Industry.

Binding Mode and Molecular Mechanism of the Two-Component Histidine Kinase Bos1 of Botrytis cinerea to Fludioxonil and Iprodione.

Gray mold caused by Botrytis cinerea is among the 10 most serious fungal diseases worldwide. Fludioxonil is widely used to prevent and control gray mold due to its low toxicity and high efficiency; however, resistance caused by long-term use has become increasingly prominent. Therefore, exploring the resistance mechanism of fungicides provides a theoretical basis for delaying the occurrence of diseases and controlling gray mold. In this study, fludioxonil-resistant strains were obtained through indoor drug domestication, and the mutation sites were determined by sequencing. Strains obtained by site-directed mutagenesis were subjected to biological analysis, and the binding modes of fludioxonil and iprodione to Botrytis cinerea Bos1 BcBos1 were predicted by molecular docking. The results showed that F127S, I365S/N, F127S + I365N, and I376M mutations on the Bos1 protein led to a decrease in the binding energy between the drug and BcBos1. The A1259T mutation did not lead to a decrease in the binding energy, which was not the cause of drug resistance. The biological fitness of the fludioxonil- and point mutation-resistant strains decreased, and their growth rate, sporulation rate, and pathogenicity decreased significantly. The glycerol content of the sensitive strains was significantly lower than that of the resistant strains and increased significantly after treatment with 0.1 μg/ml of fludioxonil, whereas that of the resistant strains decreased. The osmotic sensitivity of the resistant strains was significantly lower than that of the sensitive strains. Positive cross-resistance was observed between fludioxonil and iprodione. These results will help to understand the resistance mechanism of fludioxonil in Botrytis cinerea more deeply.

Molecular Mechanisms and Biological Characteristics of Botrytis cinerea Field Isolate Resistance to Pyrisoxazole in Liaoning Province.

Botrytis cinerea is a broad-host-range necrotrophic phytopathogen responsible for serious diseases in leading crops worldwide. The novel sterol 14α-demethylase inhibitor (DMI) pyrisoxazole was recently registered for the control of tomato gray mold caused by B. cinerea in China. One hundred fifty-seven isolates of B. cinerea were collected from tomato greenhouses in 14 cities of Liaoning Province from 2016 to 2021 and examined for sensitivity to pyrisoxazole, with a mean EC50 value of 0.151 μg/ml. Three highly resistant isolates, XD-5, DG-4, and GQ-3, were screened, and the EC50 values were 0.734, 0.606, and 0.639 μg/ml with corresponding resistance factors of 12.88, 10.63, and 11.21, respectively. Compared with field-sensitive strains, the highly resistant isolate XD-5 exhibited fitness defects in traits, including mycelial growth, conidial production, and pathogenicity, but DG-4 and GQ-3 did not experience fitness costs. Positive cross-resistance was observed only between pyrisoxazole and the DMIs tebuconazole and prochloraz but not between pyrisoxazole and the non-DMIs iprodione, procymidone, pyrimethanil, fludioxonil, fluazinam, and fluopyram. Sequence alignment of the CYP51 gene indicated that three point mutations were observed in the highly resistant mutant, namely, V24I in XD-5, G461S in GQ-3, and R464K in DG-4. When exposed to pyrisoxazole, the induced expression levels of the ABC transporter AtrD and MFS transporter Mfs1 increased in the resistant isolates compared with those in the sensitive isolates, whereas the expression level of the CYP51 gene did not change significantly. Molecular docking suggested that the G461S and R464K mutations both led to a decrease in the binding energy between CYP51 and pyrisoxazole, whereas no change was found with the V24I mutation. Thus, two point mutations in the CYP51 protein combined with induced expression of the Mfs1 and AtrD genes appeared to mediate the pyrisoxazole resistance of the highly resistant mutants DG-4 and GQ-3, while the overexpression of the Mfs1 and AtrD genes was responsible for the highly resistant mutant XD-5.

