Mingguo Zhou

A major gene for resistance to carbendazim, in field isolates of Gibberella zeae

Isolates of Gibberella zeae were collected from wheat fields at two sites in China. A total of 56 isolates were characterized for their ability to grow on the potato sucrose agar amended with various concentrations of carbendazim. Three sensitivity levels were identified among the isolates tested: sensitive (S) isolates could grow at 0.5 μg·mL−1, but were completely inhibited at 1.4 μg·mL−1 ; moderately resistant (MR) isolates grew quickly at 1.4 μg·mL−1, slowly at 50 μg·mL−1, and were completely inhibited at 100 μg·mL−1 ; highly resistant (HR) isolates were slightly inhibited at 50 μg·mL−1, but only partially inhibited at 100 μg·mL−1. Six isolates representing the three sensitivity level phenotypes were randomly selected for a study on the inheritance of carbendazim resistance by analyzing the sensitivity of hybrid F1 progeny. The nitrate nonutilizing mutant (nit) was used as a genetic marker to confirm that individual perithecia were the result of out-crossing. Five cross were assessed: S × S, MR × S, MR × MR, HR × S, and HR × MR. In crosses between the parents with different sensitivity levels, i.e., MR × S, HR × S, HR × MR, the progeny fit a 1:1 segregation ratio of the two parental phenotypes. No segregation was observed in the crosses of S × S and MR × MR. We conclude that the MR and HR phenotypes in G. zeae are conferred by different allelic mutations within the same locus. In these isolates, resistance to carbendazim was not affected by modifying genes or cytoplasmic components.

Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum

Resistance of Fusarium graminearum to carbendazim is caused by point mutations in the β2-tubulin gene. The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field resistant isolates in China. To establish a suitable method for rapid detection of the F167Y mutation in F. graminearum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed and optimized to specially distinguish the F167Y mutation genotype. The LAMP reaction was optimal at 63°C for 60 min. When hydroxynaphthol blue dye (HNB) was added prior to amplification, samples with DNA of the F167Y mutation developed a characteristic sky blue color after the reaction but those without DNA or with different DNA did not. Results of HNB staining method were reconfirmed by gel electrophoresis. The developed LAMP had good specificity, stability and repeatability and was suitable for monitoring carbendazim-resistance populations of F. graminearum in agricultural production.

Whole-genome sequencing reveals that mutations in myosin-5 confer resistance to the fungicide phenamacril in Fusarium graminearum

To determine the mechanism of resistance to the fungicide phenamacril (JS399-19) in Fusarium graminearum, the causal agent of Fusarium head blight, we sequenced and annotated the genome of the resistant strain YP-1 (generated by treating the F. graminearum reference strain PH-1 with phenamacril). Of 1.4 million total reads from an Illumina-based paired-end sequencing assay, 92.80% were aligned to the F. graminearum reference genome. Compared with strain PH-1, strain YP-1 contained 1,989 single-nucleotide polymorphisms that led to amino acid mutations in 132 genes. We sequenced 22 functional annotated genes of another F. graminearum sensitive strain (strain 2021) and corresponding resistant strains. The only mutation common to all of the resistant mutants occurred in the gene encoding myosin-5 (point mutations at codon 216, 217, 418, 420, or 786). To confirm whether the mutations in myosin-5 confer resistance to phenamacril, we exchanged the myosin-5 locus between the sensitive strain 2021 and the resistant strain Y2021A by homologous double exchange. The transformed mutants with a copy of the resistant fragment exhibited resistance to phenamacril, and the transformed mutant with a copy of the sensitive fragment exhibited sensitivity to phenamacril. These results indicate that mutations in myosin-5 confers resistance to phenamacril in F. graminearum.

A new point mutation in the iron–sulfur subunit of succinate dehydrogenase confers resistance to boscalid in Sclerotinia sclerotiorum

