| Mountain apples in northern Shaanxi are a major agricultural sector for economic development in the region.With the demand for not competing with food for land in China’s planting industry,the mountain apple production area has demonstrated obvious advantages and potential.As a result,it has become a new area for the development of the apple industry.Therefore,it is necessary to clearly establish the occurrence of diseases such as Apple Valsa Canker in the region,and conduct scientific control and prevention.This is fundamental for the sustainable and healthy development of mountain apples.Apple Valsa Canker,caused by the fungus Valsa mali,results in bark necrosis and ulceration,weakened trees,reduced fruit quantity and quality,and in severe cases,dead trees and destroyed orchards that lead to significant economic losses.In recent years,the efficacy of chemical agents such as tebuconazole for “summer liquid drenching” technology has enabled effective prevention and control of this disease.However,over-reliance on these chemicals may lead to the development of resistance.Biological control resources can be utilised to prevent and control diseases,effectively mitigating risks and contributing to the development of a green and organic apple industry in mountain regions.Based on the Bacillus amyloliquefaciens EDR4 and Bacillus subtilis Em7 biocontrol bacteria obtained from the laboratory,we measured the inhibition activity of these two strains and their fermentation filtrate against V.mali.We also examined their effectiveness in controlling Apple Valsa Canker,as well as the inhibition mechanism of two strains of bacteria.Finally,the preparation of a compound wettable powder was explored as an alternative biocontrol resource for the field control of Apple Valsa Canker in mountain orchards.The results were as follows:1.The occurrence of Apple Valsa Canker in Shaanxi mountain apple orchards.This study investigated 50 apple orchards with different ages and varieties all of which were Fuji apples,were examined in Baota District of Yan’an City,Mizhi County and Suide County of Yulin City.Results indicate that Apple Valsa Canker has become a serious concern for mountain apple production areas,with an average rate of diseased plants reaching 36.61%and a disease index of 18.74 in 2022-2023.Meanwhile,the rate of diseased plants in serious orchards can reach as high as 100%.The longer the planting time,the more serious the disease.The average disease rate of orchards with 16-25 years of age was 50.85%,which was 36.14 and 18.64 percentage points higher than that of orchards with 6-10 years of age and 10-15 years of age,respectively.There are differences in the incidence of disease among the different regions,with Baota District,Suide County and Mizhi County showing a descending order of incidence rates.2.The inhibitory effect of two strains of biocontrol bacteria on V.mali.Through measuring the mycelial growth rate,two biocontrol bacteria had a significant antagonistic effect on the growth of V.mali,inhibiting mycelial growth by 87% for EDR4 strain and82% for Em7 strain,which resulted in the marginal mycelium of V.mali turning brown.The sterile filtrate of both strains was observed to significantly inhibit the mycelial growth of V.mali,with more than 90% inhibition observed at a 2% concentration of EDR4 strain.The sterile filtrate of Em7 strain at an 8% concentration also showed no mycelial growth.Additionally,with an increase in sterile filtrate content in the medium,the inhibition effect became stronger.Observations using SEM showed that both biocontrol bacteria caused vesicular expansion at the tip of V.mali mycelium,rupture of the outer wall of mycelium and exudation of cytoplasm,flattening and crumpling of mycelium.The sterile filtrate of both strains of V.mali was also capable of inhibiting spore germination,blocking budding tube growth,and leading to spores being unable to absorb water at high concentrations.3.The inhibition mechanism of the two strains of biocontrol bacteria was studied.The results showed that both strains had the ability to secrete iron-chelating siderophores,protease,cellulase,amylase,lipopeptides,and form biofilms.Additionally,agarose gel electrophoresis was used to examine their ability to synthesize antibacterial substances.Using q RT-PCR to detect apple disease-resistance-related genes,it was found that the fermentation broth of EDR4 and Em7 strains,diluted 5 times and sprayed on apple seedlings,could increase the expression levels of apple disease-resistance-related genes Md PR1,Md PR2,Md PR4,and Md WRKY33.In addition,the growth promotion test showed that the application of the 5-fold diluted EDR4 strain fermentation solution could increase the height of tomato and tobacco by 24.40% and 24.24%,respectively.Similarly,the application of the 5-fold diluted Em7 strain fermentation solution could increase the height of tomato and tobacco by 39.05% and 21.35%,respectively,and significantly increased the fresh weight.4.The efficacy of two biocontrol bacteria strains in the control of Apple Valsa Canker.The safety of biocontrol bacteria for apple trees was established first.Indoor control efficacy of the biocontrol bacteria strains and their sterile filtrate on Apple Valsa Canker was determined by isolated branch tests.Results showed that the protection effect of EDR4 strain and sterile filtrate on Apple Valsa Canker was 72.35% and 50.42%,with treatment effect of 32.86% and 14.18%,respectively.The protection effect of Em7 strain and sterile filtrate on Apple Valsa Canker was 73.04% and 42.53%,with treatment effect of 30.72%and 10.35%,respectively.Preliminary findings suggested that prevention had better protection effect.A field trial of fermentation solution was carried out investigation results all agents were effective in reducing disease scars.Specifically,average field efficacy for strains EDR4 and Em7 diluted 5 times were 64.05% and 53.07%.5.Preparation of complex wettable powders of EDR4 and Em7 strains was carried out.The affinity assay and blood agar plate test did not show any transparent circles indicating that the two biocontrol bacteria strains had no incompatibility and were safe for humans and animals,making them suitable for bacterial agent preparation.The selection of carriers,wetting agents,dispersants,stabilizers,UV protectants was carried out using single-factor tests based on maximum adsorption,wetting time,mass suspension rate,and viable bacteria number as indicators.An orthogonal test was further conducted to determine the dosage and best additive combination in the study,resulting in an optimal formulation of the wettable powder with 8% Polyethylene glycol,10% SDS,1% dipotassium hydrogen phosphate,1%humic acids,70% master powder,5% Component A and 100% carrier complement.Quality evaluation of the wettable powder was performed according to national standards,all indexes except the suspension rate met the standard.In isolated branch tests,the protection effect of the wettable powder on Apple Valsa Canker was 72.67% and the treatment effect was 31.35%.These results can lay the foundation for field application of the wettable powder. |
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