Indoor fungicide screening and identification of antagonistic strains against Wilsonomyces carpophilus

Abstract: 【Objective】Prunus armeniaca is one of the main tree crops in the Tianshan wild fruit forest, which plays an important role in maintaining the stability of the Tianshan wild fruit forest ecosystem. The occurrence of wild apricot perforation disease caused by Wilsonomyces carpophilus has become an important factor endangering the healthy growth of P. armeniaca. W. carpophilus mainly harms the leaves and fruits of P. armeniaca, causing leaf perforation and fruit browning. Screening of fungicides and identification of antagonistic fungi against W. carpophilus can effectively control fungal perforation of wild apricot.【Methods】Based on the previous research, 8 types of commonly used low-toxicity and high-efficiency fungicides on the market were selected as the test agents, and the concentrations of each fungicide were adjusted according to the recommended dilution ratio and pre-experiment results of the commercial agents. Using the mycelial growth rate method to determine the toxicity of different fungicides for the mycelial growth of W. carpophilus. Mix the fungicide and PDA medium in a 1∶9 ratio to form a medicated medium, inoculate the bacterial cake into the center of the plate, and use the non-medicated medium as the control. Measure the colony diameter using the cross over method to calculate the inhibition rate of mycelial growth. Prepare a suspension of conidia of W. carpophilus, mix the prepared suspension with the medicinal solution and incubate at a constant temperature for 14 hours before ob-serving the results. The spore germination standard is set below: when the length of the bud tube exceeds half of the maximum diameter length of the spore, it is considered as initial germination. The effective result is to control the germination rate to reach 90% or above. Use the spore germination method to compare the sensitivity of the conidia of W. carpophilus to the toxicity of eight fungicides. The toxicity regression equation (y=ax+b) was established by using the least square method with the natural logarithm of the concentration of the agent as the independent variable (x) and the probability values of the inhibition rate and the inhibition rate of spore germination as the dependent variable (y). Observe and record the size, color, transparency, surface texture, and other cultural characteristics of individual colonies of antagonistic strains, as well as the results of Gram staining and physiological and biochemical characteristics measurement; Using the Neighbor- jioning method, we selected known sequences with high homology on the NCBI website to construct a phylogenetic tree of antagonistic strains, and determined the taxonomic status of the strains based on comprehensive cultural characteristics and molecular biology results; Determinate the inhibitory effect of antagonistic strains on different pathogens using plate confrontation method.【Results】Different fungicides had inhibitory effects on the growth of the hyphae of W. carpophilus. Among them, 50% carbendazim at different concentration gradients had a strong inhibitory effect on the growth of the hyphae. After being inoculated into the medicated medium, the hyphae did not grow. 722 g ·L^-1 propamocarb hydrochloride and 36% quinoline ·tebuconazole had strong inhibitory effects on the growth of W. carpophilus hyphae, with EC50 values being 0.322 5 mg ·L^-1 and 0.329 8 mg ·L^-1 , respectively; The inhibitory effect of 75% chlorothalonil on the growth of W. carpophilus hyphae was poor, with an EC50 value of 918.8 mg ·L^-1 . The results of inhibiting the germination of conidia of W. carpophilus using different fungicides showed that among the 8 selected fungicides, 27% pentazole·thiamethoxazole and 722 m ·L^-1 propamocarb hydrochloride had better inhibitory effects on the germination of conidia of W. carpophilus, with EC50 values being 0.060 5 mg ·L^-1 and 0.164 mg·L-1 , respectively. The inhibitory effect of 75% chlorothalonil on the germination of conidia of W. carpophilus was poor, with an EC50 of 1103 mg ·L^-1 . After incubating the antagonistic strain XHG-1-3m2 on LB solid culture medium at a constant temperature for 3 d, the single colony was circular, with irregular edges and milky white color in the early stage, but showed gradually deepened, opaque, and slightly raised in the later stage, and the surface was not smooth. Gram staining was positive, V-P and nitrate reduction reactions were both positive and aerobic, and can liquefy gelatin and hydrolyze starch. The similarity between strain XHG-1-3m2 and B. atrophaeus sequence reached 100% in the NCBI database BLAST results. The phylogenetic tree results showed that strain XHG-1-3m2 and B. atrophaeus were clustered into the same branch. Based on comprehensive cultural characteristics and molecular biology analysis, strain XHG- 1- 3m2 was identified as B. atrophaeus. The antagonistic strain XHG-1-3m2 had an inhibitory effect of 88.88% on W. carpophilus, which can inhibit the growth of W. carpophilus hyphae, cause deformities, shorten internodes and affect the normal growth of hyphae. Simultaneously, it had inhibitory effects on all 17 other pathogenic fungi.【Conclusion】50% carbendazim had a good inhibitory effect on the mycelial growth and conidial germination of W. carpophilus. The antagonistic strain XHG-1-3m2 was B. atrophicus, which can not only inhibit the growth of W. carpophilus, but also have good antagonistic effects on other fungi, with broad-spectrum antifungal properties.