Mechanism of Pydiflumetofen Resistance in Fusarium graminearum in China

Abstract

Fusarium head blight (FHB), which is primarily caused by Fusarium graminearum, is a widespread and devastating disease of wheat. In the absence of resistant varieties, the control of FHB relies heavily on the application of fungicides, and the new generation SDHI fungicide, pydiflumetofen, has recently been registered in China for the control of FHB in wheat. The current study explored three genetically stable, highly resistant laboratory mutants (S2-4-2R, S27-3R, and S28-2R, with EC50 values of 25.10, 28.57, and 19.22 μg/mL, respectively) to investigate the potential risks associated with pydiflumetofen resistance. Although the mycelial growth of the mutants differed little compared to their parental isolates, the study found that the resistant mutants exhibited significantly reduced (p < 0.05) levels of sporulation and pathogenicity, which suggests a significant fitness cost associated with pydiflumetofen resistance in F. graminearum. Sequence analysis of the Sdh target protein identified numerous amino acid substitutions in the predicted sequences of the four subunits: FgSdhA, FgSdhB, FgSdhC, and FgSdhD. Indeed, the mutants were found to have a series of substitution in multiple subunits such that all three exhibited five identical changes, including Y182F in the FgSdhA subunit; H53Q, C90S, and A94V in FgSdhB; and S31F in FgSdhC. In addition, gene expression analysis revealed that all of the FgSdh genes had significantly altered expression (p < 0.05), particularly FgSdhA and FgdhC, which exhibited remarkably low levels of expression. However, the study found no evidence of cross-resistance between pydiflumetofen and tebuconazole, fludioxonil, prochloraz, fluazinam, carbendazim, pyraclostrobin, or difenoconazole, which indicates that these fungicides, either in rotation or combination with pydiflumetofen, could mitigate the risk of resistance emerging and provide ongoing control of FHB to ensure high and stable wheat yields.

Baseline tebuconazole sensitivity and potential resistant mechanisms in Fusarium graminearum

“…The relative expression of the three FgCYP51 genes was assessed in both the tebuconazole-resistant mutants and the wild-type parental isolates, in either the absence or presence of tebuconazole (0.5 μg/mL) using the method of a previous study [41]. Total RNA was first extracted from fresh mycelium samples using a fungal RNA kit (Omegabio-tek, Basel, Switzerland), before the cDNA was synthesized using the PrimeScriptRT kit (TaKaRa, Kusatsu, Japan).…”

Exploring the Potential Mechanism of Prothioconazole Resistance in Fusarium graminearum in China

“…All of the wild-type isolates were highly sensitive to prothioconazole, with EC 50 values of 0.579, 0.311, and 0.251 µg/mL, respectively. After repeated exposure of these isolates to prothioconazole under laboratory conditions following the protocol of a previous study [29]), three genetically stable, highly resistant mutants (3-aR, 4-aR, and SZ-1-3R) were recovered, which had EC 50 values of 10.10, 12.34, and 21.24 µg/mL, respectively (Table 1). Both the wild-type isolates and the resistant mutants were routinely maintained on potato dextrose agar (PDA: 20 g/L glucose, 200 g/L potato, and 20 g/L agar) at 24 • C.…”

“…The biological characteristics of the three resistant F. graminearum mutants (3-aR, 4-aR, and SZ-1-3R) were compared to those of their wild-type parental isolates (3-a, 4-a, and SZ-1-3), by assessing mycelial growth on PDA, sporulation in mung bean broth, spore germination in sterilized water, and pathogenicity on wheat coleoptiles using the methods detailed in a previous study [29]. Each isolate was represented by at least eight individual plates in the growth assay, six flasks in the sporulation and spore germination assays, and ten wheat coleoptiles in the pathogenicity tests, with each experiment being performed twice.…”

“…Mycelium samples were prepared following the method of a previous study [29], and total genomic DNA was extracted using the Omega bio-tek Fungal DNA Kit (Omega bio-tek lnc., Guangzhou, China) according to the instructions of the manufacturer. The resulting DNA was used as a template for amplification of the full-length FgCYP51 genes (FgCYP51A, FGSG_04092; FgCYP51B, FGSG_01000, and FgCYP51C, FGSG_11024), with primer sets designed to the three different homologues using primer premier software (ver.6.0., PREMIER Biosoft, Canada): FgCYP51A-F/FgCYP51A-R, FgCYP51B-F/FgCYP51B-R, and FgCYP51C-F/FgCYP51C-R, respectively (Table S2).…”

“…The qPCR itself was performed using the QuantStudio 6 Flex PCR detection system (ThermoFisher, Waltham, MA, USA), with reaction mixtures containing SYBR Green I fluorescent dye, and the following program: initial denaturation at 95 • C for 10 s; followed by 40 cycles of 95 • C for 5 s, and 60 • C for 32 s; and dissociation at 95 • C for 15 s, 60 • C for 60 s, and 95 • C for 15 s. The relative expression of each gene was then determined following the protocol of a previous study using actin as the reference gene. Each isolate or mutant was represented by three biological replicates, and the entire experiment was performed twice [29].…”