Apple Leaves Early Disease Biological Control Of Key Technology And Its Field Applications

Nowadays Apple early defoliation diseases have becoming the most serious diseases in most apple-growing areas in China,decreasing the yield and quality of apple.And the incidence was rising year by year.Until now,there is no a very effective control methods for the disease,the chemical pesticides were employed to suppress the diseases.However,the overuse of chemical pesticides inevitably brought about pesticide residues in apple fruit and environmental pollution,directly or indirectly affecting the health of human.Aimed at obtaining the broad-spectrum antagonistic strains and exploring a green and efficient,and sustainable control methods towards Apple early defoliation diseases,the present study was undertaken in antagonistic Bacillus stains screening,identification,inhibitory action, purification and characteristic of antifungal substance,optimization of fermentation process and field experiment.173 isolates of bacterial strains were isolated from 11 samples of different sources by Plate Dilution method.20 isolates with broad-spectum antagonistic activity against eight phytopathogens such as Alternaria alternata f.sp.Mali,Marssonina coronaria(Ell Et Davis) Davis,Phyllosticta pirina,Bolyosphoma berengeriana and so on were screening from these 173 isolates by Plate inhibition method.10%of the twelve bacterial strains exhibited obvious antagonistic activity against all of the eight phyopathogens and 70%exhibited obvious antagonistic activity against at least 3 phytopathogens.Seven strains could effectively inhibit Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis,the width of inhibition zone both exceeded 5 mm.The potential antagonistic strains against Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis were screening from the seven broad-spectum antagonistic strains by In vitro leavesf plate method.Compared with the control,the rate of diseased leaves in treatment group were significantly decreased(p＜0.05), and strain BS24 and D4 were proved to be the most effective ones with inhibition rate of 100%and 90.48%.This is the first report on the antagonistic strain with broad-spectum antagonistic activity against Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis.Strain BS24 and D4 were respectively identified as Bacillus subtilis and Bacillus megaterium though the traditional bacterial systemic identification method and the analysis of 16S rDNA sequence.Genbank accession number was GQ213991 and FJ755786,repectively. This is the first report on Bacillus megaterium with broad-spectum antagonistic activity against kinds of phyopathogens.Antagonism of antagonistic strains on phyopathogens was always achieved by the synergistic effect of various action modes.Strain BS24 could strongly inhibit the mycelial growth of Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis, and the width of inhibition zone were 9.5 mm and 9.2 mm,respectively.It could colonize rapidly on apple leaves in vitro and occupy the living space,which inhibit the colonization of phyopathogens and effectively block their invasion of apple leaves.Cell-free culture filtrate could also strongly inhibit the growth of Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis,the inhibition rate was 85.48%and 82.86%,respectively.It could also result in the abnormity and tortuosity of mycelial,cytoplasmic aggregation,the leak of cytoplasmic substances,the abnormal growth of germ tube and the inhibition of Conidia germination.After acid precipitation,methanol extraction and RP-HPLC,BSP,an antifungal substance, was obtained and initially determined as a small antifungal peptide.BSP had broad-spectrum angonistic activity,such as Alternaria alternata f.sp.Mali,Marssonina coronaria(Ell Et Davis) Davis,Phyllosticta pirina,Bolyosphoma berengeriana,Fusarim oxysporium f.sp. Melonis,Glanerella cingulata,Alternaria alternata fries Keissler and Alternaria solani and Escherichia coli.And the EC₅₀ towards Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis was 5.03μg/mL and 5.60μg/mL,respectively.BSP had good thermal stability,pH stability and protease stability.Strain BS24 can produce protease.Considering viable count in fermentation broth and the requirements for industrial production,the initial fermentation medium was screening from six fermentation medium. The further optimization was undertaken though Plackett-Burman Design,path steepest ascent and Central Composite Design.And a good regression equation was obtained;the determination coefficient(R²) was 0.96,which ensure an adequate credibility of the model. Verification of the predicted values was conducted by using optimal conditions in inoculation experiments.Result showed that the predicted value was much closer to practical value, which corroborated the validity and the effectiveness of this model.The viable count in fermentation broth after optimization was 15.08×10⁹ cfu/mL,which increased by 75.35% compared with initial count 8.6×10⁹ cfu/mL.The fermentation medium after optimization was as followed,corn flour 1.5 g,bean flour 2.16 g,yeast extract 0.3 g,sucrose 0.5 g,urea 0.3 g, MnSO₄ 0.01 g,MgSO₄ 0.01 g,K₂HPO₄ 0.2 g,distilled water 100 mL,pH 7.0,300 r/min and 7.8%of inoculation.Field experiment showed that the three concentration of antagonistic inoculum have both achieved the desirable control efficacy towards Apple early defoliation diseases.The control efficacy were 83.14%,79.21%and 76.99%respectively,which both exceeded that of 10% multiantimycin.The antagonistic inoculum had no harmful effects on plants under the three testing concentration.Residues of chemical pesticides and heavy metals were in all accord with national request for food.Due to its high control efficacy and safety,the antagonistic agent had great potential of application in the field control of Apple early defoliation diseases.