Research has established that mutations in highly conserved amino acids of the succinate dehydrogenase (SDH) complex in various fungi confer SDH inhibitor (SDHI) resistance. For Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic fungus with a broad host range and a worldwide distribution, boscalid resistance has been attributed to the mutation H132R in the highly conserved SdhD subunit protein of the SDH complex. In our previous study, however, only one point mutation, A11V in SdhB (GCA to GTA change in SdhB), was detected in S. sclerotiorum boscalid-resistant (BR) mutants. In the current study, replacement of the SdhB gene in a boscalid-sensitive (BS) S. sclerotiorum strain with the mutant SdhB gene conferred resistance. Compared with wild-type strains, BR and GSM (SdhB gene in the wild-type strain replaced by the mutant SdhB gene) mutants were more sensitive to osmotic stress, lacked the ability to produce sclerotia and exhibited lower expression of the pac1 gene. Importantly, the point mutation was not located in the highly conserved sequence of the iron-sulfur subunit of SDH. These results suggest that resistance based on non-conserved vs. conserved protein domains differs in mechanism. In addition to increasing our understanding of boscalid resistance in S. sclerotiorum, the new information will be useful for the development of alternative antifungal drugs.

Development of a rapid and high-throughput molecular method for detecting the F200Y mutant genotype in benzimidazole-resistant isolates of Fusarium asiaticum

BACKGROUND The point mutation at codon 200 (TTC→TAC, F200Y) of the β2 -tubulin gene confers resistance to benzimidazole fungicide in Fusarium asiaticum. These isolates with this mutation have been detected mainly by determining the minimum inhibitory concentration (MIC) of fungicides, which is always time consuming, tedious and inefficient. RESULTS A visual, rapid and efficient method with high specificity was developed, based on loop-mediated isothermal amplification (LAMP). Six sets of LAMP primers were designed, and one set was optimised specifically to distinguish the F200Y mutant genotype. With the optimal LAMP primers, concentrations of LAMP components were optimised. The optimal reaction conditions were 57-64 °C for 75 min. The feasibility of the LAMP assay for detection of the F200Y mutant genotype of F. asiaticum was demonstrated by assaying diseased wheat spikelets that were artificially inoculated in the field. CONCLUSION The new LAMP assay had good specificity, sensitivity, stability and repeatability. It will be useful for assessing the risk of F. asiaticum populations with carbendazim resistance developing in the field, and will also provide important reference data for integrated control of Fusarium head blight caused by F. asiaticum.

Control of Sclerotinia sclerotiorum infection in oilseed rape with strobilurin fungicide SYP-7017

Abstract: SYP-7017, a broad-spectrum fungicide, belongs to the chemical group of strobilurins. Baseline sensitivity of Sclerotinia sclerotiorum (Lib) de Bary to SYP-7017 was determined using 120 strains collected during 2010 and 2011 from rapeseed fields without a previous history of strobilurin usage in Jiangsu Province, China. The median effective concentration (EC50) values for SYP-7017 that inhibited mycelial growth ranged from 0.006 to 0.047 µg mL−1 (mean of 0.016 µg mL−1). No cross-resistance between SYP-7017 and carbendazim or iprodione was detected. On detached rapeseed leaves, SYP-7017 at 100 µg mL−1 provided over 90% control efficacy. Moreover, SYP-7017 exhibited good characteristics of absorption and translocation, and also exhibited excellent protective activity in pot experiments. In field trials, control efficacies of SYP-7017 at 75 g a.i. ha−1 and 187.5 g a.i. ha−1 were 80.2% and 91.58%, respectively, higher than other traditional fungicides. These results suggest that SYP-7017 has strong antifungal activity and a potential application in controlling S. sclerotiorum.

The autophagy‐related gene BcATG1 is involved in fungal development and pathogenesis in Botrytis cinerea

Autophagy, a ubiquitous intracellular degradation process, is conserved from yeasts to humans. It serves as a major survival function during nutrient depletion stress and is crucial for correct growth and differentiation. In this study, we characterized an atg1 orthologue Bcatg1 in the necrotrophic plant pathogen Botrytis cinerea. Quantitative real-time polymerase chain reaction (qRT-PCR) assays showed that the expression of BcATG1 was up-regulated under carbon or nitrogen starvation conditions. BcATG1 could functionally restore the survival defects of the yeast ATG1 mutant during nitrogen starvation. Deletion of BcATG1 (ΔBcatg1) inhibited autophagosome accumulation in the vacuoles of nitrogen-starved cells. ΔBcatg1 was dramatically impaired in vegetative growth, conidiation and sclerotial formation. In addition, most conidia of ΔBcatg1 lost the capacity to form the appressorium infection structure and failed to penetrate onion epidermis. Pathogenicity assays showed that the virulence of ΔBcatg1 on different host plant tissues was drastically impaired, which was consistent with its inability to form an appressorium. Moreover, lipid droplet accumulation was significantly reduced in the conidia of ΔBcatg1, but the glycerol content was increased. All of the defects of ΔBcatg1 were complemented by re-introduction of an intact copy of the wild-type BcATG1 into the mutant. These results indicate that BcATG1 plays a critical role in numerous developmental processes and is essential to the pathogenesis of B. cinerea.

