Fusarium graminearum is the primary causal agent of Fusarium head blight (FHB) of wheat. The phenylpyrrole fungicide fludioxonil is not currently registered for the management of FHB in China. The current study assessed the fludioxonil sensitivity of a total of 53 F. graminearum isolates collected from the six most important wheat-growing provinces of China during 2018 and 2019. The baseline fludioxonil sensitivity distribution indicated that all of the isolates were sensitive, exhibiting a unimodal cure with a mean effective concentration for 50% inhibition value of 0.13 ± 0.12 μg/ml (standard deviation). Five fludioxonil-resistant mutants were subsequently induced by exposure to fludioxonil under laboratory conditions. Ten successive rounds of subculture in the absence of the selection pressure indicated that the mutation was stably inherited. However, the fludioxonil-resistant mutants were found to have reduced pathogenicity, higher glycerol accumulation, and higher osmotic sensitivity than the parental wild-type isolates, indicating that there was a fitness cost associated with fludioxonil resistance. In addition, the study also found a positive cross resistance between fludioxonil, procymidone, and iprodione, but not with other fungicides such as boscalid, carbendazim, tebuconazole, and fluazinam. Sequence analysis of four candidate target genes (FgOs1, FgOs2, FgOs4, and FgOs5) revealed that the HBXT2R mutant contained two point mutations that resulted in amino acid changes at K223T and K415R in its FgOs1 protein, and one point mutation at residue 520 of its FgOs5 protein that resulted in a premature stop codon. Similarly, the three other mutants contained point mutations that resulted in changes at the K192R, K293R, and K411R residues of the FgOs5 protein but none in the FgOs2 and FgOs4 genes. However, it is important to point out that the FgOs2 and FgOs4 expression of all the fludioxonil-resistant mutants was significantly (P < 0.05) downregulated compared with the sensitive isolates (except for the SQ1-2 isolate). It was also found that one of the resistant mutants did not have changes in any of the sequenced target genes, indicating that an alternative mechanism could also lead to fludioxonil resistance.

Sclerotinia sclerotiorum is one of the most damaging and economically important necrotrophic plant pathogens, infecting more than 400 plant species globally. Although the phenylpyrrole fungicide fludioxonil has high activity against S. sclerotiorum, recent reports have indicated that there is also a substantial potential for the development of fungicide resistance. However, the current study investigating five fludioxonil-resistant laboratory mutants found a significant fitness cost associated with fludioxonil resistance resulting in significantly (p < 0.005) reduced mycelial growth and sclerotia formation on PDA, as well as significantly (p < 0.05) lower pathogenicity on detached tomato leaves, with one mutant, LK-1R, completely losing the capacity to cause infection. In addition, all of the fludioxonil-resistant mutants had significantly (p < 0.05) increased sensitivity to osmotic stress (0.5 M KCl and 1.0 M Glucose), which is consistent with the proposed fludioxonil target sites within the High Osmolarity Glycerol (HOG1) stress response mitogen-activated protein kinase (MAPK) (HOG1-MAPK) signaling transduction pathway. Sequence analysis of six genes from this two-component pathway, including SsHk, SsYpd, SsSk1, SsSk2, SsPbs, and SsHog, revealed several mutations which maybe associated fludioxonil resistance. For example, six separate point mutations were found in the SsHk gene that lead to changes in the predicted amino acid sequence, including A136G, F249V, G353A, E560K, M610K, and K727R. Similarly, the SsPbs gene had three mutations (D34G, S46L, and L337E); the SsSk1 and SsYpd genes two (S53G and A795V, and E67G and Y141H, respectively) and the SsHog and SsSk2 genes one each (V220A and S763P, respectively). To the best of our knowledge, these constitute the first reports of amino acid changes in the proteins of the HOG1-MAPK pathway being associated with fludioxonil resistance in S. sclerotiorum. The study also found a positive cross-resistance between fludioxonil and dimethachlone and procymidone, but none with tebuconazole or carbendazim, indicating that the inclusion of tebuconazole within an integrated pest management program could reduce the risk of fludioxonil developing in field populations of S. sclerotiorum, and ensure the sustainable production of soybeans in China into the future.