Resistance mechanism of Phomopsis longicolla to fludioxonil is associated with modifications in PlOS1, PlOS4 and PlOS5

Phomopsis longicolla, a causal agent of soybean root rot, stem blight, seed decay, pod and stem canker, which seriously affects the yield and quality of soybean production worldwide. The phenylpyrrole fungicide fludioxonil exhibits a broad spectrum and high activity against phytopathogenic fungi. In this study, the baseline sensitivity of 100 P. longicolla isolates collected from the main soybean production areas of China to fludioxonil were determined. The result showed that the EC50 values of all the P. longicolla isolates ranged from 0.013 to 0.035 μg/ml. Furthermore, 12 fludioxonil-resistance (FluR) mutants of P. longicolla were generated from 6 fludioxonil-sensitive (FluS) isolates. and the resistance factors (RF) of 12 FluR mutants were >3500. Sequence alignment showed that multiple mutation types were found in PlOS1, PlOS4 or/and PlOS5 of FluR mutants. All the FluR mutants exhibited fitness penalty in mycelial growth, conidiation, virulence and osmo-adaptation. Under fludioxonil or NaCl treatment condition, the glycerol accumulation was significantly increased in FluS isolates, but was slightly increased in FluR mutants, and the phosphorylation level of most FluR mutants was significantly decreased when compared to the FluS isolates. Additionally, positive cross-resistance was observed between fludioxonil and procymidone but not fludioxonil and pydiflumetofen, pyraclostrobin or fluazinam. This is first reported that the baseline sensitivity of P. longicolla to fludioxonil, as well as the biological and molecular characterizations of P. longicolla FluR mutants to fludioxonil. These results can provide scientific directions for controlling soybean diseases caused by P. longicolla using fludioxonil.

Bioactivity and Resistance Risk of Fluxapyroxad, a Novel SDHI Fungicide, in Didymella bryoniae.

Gummy stem blight, caused by Didymella bryoniae, is an important disease in watermelon in China. Fluxapyroxad, a new succinate dehydrogenase inhibitor fungicide, shows strong inhibition of the mycelia growth of D. bryoniae. However, its resistance risk in D. bryoniae is unclear. In this research, the sensitivities of 60 D. bryoniae strains to fluxapyroxad were investigated. The average EC50 value and MIC values of 60 D. bryoniae strains against fluxapyroxad were 0.022 ± 0.003 μg/ml and ≤0.1 μg/ml for mycelial growth, respectively. Eight fluxapyroxad-resistant mutants with medium resistance levels were acquired from three wild-type parental strains. The mycelial growth and dry weight of mycelia of most mutants were significantly lower than those of their parental strains. However, four resistant mutants showed a similar phenotype in pathogenicity compared with their parental strains. The above results demonstrated that there was a medium resistance risk for fluxapyroxad in D. bryoniae. The cross-resistance assay showed that there was positive cross-resistance between fluxapyroxad and pydiflumetofen, thifluzamide, and boscalid, but there was no cross-resistance between fluxapyroxad and tebuconazole and mepronil. These results will contribute to evaluating the resistance risk of fluxapyroxad for managing diseases caused by D. bryoniae and further increase our understanding about the mode of action of fluxapyroxad.

The HOG-pathway related AaOS1 leads to dicarboximide-resistance in field strains of Alternaria alternata and contributes, together with the Aafhk1, to mycotoxin production and virulence.

Garlic leaf spot (GLS) caused by Alternaria alternata is one of the main diseases in the garlic production areas, and its management heavily relies on dicarboximide fungicides. However, the efficacy of dicarboximides against the GLS disease has decreased year on year. In the present study, 10 of 148 A. alternata strains separated from Jiangsu Province were moderately resistant (MR) to a dicarboximide fungicide procymidone (ProMR). Positive cross-resistance was observed between Pro and iprodione (Ipro) or fludioxonil (Fld), but not between Pro and fluazinam or azoxystrobin. Mutations at AaOS1, but not Aafhk1, were confirmed to confer the Pro resistance by constructing replacement mutants, whereas mutations at both AaOS1 and Aafhk1 decreased the gene expression level of AapksI, as well as the ability to produce mycotoxin AOH (polyketide-derived alternariol) and virulence. Additionally, more genes (AaOS1 and Aafhk1) harboring the mutations experienced a larger biological fitness penalty. To our knowledge, this is the first report on Pro resistance selected in garlic fields, and mutations at AaOS1 of A. alternata causing a decreased ability to produce the mycotoxin AOH. © 2024 Society of Chemical Industry.