Genotypes and Characteristics of Phenamacril-Resistant Mutants in Fusarium asiaticum

Fusarium asiaticum is a critical pathogen of Fusarium head blight (FHB) in the southern part of China. The fungicide phenamacril has been extensively used for controlling FHB in recent years, which reduced both FHB severity and mycotoxin production. Our previous report indicated that resistance of F. asiaticum to phenamacril was related to mutations in myosin5. A recent article revealed that the resistance level of phenamacril-resistant mutants was associated with the genotypes of myosin5 in these mutants. In total, we obtained 239 resistant isolates by fungicide domestication, and 82 resistant mutants were randomly selected for further study. Of these mutants, 25.6, 7.3, and 67.1% showed low resistance (LR), moderate resistance (MR), and high resistance (HR), respectively, to phenamacril determined by 50% effective concentration values. Point mutations A135T, V151M, P204S, I434M, A577T, R580G/H, or I581F led to LR. Point mutations S418R, I424R, and A577G were responsible for MR and point mutations K216R/E, S217P/L, or E420K/G/D conferred HR. Interestingly, all of the mutations concentrated in the myosin5 motor domain and mutations conferring HR occurred at codon 217 and 420, which we called the core region. Homology modeling revealed that mutations far from the core region led to a lower resistance degree. Phenotype assays revealed that the most highly resistant mutants did not significantly change pathogenicity but decreased conidia production compared with the wild type, which may slow down the formation of the resistant pathogen population in the fields.

Myosins FaMyo2B and Famyo2 Affect Asexual and Sexual Development, Reduces Pathogenicity, and FaMyo2B Acts Jointly with the Myosin Passenger Protein FaSmy1 to Affect Resistance to Phenamacril in Fusarium asiaticum.

We previously reported that mutations occurred in the gene myosin5 were responsible for resistance to the fungicide phenamacril in Fusarium graminearum. Here, we determined whether there is a functional link between phenamacril resistance and the myosin proteins FaMyo2B and Famyo2 in Fusarium asiaticum, which is the major causal agent of Fusarium head blight in China. We found that FaMyo2B acts jointly with FaSmy1 to affect resistance to phenamacril in F. asiaticum. We also found that FaMyo2B disruption mutant and Famyo2 deletion mutant were defective in hyphal branching, conidiation, and sexual reproduction. ΔFamyo2 also had an enhanced sensitivity to cell wall damaging agents and an abnormal distribution of septa and nuclei. In addition, the FaMyo2B and Famyo2 mutants had reduced pathogenicity on wheat coleoptiles and flowering wheat heads. Taken together, these results reveal that FaMyo2B and Famyo2 are required for several F. asiaticum developmental processes and activities, which help us better understand the resistance mechanism and find the most effective approach to control FHB.

Activity of a novel succinate dehydrogenase inhibitor fungicide pyraziflumid against Sclerotinia sclerotiorum

Pyraziflumid is a novel member of succinate dehydrogenase inhibitor fungicides (SDHI). In this study, baseline sensitivity of Sclerotinia sclerotiorum (Lib.) de Bary to pyraziflumid was determined using 105 strains collected during 2015 and 2017 from different geographical regions in Jiangsu Province of China, and the average EC50 value was 0.0561 (±0.0263)μg/ml for mycelial growth. There was no cross-resistance between pyraziflumid and the widely used fungicides carbendazim, dimethachlon and the phenylpyrrole fungicide fludioxonil. After pyraziflumid treated, hyphae were contorted with offshoot of top increasing, cell membrane permeability increased markedly, oxalic acid content significantly decreased and mycelial respiration was strongly inhibited. But the number and dry weight of sclerotia did not change significantly. The protective and curative activity test of pyraziflumid suggested that pyraziflumid had great control efficiency against S. sclerotiorum on detached rapeseed leaves, and protective activity was better than curative activity. These results will contribute to us on evaluating the potential of the new SDHI fungicide pyraziflumid for management of diseases caused by S. sclerotiorum and understanding the mode of action of pyraziflumid against S. sclerotiorum.