Resistance Risk Assessment Of Valsa Mali To Pyraclostrobin And Antifungal Activity Of Biological Fungicides

The apple Valsa canker?AVC?caused by Valsa mali is a devastating branch disease that has seriously threatened the healthy and sustainable development of the apple industry in China.The pathogen can infect branches asymptomatically through a variety of ways and the pathogenic mechanism is complex,making it difficult to prevent and control.In practice,the application of fungicides has been the main method to control AVC.However,repeated use of chemical agents can lead to the development of resistance to pathogens and serious environmental pollution.Therefore,the assessment of chemical agents resistance risk and searching for high-efficiency,low-toxicity and low residue alternative biological fungicides are of great significance for the formulation and implementation of AVC prevention and control measures and scientific pesticides used in practice.The objectives of this study were to?i?determine the baseline sensitivity of natural populations of V.mali to pyraclostrobin which belongs to the quinol oxidation inhibitor?QoI?class with highly efficient,low-toxicity,broad-spectrum;?ii?generate pyraclostrobin-resistant strains?PR strains?of V.mali;?iii?measure the biofitness of these resistant strains in vitro;?iv?assay the cross-resistance relationships between pyraclostrobin and other fungicides,and?v?screen of biological fungicides to improve our understanding of resistance development to pyraclostrobin of V.mali and provide a scientific basis for the security prevention of AVC.The main results are as follows:1.In total,120 isolates of Valsa mali were collected from six provinces?Liaoning,Shandong,Gansu,Shanxi,Shaanxi,and Xinjiang?of China.The baseline sensitivity of these isolates to pyraclostrobin was determined.All collected strains showed similar sensitivity to pyraclostrobin,which had strong antifungal activity with EC₅₀ values from 0.00137 to 0.0240?g/ml.The baseline sensitivity was distributed as a unimodal curve with a mean EC₅₀ value of0.00909?±0.00552??g/ml.The difference in EC₅₀ values among different strains is so small that the ratio of the maximum to the minimum EC₅₀ values was 17.5.The mean EC₅₀ values differed among different geographic locations.The most sensitive strain is from Xinjiang and the most insensitive strain is from Liaoning.No sub-population of V.mali with reduced sensitivity.2.Three pyraclostrobin-resistant strains?PR strains?were obtained by domestication of pyraclostrobin through fourteen isolates of V.mali randomly selected.The resistance factor of resistant strains was 41.0,56.8 and 22.0-fold compared with their parental isolates,respectively.The resistance of the strains was found to be stable after 10 serial transfers on pyraclostrobin-free medium.Moreover,the phenotypes of PR strains were observed.The number of propagules between the PR strains and their parental isolates showed no significant differences,but the hyphal growth on PDA,mycelial dry weight and pathogenicity were significantly lower than those of their parental isolates.Sensitivity to the alternative oxidase inhibitor salicylhydroxamic acid?SHAM?and NaCl were increased in the PR strains.Furthermore,no cross-resistance existed between pyraclostrobin and tebuconazole,difenoconazole,imazalil,and thiophanate methyl.Therefore,pyraclostrobin can currently be recommended to control apple Valsa canker with a low-to-moderate risk of resistance.3.Evaluating antifungal activity of six biological fungicides against V.mali in vitro.The results showed that all the tested agents displayed inhibitory effects on V.mali.They presented stronger inhibitory effects on conidial germination than mycelial growth with the exception of 3%Zhongshengmycin WP.Among them,3.0×10 ¹⁰ cfu/g Bacillus amyloliquefaciens WP and 1.0×10 ¹¹ cfu/g B.subtilis WP were the most toxic with EC₅₀values of 0.69?g/mL,0.89?g/mL for mycelial growth and 0.19?g/mL,0.039?g/mL for conidial germination,respectively.The EC₅₀ values of 3%Zhongshengmycin WP,0.5%berberine AS,and 0.4%osthol SL were ranged from 26.06?g/mL to 290.7?g/mL for mycelial growth and 8.29?g/mL to 65.22?g/mL for conidial germination.However,2%Nongkang 120 AS was the lowest toxic since the EC₅₀ of mycelial growth was higher than5000?g/mL.The protective measures for protecting inoculation on excised twigs showed that3.0×10 ¹⁰ cfu/g Bacillus amyloliquefaciens WP and 1.0×10 ¹¹ cfu/g B.subtilis WP had the highest protection effect on shoot growth with 92.7%and 86.7%of control effect,but 2%Nongkang 120 AS showed the worst protection effect,which was consistent with the virulence test results.Therefore,3.0×10 ¹⁰ cfu/g B.amyloliquefaciens WP and 1.0×10 ¹¹ cfu/g B.subtilis WP could be used as alternative biological fungicides for controlling AVC.