Resistance risk, resistance mechanism and the effect on DON production of a new SDHI fungicide cyclobutrifluram in Fusarium graminearum

Fusarium head blight in wheat is caused by Fusarium graminearum, resulting in significant yield losses and grain contamination with deoxynivalenol (DON), which poses a potential threat to animal health. Cyclobutrifluram, a newly developed succinate dehydrogenase inhibitor, has shown excellent inhibition of Fusarium spp. However, the resistance risk of F. graminearum to cyclobutrifluram and the molecular mechanism of resistance have not been determined. In this study, we established the average EC50 of a range of F. graminearum isolates to cyclobutrifluram to be 0.0110 μg/mL. Six cyclobutrifluram-resistant mutants were obtained using fungicide adaptation. All mutants exhibited impaired fitness relative to their parental isolates. This was evident from measurements of mycelial growth, conidiation, conidial germination, virulence, and DON production. Interestingly, cyclobutrifluram did not seem to affect the DON production of either the sensitive isolates or the resistant mutants. Furthermore, a positive cross-resistance was observed between cyclobutrifluram and pydiflumetofen. These findings suggest that F. graminearum carries a moderate to high risk of developing resistance to cyclobutrifluram. Additionally, point mutations H248Y in FgSdhB and A73V in FgSdhC1 of F. graminearum were observed in the cyclobutrifluram-resistant mutants. Finally, an overexpression transformation assay and molecular docking indicated that FgSdhBH248Y or FgSdhC1A73V could confer resistance of F. graminearum to cyclobutrifluram.

Mefentrifluconazole-Resistant Risk and Resistance-Related Point Mutation in FpCYP51B of Fusarium pseudograminearum.

Mefentrifluconazole, a triazole fungicide, exhibits remarkable efficacy in combating Fusarium spp. The mean EC50 value of mefentrifluconazole against 124 isolates of Fusarium pseudograminearum was determined to be 1.06 μg/mL in this study. Fungicide taming produced five mefentrifluconazole-resistant mutants with resistance factors ranging from 19.21 to 111.34. Compared to the original parental isolates, the fitness of three resistant mutants was much lower, while the remaining two mutants displayed enhanced survival fitness. There was evidence of positive cross-resistance between tebuconazole and mefentrifluconazole. Mefentrifluconazole resistance in F. pseudograminearum can be conferred by FpCYP51BL144F, which was identified in four mutants according to molecular docking and site-directed transformation experiments. Overexpression of FpCYP51s was also detected in the resistant mutants. In conclusion, mefentrifluconazole has a low-to-medium resistance risk in F. pseudograminearum, and the L144F mutation in FpCYP51B and the increased expression level of FpCYP51s may be responsible for mefentrifluconazole resistance in F. pseudograminearum.

Activity of fungicide cyclobutrifluram against Fusarium fujikuroi and mechanism of the pathogen resistance associated with point mutations in FfSdhB, FfSdhC2 and FfSdhD

Rice bakanae disease (RBD) is a devastating plant disease caused by Fusarium fujikuroi. This study aimed to evaluate the potential of cyclobutrifluram, a novel succinate dehydrogenase inhibitor (SDHI), to control RBD, and determine the risk and mechanism of resistance to cyclobutrifluram in F. fujikuroi. In vitro experiments showed that cyclobutrifluram significantly inhibited mycelial growth and spore germination, and altered the morphology of mycelia and conidia. Treatment with cyclobutrifluram significantly decreased mycotoxin production and increased cell membrane permeability in F. fujikuroi. The baseline sensitivity of 72 F. fujikuroi isolates to cyclobutrifluram was determined using mycelial growth and spore germination inhibition assays, which revealed EC50 values of 0.0114 – 0.1304 μg mL-1 and 0.0012 – 0.016 μg mL-1, with mean EC50 values of 0.0410 ± 0.0470 μg mL-1 and 0.0038 ± 0.0015 μg mL-1, respectively. Pot experiments demonstrated that the protective effect of cyclobutrifluram against F. fujikuroi was more significant than that of phenamacril and azoxystrobin, indicating that cyclobutrifluram is a promising antifungal agent for the control of RBD. Six cyclobutrifluram-resistant mutants of F. fujikuroi were obtained via fungicide adaptation. Moreover, these mutants exhibited weaker fitness than their parental isolate and positive cross-resistance with other SDHI fungicides, including pydiflumetofen and penflufen; however, no cross-resistance was detected with other classes of fungicides, including phenamacril, fludioxonil, prochloraz, or azoxystrobin. These results indicated that the resistance risk of F. fujikuroi to cyclobutrifluram might be moderate. Sequencing analysis revealed that mutations, including H248D in FfSdhB, A83V in FfSdhC2, and S106F and E166K in FfSdhD, contributed to resistance, which was confirmed by molecular docking and homologous replacement experiments. The results suggest a high potential for cyclobutrifluram to control RBD and a moderate resistance risk of F. fujikuroi to cyclobutrifluram, which are meaningful findings for the scientific application of cyclobutrifluram.

