Composition Analysis, Identification And Screening Of Antagonistic Bacterium Against Botrytis Cinerea And Its Control Effect In The Field

Gray mold, a disease caused by the fungus Botrytis cinerea, influences the yield and quality of strawberry. This disease mainly infects plants, such as cucumber, tomato, hot pepper, and strawberry, and has a wide host range. Given its strong selectivity, good adaptability, rapid breeding speed, and fine variability, B. cinerea displays drug resistance toward many chemical pesticides. Thus, preventing and controlling the diseases caused by B. cinerea are relatively difficult. The long-term wide usage of chemical agents, including procymidone, iprodione, pyrimethanil, diethofencarb, and carbendazim, has enhanced the drug resistance of B. cinerea. Hence, finding new strategies to prevent and control gray mold of strawberry is necessary. Biological control, which features advantages such as high efficiency, low toxicity, and low cost, avoids the negative effects of chemical agents. This technique has become a research hot spot in the past few years. Some biocontrol bacteria have become the focus of basic research indoors, and some have been registered. However, the strains of other bacteria easily degenerate. Consequently, new bacterial strains should be determined. Aiming at these problems, this research isolates bacteria with a relatively strong antagonism against B. cinerea from soil that supports strawberry growth. The main research results are as follows:1) Isolation and screening of antagonistic bacteria against B. cinereaIn total,102 bacterial strains were isolated from soil through attenuation and isolation. Strains W2, W4, W7, W11, and W20, occupying 4.9% of the screened strains, exhibit resistance against gray mold of strawberry. The colonial morphology of W2 is in a radial pattern, that of W20 is irregular, and those of W4, W7, and antagonistic bacterium W11 are round. The colony surfaces of W2 and W11 are smooth, whereas W4 and W20 have rough surfaces. The edges of W2 and W20 are irregular, whereas those of the remaining three strains are regular. The color of the colony of W4 is pale, whereas those of the three other strains are milky white. Among the five antagonistic bacteria, W11 exerts the strongest antagonistic effect on gray mold of strawberry, whereas the other four strains demonstrate a relatively weak antagonistic effect. Hence, W11 is selected as the antagonistic bacterium against gray mold of strawberry in this research for further study.2) Effect of W11 on B. cinereaThe antagonistic effect of W11 against various plant pathogenic fungi is evaluated using the agar disk diffusion method. The results indicate that W11 displays the strongest antagonism against the pathogenic bacteria of gray mold of strawberry, followed by Rhizoctonia solani, Colletotrichum capsici, Sclerotinia sclerotiorum, Verticillium dahliae, and Phytophthora capsici. The strain has a wide antimicrobial spectrum, can control many plant diseases, and has a potential to develop biopesticides. The toxicity of W11 on B. cinerea is studied through the growth rate method. A high concentration of the active matter in B. cinerea corresponds to a significant inhibitory effect. The best inhibitory effect (69.04%) is obtained when the concentration reaches 7.5 ?g/mL, and the EC50 of the active matter on B. cinerea is 4.3363 ?g/mL. The regression equation is y= 0.1706+1.9607x, and the correlation coefficient is 0.9622.A series of physiological and biochemical measurements indicates that this strain can make glucose produce acid, liquefy gelatin, and hydrolyze starch. The strain tests positive for methyl red, Voges-Proskauer, catalase, nitrate reduction, and methyl response but negative for indol, hydrogen sulfide, anaerobic growth, malonate utilization, and phenylalanine deaminase. The results of molecular biological identification show that the 16S rDNA sequence length of this strain is 1477 bp. Comparison of this sequence and that in the online database of the National Center for Biotechnology Information indicates that the 16S rDNA sequence of W11 is relatively highly homologous (reaching 100%) to that of Bacillus subtilis. The established phylogeny tree also presents that W11 is the closest to B. subtilis in the aspect of evolutionary status. W11 is identified to be B. subtilis after combining the results of the molecular biological identification of 16S rDNA with the results of morphological, physiological, and biochemical tests.3) Antifungal active component analysis of W11The total ion current diagram is obtained through preliminary analysis of the active matter of W11 by using gas chromatography-mass spectrometry. The mass fraction of each ingredient in the total ingredient is solved using the peak area normalization method. A total of 44 compounds are detected after analyzing the active matter of W11 on the basis of the similarity of over 80% and the content of over 2%. Of these 44 compounds,43 occupy 97% of the total ingredients. The active components of W11 mainly include lipoid materials, ketones, esters, alcohols, acids, phenols, peptides, alkenes, amines, and aldehydes. Lipoid materials (23.68%) are the major components of the active components of W11, followed by ketones (18.42%), esters (13.16%), alcohols and acids (7.9%), and phenols (5.26%). Other active components, in order of large to small proportion, are loops, peptides, aldehydes, anhydrides, amines, pyrimidines, and pyrrole.4) Control effect of the active substance of W11 on gray mold of strawberry in the fieldThe active substance of W11 can prevent and control gray mold of strawberry in a dose-dependent manner. The weakest disease prevention effect (i.e.,<60%) is observed when the concentration reaches 6.25-25 ?g/mL. The disease prevention effect is between 62% and 77% when the concentration reaches 50-70 ?g/mL. The strongest disease prevention effect of 81.69% is obtained when the concentration reaches 100 ?g/mL. This value is significantly higher than that of the control group of agentia procymidone. The results of variance analysis indicate that significant differences exist among different concentrations. In addition, the active substance of W11 exerts no influence on the normal growth of strawberry. No spots of phytotoxicity are observed on the leaves, flowers, fruits, and roots of strawberry. This finding proves that the active matter of W11 ensures the growth safety of strawberry and thus can be widely applied.