Introduction: Alternaria solani is a challenging pathogen in the tomato crop globally. Chemical control is a rapid approach, but emerging fungicide resistance has become a severe threat. The present study investigates the use of culture filtrates (CFs) of three species of Trichoderma spp. to control this disease.Methods: Highly virulent A. solani strain and three Trichoderma fungal strains viz., T. harzianum (Accession No: MW590687), T. atroviride (Accession No: MW590689) and T. longibrachiatum (Accession No: MW590688) previously isolated by authors were used in this study. The efficacy of culture filtrates (CFs) to mitigate early blight disease were tested under greenhouse and field conditions, experiments were conducted in different seasons of 2020 using a tomato variety “doucen”.Results and discussion: The CFs of T. harzianum, T. longibrachiatum, and T. atroviride significantly inhibited the in vitro mycelial growth of A. solani (62.5%, 48.73%, and 57.82%, respectively, followed by control 100%). In the GC–MS analysis of Trichoderma CF volatile compounds viz., harzianic acid (61.86%) in T. harzianum, linoleic acid (70.02%) in T. atroviride, and hydroxymethylfurfural (68.08%) in the CFs of T. longibrachiatum, were abundantly present. Foliar application of CFs in the greenhouse considerably reduced the disease severity (%) in all treatments, viz., T. harzianum (18.03%), T. longibrachiatum (31.91%), and T. atroviride (23.33%), followed by infected control (86.91%), and positively affected the plant biomarkers. In the greenhouse, the plants treated with CFs demonstrated higher flavonoids after 6 days of inoculation, whereas phenolic compounds increased after 2 days. The CF-treated plants demonstrated higher antioxidant enzymes, i.e., phenylalanine ammonia-lyase (PAL) and peroxidase (POD), after 4 days, whereas polyphenol oxidase (PPO) was higher after 6 days of inoculation, followed by healthy and infected controls. In open field conditions, disease severity in CF-treated plants was reduced in both seasons as compared to naturally infected plants, whereas CF-treated plants exhibited a higher fruit yield than controls. The present results conclude that CFs can be a potential biocontrol candidate and a promising alternative to the early blight pathogen for sustainable production.

The genus Colletotrichum contains a wide variety of important plant pathogens, and C. 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. Plots of frequency had unimodal curves for prochloraz, epoxiconazole, and difenoconazole with mean (± SE) EC50 values of 0.053 ± 0.023, 1.956 ± 0.815, and 1.027 ± 0.644 µg/ml, respectively. Eleven stable DMI-resistant mutants all had lower fitness levels than their wild-type parents, suggesting a low risk of DMI resistance in C. truncatum. By inducing gene expression, CtCYP51 gene was confirmed to play a partial role in the resistance to DMIs in C. truncatum mutants. Molecular docking was used to explain the sensitivity differentiation to five DMI fungicides in C. truncatum. It showed that M376L/H373N mutations in CYP51 changed the conformation of DMIs in the binding pocket. This change 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 myclobutani insensitivity.

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.

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 resistance status after long-term use in the field is unclear. The baseline sensitivity to difenoconazole of 142 B. cinerea isolates from China with no history of DMI usage was characterized, with a mean effective concentration for 50% mycelial growth inhibition (EC₅₀) of 0.97 ± 0.50 μg/ml. EC₅₀ values for difenoconazole sensitivity 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 nonnormal distribution curve. Detached fruit studies revealed that isolates with EC₅₀ values of approximately 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 isolate and one triple resistant isolate 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 EC₅₀ 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.