Resistance to the DMI fungicide mefentrifluconazole in Monilinia fructicola: risk assessment and resistance basis analysis.

Brown rot disease, caused by Monilinia fructicola, poses a significant challenge to peach production in China. The efficacy of mefentrifluconazole, a new triazole fungicide, in controlling brown rot in peaches has been remarkable. However, the resistance risk and mechanism associated with this fungicide remain unclear. This study was designed to assess the resistance risk of M. fructicola to mefentrifluconazole and reveal the potential resistance mechanism. The mean median effective concentration (EC50 ) of 101 M. fructicola isolates to mefentrifluconazole was 0.003 μg mL-1 , and the sensitivity exhibited a unimodal distribution. Seven mefentrifluconazole-resistant mutants were generated from three parental isolates in the laboratory through fungicide adaption. The biological characteristics of the resistant mutants revealed that three of them exhibited enhanced survival fitness compared to the parental isolates, whereas the remaining four mutants displayed reduced survival fitness. Mefentrifluconazole showed strong positive cross-resistance with fenbuconazole, whereas no cross-resistance was observed with pyrimethanil, procymidone or pydiflumetofen. No overexpression of MfCYP51 gene was detected in the resistant mutants. Multiple sequence alignment revealed that three resistant mutants (MXSB2-2, Mf12-1 and Mf12-2) had a point mutation (G461S) in MfCYP51 protein. Molecular docking techniques confirmed the contribution of this point mutation to mefentrifluconazole resistance. The risk of M. fructicola developing resistance to mefentrifluconazole is relatively low-to-medium and point mutation G461S in MfCYP51 could confer mefentrifluconazole resistance in M. fructicola. This study provided essential data for monitoring the emergence of resistance and developing resistance management strategies for mefentrifluconazole. © 2023 Society of Chemical Industry.

Sensitivity of Botrytis cinerea from Vineyards to Boscalid, Isofetamid, and Pydiflumetofen in Shandong Province, China.

Succinate dehydrogenase inhibitor (SDHI) fungicides are the most commonly and effectively used class of fungicides for controlling gray mold. Among them, only boscalid has been registered in China for controlling grape gray mold, whereas isofetamid and pydiflumetofen are two new SDHI fungicides that have demonstrated high efficacy against various fungal diseases. However, the sensitivity of Botrytis cinerea isolates from vineyards in China to these three fungicides is currently unknown. In this study, the sensitivity of 55 B. cinerea isolates from vineyards to boscalid, isofetamid, and pydiflumetofen was determined, with the effective concentrations for inhibiting 50% of spore germination (EC50) values ranging from 1.10 to 393, 0.0300 to 42.0, and 0.0990 to 25.5 μg ml-1, respectively. The resistance frequencies for boscalid, isofetamid, and pydiflumetofen were 60.0, 7.2, and 12.8%, respectively. Three mutations (H272R, H272Y, and P225F) were detected in the SdhB subunit, with H272R being the most prevalent (75.7%), followed by H272Y (16.2%) and P225F (8.1%). All three mutations are associated with resistance to boscalid, and of them, H272R mutants exhibited high resistance. Only P225F and H272Y mutants exhibited resistance to isofetamid and pydiflumetofen, respectively. A weakly positive cross-resistance relationship was observed between boscalid and pydiflumetofen (r = 0.38, P < 0.05). Additionally, the H272R mutants showed no significant fitness costs, whereas the remaining mutants exhibited reduced mycelial growth (P225F) and sporulation (H272Y and P225F). These results suggest that isofetamid and pydiflumetofen are effective fungicides against B. cinerea in vineyards, but appropriate rotation strategies must be implemented to reduce the selection of existing SDHI-resistant isolates.

