The genus Colletotrichum contains a wide variety of important plant pathogens, and Colletotrichum truncatum is one of the most prevalent species of Colletotrichum on chili in China. Demethylation-inhibitor fungicides (DMIs) are currently registered chemical agents for the management of the anthracnose disease caused by Colletotrichum spp. To assess the risk for DMI resistance development, 112 C. truncatum isolates were collected from infected pepper in 13 regions of China. The sensitivity of C. truncatum isolates to five DMI fungicides was determined based on mycelial growth inhibition assay. C. truncatum was sensitive to prochloraz, epoxiconazole, and difenoconazole, but not to tebuconazole or myclobutanil. Baseline sensitivity using the 112 C. truncatum isolates was established for the first three effective DMIs. Prochloraz, epoxiconazole, and difenoconazole EC50 values were 0.053 ± 0.023, 1.956 ± 0.815, and 1.027 ± 0.644 μg/ml, respectively. Eleven stable DMI-resistant mutants all exhibited lower fitness levels than their wild-type parents, suggesting a low risk of DMI resistance in C. truncatum. By inducing gene expression, CtCYP51 expression increased slightly in the resistant mutants as compared to wild-types when exposed to DMI fungicides and thus contributed at least partially to resistance. Molecular docking with CYP51 structure models was used to explain differential sensitivity of the DMI fungicides in C. truncatum. Our results suggest that the M376L/H373N mutations in CYP51 changed the conformation of DMIs in the binding pocket. These changes prevented the formation of the Fe – N coordinate bond between the heme iron active site and tebuconazole or myclobutanil, and apparently contributed to tebuconazole and myclobutanil insensitivity of C. truncatum.

Gray mold caused by Botrytis cinerea is one of the most important diseases in tomato. It can be controlled effectively by demethylation inhibitor (DMI) fungicides, but their resistant status is unclear after long-term use in the field. Baseline sensitivity to difenoconazole of 142 B. cinerea isolates from China with no history of DMI usage was characterized, with a mean EC50 of 0.97 ± 0.50 μg/mL. EC50 values to difenoconazole of another 248 isolates collected in 2011 and 2016 ranged from 0.04 to 11.99 μg/mL, and the frequency of difenoconazole sensitivity formed a non-normal distribution curve. Detached fruit studies revealed that isolates with EC50 values of ~6.00 μg/ml were not controlled effectively. The mean EC50 of the resistant isolates changed from 6.74 to 8.65 μg/mL between 2011 and 2016. Positive cross-resistance was only observed between difenoconazole and two DMIs. One dual resistant and one triple resistant isolates were found among the difenoconazole-resistant isolates collected in 2016, associated with point mutations in corresponding target proteins of the fungicides azoxystrobin and fludioxonil. This indicated that B. cinerea not only showed higher difenoconazole resistance levels but gradually changed from single to multiple fungicide resistance over time. No amino acid variation was found in the CYP51 protein. In the absence of difenoconazole, the relative expression of CYP51 was not significantly different in sensitive and resistant isolates. Induced expression of CYP51 is an important determinant of DMI resistance in B. cinerea from tomato. However, nucleotide variants found in the upstream region had no association with the fungicide resistance phenotype. These results will be helpful for the management of B. cinerea in the field.

Botrytis cinerea is a destructive plant pathogenic ascomycete that causes serious pre-and post-harvest losses 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. Baseline sensitivity of 110 B. cinerea isolates collected from nine provinces in China to pyrisoxazole was demonstrated, with a mean EC 50 of 0.057 ± 0.029 µg/ml. Eleven stable mutants resistant to pyrisoxazole were generated via UV irradiation (RU-mutants) and spontaneous selection (RS-mutants) of conidia. The efficacy of pyrisoxazole against the resistant mutants was significantly lower than that of the sensitive isolates. Most of the pyrisoxazole-resistant mutants were less fit than the sensitive isolates, with reduced sporulation, conidia germination, sclerotium production, and pathogenicity, which was confirmed by the competitive ability test. Positive cross-resistance was only observed between pyrisoxazole and the DMIs tebuconazole and prochloraz, but not between pyrisoxazole and non-DMIs iprodione, procymidone, diethofencarb, fluazinam, pyrimethanil, or fludioxonil. A two-point mutation, at G476S and K104E in the RU-mutants, and a one point mutation, M231T, in the RS-mutants, were detected in the CYP51 protein of the resistant mutants. When exposed to pyrisoxazole, the induced expression level of CYP51 increased in the resistant isolates as compared to sensitive ones. Molecular docking suggested that G476S and M231T mutations both led to the loss of electrostatic interactions between CYP51 and pyrisoxazole, while no change was found with the K104E mutation. Thus, two point mutations on CYP51 protein combined with induced expression of its target gene appeared to mediate the pyrisoxazole resistance of B cinerea.

Botrytis cinerea is a destructive necrotrophic pathogen that can infect many plant species. The control of gray mold mainly relies on the application of fungicides, and the fungicide fludioxonil is widely used in China. However, the field fungicide resistance of B. cinerea to this compound is largely unknown. In this study, B. cinerea isolates were collected from different districts of Shanghai province in 2015–2017, and their sensitivity to fludioxonil was determined. A total of 65 out of 187 field isolates (34.76%) were found to be resistant to fludioxonil, with 36 (19.25%) showing high resistance and 29 (15.51%) showing moderate resistance. Most of these resistant isolates also showed resistance to iprodione, and some developed resistance to fungicides of other modes of action. AtrB gene expression, an indicator of MDR1 and MDR1h phenotypes, was not dramatically increased in the tested resistant isolates. Biological characteristics and osmotic sensitivity investigations showed that the fitness of resistant isolates was lower than that of sensitive ones. To investigate the molecular resistance mechanisms of B. cinerea to fludioxonil, the Bos1 amino acid sequences were compared between resistant and sensitive isolates. Resistant isolates revealed either no amino acid variations or the mutations I365S, I365N, Q369P/N373S, and N373S.

Background Early blight disease of tomato caused by pathogenic fungi Alternaria solani is the most significant and common disease throughout the world as well as in Kingdom of Saudi Arabia. The aim of this study was to isolate and identify native Trichoderma species from the Jeddah region in Saudi Arabia; evaluate their antagonistic potential against A. solani; and study their influence early blight disease severity in greenhouse and in open field. Results The present study focused to explore the biocontrolling potential of native Trichoderma spp. against A. solani strain to compare with a conventional fungicide. Out of 21, 3 Trichoderma isolates showed an antifungal activity and significantly inhibited the mycelial growth of pathogen that were identified as Trichoderma atroviride, T. harzianum and T. longibrachiatum by their ITS region sequence analysis. Strong in vitro mycelial growth suppression (70.66%) was also recorded at 400 ppm Mancozeb (90%WP®) fungicide. Further, these Trichoderma bioagents and fungicide were further evaluated in greenhouse (artificially inoculated) and in field on naturally infected tomato plants. In greenhouse, (13.74%) disease severity after T. harzianum treatment was recorded, followed by T. longibrachiatum (25.83%) and T. atroviride (21.67%). The disease severity after fungicide (50 mg/L; 10 ml per plant) application was (7.91%). Further, positive impact on the plant biomarkers was demonstrated by all selected Trichoderma isolates in greenhouse. Under natural infection in season I, the disease severity (%) after T. longibrachiatum, T. atroviride and T. harzianum treatments was 11.5, 13.26 and 16.81%, respectively, followed by control (32.12%), whereas 7.18% disease severity was recorded after fungicide application. Conclusions The results revealed that native Trichoderma of this region had potential to mitigate the early blight disease intensity in field.