Risk assessment and molecular mechanism of Sclerotium rolfsii resistance to boscalid.

Boscalid, a widely used SDHI fungicide, has been employed in plant disease control for over two decades. However, there is currently no available information regarding its antifungal activity against Sclerotium rolfsii and the potential risk of resistance development in this pathogen. In this study, we evaluated the sensitivity of 100 S. rolfsii strains collected from five different regions in China during 2018-2019 to boscalid using mycelial growth inhibition method and assessed the risk of resistance development. The EC50 values for boscalid ranged from 0.2994 µg/mL to 1.0766 µg/mL against the tested strains, with an average EC50 value of 0.7052 ± 0.1473 µg/mL. Notably, a single peak sensitivity baseline was curved, indicating the absence of any detected resistant strains. Furtherly, 10 randomly selected strains of S. rolfsii were subjected to chemical taming to evaluate its resistance risk to boscalid, resulting in the successful generation of six stable and inheritable resistant mutants. These mutants exhibited significantly reduced mycelial growth, sclerotia production, and virulence compared to their respective parental strains. Cross-resistance tests revealed a correlation between boscalid and flutolanil, benzovindiflupyr, pydiflumetofen, fluindapyr, and thifluzamide; however, no cross-resistance was observed between boscalid and azoxystrobin. Thus, we conclude that the development risk of resistance in S. rolfsii to boscalid is low. Boscalid can be used as an alternative fungicide for controlling peanut sclerotium blight when combined with other fungicides that have different mechanisms of action. Finally, the target genes SDHB, SDHC, and SDHD in S. rolfsii were initially identified, cloned and sequenced to elucidate the mechanism of S. rolfsii resistance to boscalid. Two mutation genotypes were found in the mutants: SDHD-D111H and SDHD-H121Y. The mutants carrying SDHD-H121Y exhibited moderate resistance, while the mutants with SDHD-D111H showed low resistance. These findings contribute to our comprehensive understanding of molecular mechanisms underlying plant pathogens resistance to SDHI fungicides.

Risk assessment and molecular mechanism of Fusarium incarnatum resistance to phenamacril.

BACKGROUND: Cucumber fruit rot (CFR) caused by Fusarium incarnatum is a devastating fungal disease in cucumber. In recent years, CFR has occurred frequently, resulting in serious yield and quality losses in China. Phenamacril exhibits a specific antifungal activity against Fusarium species. However, no data for phenamacril against F. incarnatum is available. RESULTS: The sensitivity of 80 F. incarnatum strains to phenamacril was determined. The half maximal effective concentration (EC50 ) values ranged from 0.1134 to 0.3261 µg mL-1 with a mean EC50 value of 0.2170 ± 0.0496 µg mL-1 . A total of seven resistant mutants were obtained from 450 mycelial plugs by phenamacril-taming on potato dextrose agar (PDA) plates with 10 µg mL-1 of phenamacril, and the resistant frequency was 1.56%. Phenamacril-resistant mutants showed decreased mycelial growth, conidiation and virulence as compared with the corresponding wild-type strains, indicating that phenamacril resistance suffered a fitness penalty in F. incarnatum. In addition, using sequence analysis, the point mutations of S217P or I424S were discovered in Fimyosin-5 (the target of phenamacril). The site-directed mutagenesis of the S217P, P217S, I424S and S424I substitutions were constructed to reveal the relationship between the point mutations and phenamacril resistance. The results strongly demonstrated that the mutations of S217P and I424S in Fimyosin-5 conferred phenamacril-resistance in F. incarnatum. CONCLUSION: Phenamacril-resistant mutants were easily induced and their resistance level was high. The S217P or I424S substitutions in Fimyosin-5 conferring phenamacril resistance were detected and futherly verified by transformation assay with site-directed mutagenesis. Thus, we proposed that the resistance development of F. incarnatum to phenamacril is high risk. © 2022 Society of Chemical Industry.

Resistance risk assessment for a novel succinate dehydrogenase inhibitor pydiflumetofen in Fusarium asiaticum.

BACKGROUND: Fusarium asiaticum is one of predominant pathogens of Fusarium head blight (FHB) in China. Pydiflumetofen (Pyd) is a novel succinate dehydrogenase inhibitor (SDHI) which has been commercialized in China for the controlling of wheat FHB since 2019. In the current study, a risk assessment of the pydiflumetofen-resistance selected in Fusarium asiaticum was investigated. RESULTS: One PydMR mutant [resistance factor (RF) < 80] and four PydHR mutants (RF > 3000) were generated by fungicide-taming from 1000 mycelial discs of the wild-type strain 2021. Nucleotide sequences alignment results of FaSdh from the wild-type strain and resistant mutants showed that all the mutations were categorized into three genotypes, i.e. FaSdhBH248Y from PydMR mutant, both FaSdhC1 A64V and FaSdhC1 R67K from PydHR mutants. All the resistant mutants possessed no fitness penalty based on the data of mycelial linear growth, conidiation and virulence. In addition, the FaSdhC1 A64V mutants showed positive cross-resistance between pydiflumetofen and boscalid or thifluzamide, but no cross-resistance between pydiflumetofen and Y13149 or Y12196, while the FaSdhC1 R67K mutants exhibited positive cross-resistance between pydiflumetofen and boscalid, thifluzamide or Y12196, and no cross-resistance between pydiflumetofen and Y13149. Furthermore, positive cross-resistance between the five tested SDHIs was detected in the FaSdhBH248Y mutants. CONCLUSION: The results suggest a moderate to high resistance risk of F. asiaticum to pydiflumetofen, and provide essential data for monitoring the emergence of resistance and resistance management strategies for pydiflumetofen, which will be useful for scientific application of this fungicide in China.

Resistance risk assessment for fluazinam in Sclerotinia sclerotiorum.

In the current study, sensitivity distribution of Sclerotinia sclerotiorum populations to fluazinam was determined using 103 strains collected from the fields of Jiangsu Province of China in 2016-2017 and the resistance risk of fluazinam was assessed. The average EC50 (50% effective concentration) values and MIC (minimum inhibitory concentration) values of 103 S. sclerotiorum strains against fluazinam were 0.0073±0.0045µg/ml and <0.3µg/ml for mycelial growth, respectively. Nine mutants with low resistance level were obtained from wild type sensitive strains exposed on PDA medium amended with fluazinam and the resistance was stable after their ten transfers on PDA without the fungicide. Compared with the parental strains, the nine fluazinam-resistant mutants decreased in mycelial growth, sclerotial production, pathogenicity and were more sensitive to 0.7M NaCl. In addition, cell membrane permeability of resistant mutants was higher than that of their parental strains. Cross resistance assay showed that there was no cross-resistance between fluazinam and fludioxonil, dimetachlone, prochloraz, tebuconazole, azoxystrobin, or procymidone in S. sclerotiorum. The above results indicated that there was a low resistance risk for fluazinam in S. sclerotiorum. However, the sensitivity of all fluazinam-resistant mutants to fludioxonil decreased. Sequencing alignment results showed that there were no mutations in the two-component histidine kinase gene (Shk1) of the resistant mutants and the expression levels of Shk1 of three resistant mutants were significantly up-regulated while others were almost the same as their parental strains. These results will contribute to evaluating the resistance risk of fluazinam for management of diseases caused by S. sclerotiorum and further increase our understanding about the mode of action of fluazinam.