Heterogeneous Phenotypic Responses of Antibiotic-Resistant Salmonella Typhimurium to Food Preservative-Related Stresses.

This study was designed to evaluate the response of antibiotic-resistant Salmonella Typhimurium to food preservative-related stresses, such as lactic acid and sodium chloride (NaCl). S. Typhimurium cells were exposed to 1 µg/mL of ciprofloxacin (CIP), 0.2% lactic acid (LA), 6% NaCl, CIP followed by LA (CIP-LA), and CIP followed by NaCl (CIP-NaCl). The untreated S. Typhimurium cells were the control (CON). All treatments were as follows: CON, CIP, LA, NaCl, CIP-LA, and CIP-NaCl. The phenotypic heterogeneity was evaluated by measuring the antimicrobial susceptibility, bacterial fluctuation, cell injury, persistence, and cross-resistance. The CIP, CIP-LA, and CIP-NaCl groups were highly resistant to ciprofloxacin, showing MIC values of 0.70, 0.59, and 0.54 µg/mL, respectively, compared to the CON group (0.014 µg/mL). The susceptibility to lactic acid was not changed after exposure to NaCl, while that to NaCl was decreased after exposure to NaCl. The Eagle phenomenon was observed in the CIP, CIP-LA, and CIP-NaCl groups, showing Eagle effect concentrations (EECs) of more than 8 µg/mL. No changes in the MBCs of lactic acid and NaCl were observed in the CIP, LA, and CIP-LA groups, and the EECs of lactic acid and NaCl were not detected in all treatments. The bacterial fluctuation rates of the CIP-LA and CIP-NaCl groups were considerably increased to 33% and 41%, respectively, corresponding to the injured cell proportions of 82% and 89%. CIP-NaCl induced persister cells as high as 2 log cfu/mL. The LA and NaCl treatments decreased the fitness cost. The CIP-NaCl treatment showed positive cross-resistance to erythromycin (ERY) and tetracycline (TET), while the LA and NaCl treatments were collaterally susceptible to chloramphenicol (CHL), ciprofloxacin (CIP), piperacillin (PIP), and TET. The results provide new insight into the fate of antibiotic-resistant S. Typhimurium during food processing and preservation.

Diversity and Characterization of Resistance to Pyraclostrobin in Colletotrichum spp. from Strawberry

Strawberry crown rot poses a significant menace to strawberry production during the seedling stage, and the main pathogen is Colletotrichum spp. Pyraclostrobin is one of the main fungicides that have been registered to control anthracnose caused by Colletotrichum spp. The diversity of pathogens and the risk of fungicide resistance may change from year to year. In order to explore the diversity of pathogens causing crown rot and evaluate the resistance risk of pathogens to pyraclostrobin in different years, crown rot samples were collected in Jiande, Zhejiang Province in 2019 and 2021, and the pathogens were identified. Based on the morphological identification and phylogenetic analysis based on ACT, CAL, CHS, GAPDH, and ITS, all 55 strains were identified as C. gloeosporioides species complexes, including 23 C. siamense isolates and 2 C. fructicola isolates in 2019, and all isolates were identified as C. siamense in 2021. C. siamense was the dominant pathogen of strawberry crown rot in 2019 and 2021. The resistance frequencies of the isolates collected in 2019 and 2021 to pyraclostrobin were 69.57% and 100%, respectively. In general, compared to that in 2019, the resistance frequencies of the pathogen to pyraclostrobin increased in 2021. In terms of fitness, there was no significant difference between resistant strains and sensitive strains in the mycelium growth rate, sporulation and spore germination rate. In addition, the resistant mutants exhibited positive cross-resistance to kresoxim-methyl and azoxystrobin. A sequential analysis of cytochrome b gene showed that C. siamense resistance to pyraclostrobin is linked to the G143A point mutation. Our study indicated that the risk of resistance a fungicide gradually increases with the increase in use years, and in order to reduce the emergence and spread of resistant populations, we should choose fungicides of different mechanisms of action for rotation to reduce the risk of resistance development.

Three point mutations in AaCYP51 combined with induced overexpression of AaCYP51 conferred low-level resistance to mefentrifluconazole in Alternaria alternata

Tomato early blight is a significant disease that causes substantial losses to tomato yield and quality. Mefentrifluconazole, an isopropanol-azole subgroup of triazole fungicides, has been registered in China for controlling various plant diseases, including tomato early blight, grape anthracnose, and apple brown spot. However, limited information is available on the mefentrifluconazole resistance risk and mechanism in plant pathogens. The sensitivity to mefentrifluconazole of 122 isolates of Alternaria alternata, one of the causal agents of tomato early blight, collected from different provinces in China, was evaluated. The results showed a unimodal curve for the sensitivity frequency, with an average EC50 of 0.306 μg/mL. Through fungicide adaption, six resistant mutants (N4, N5, T4, T5, NG1, and NG10) were obtained from three parental isolates, with a mutation frequency of 3.28 × 10−4 and resistance factors ranging between 19 and 147. The survival fitness of the resistant mutants, except for NG1, was significantly lower than that of their parental isolates. Positive cross-resistance was observed between mefentrifluconazole and difenoconazole or fenbuconazole, whereas no cross-resistance was found with three non-DMI fungicides. Furthermore, three distinct point mutations were detected in the AaCYP51 protein of the resistant mutants: I300S in T4 and T5; A303T in N4, NG1, and NG10; and A303V in N5. Compared to the parental isolates, the AaCYP51 gene was overexpressed in all six resistant mutants when treated with mefentrifluconazole. In summary, the resistance risk of A. alternata to mefentrifluconazole was low, and point mutations and overexpression of the AaCYP51 gene were identified as contributing factors to mefentrifluconazole resistance in A. alternata.

Helicoverpa armigera (Hubner) Associated Cross Resistance Patterns in South Indian Crop Ecosystem

Aim: To study the cross resistance patterns associated with Mahaboobnagar, Raichur, Nagpur populations of Helicoverpa armigera (Hubner).&#x0D; Study design: Bioassay&#x0D; Place and Duration of Study: The experiment was carried out from February 2010 to May 2011 at Department of Entomology, College of Agriculture, Rajendranagar, Hyderabad, Telangana.&#x0D; Methodology: Helicoverpa armigera was selected for indoxacarb in F1 and F2 continuously then the population subjected to different selected insecticides to know the cross resistance patterns associated.&#x0D; Results: Mahaboobnagar population recorded 1.109 and 0.816 fold resistance at LD50 and LD90, respectively, while Raichur population has developed still higher levels of relative resistance by 1.591 and 0.846 fold when compared with the Nagpur population at LD50 and LD90, respectively. Similarly, the Raichur population has developed 1.435 and 1.037 folds relative resistance at LD50­ and LD90, respectively as compared with the Mahaboobnagar population.&#x0D; The Mahaboobnagar population resistant to indoxacarb at F3, when subjected to selected insecticides like cypermethrin, methomyl, spinosad showed a negative cross resistance ratio of 0.665, 0.830, 0.916 to cypermethrin, methomyl, spinosad respectively, and a positive cross resistance ratio of 1.019 to indoxacarb, while similar trend was displayed by Raichur population showing a negative cross resistance ratio of 0.932, 0.565, 0.803 to cypermethrin, methomyl, spinosad respectively and positive cross resistance of 1.036 indoxacarb further, same trend was shown by Nagpur population by displaying a negative cross resistance ratio of 0.610, 0.735, 0.519 to cypermethrin, methomyl, spinosad and positive cross resistance ratio of 1.026 to indoxacarb.&#x0D; Conclusion: Continuous application of single insecticide belonging to a specific group across the generations increases the resistance from F1 to F3. Alternating the new chemistries with old conventional chemicals resulted in no cross resistance development as it was observed in all test populations.