Aspergillus flavus biocontrol strain realizing resistance on bactericides without producing aflatoxin and application of aspergillus flavus biocontrol strain
A kind of technology of aflatoxin and bio-control strains, applied in the direction of application, biocides, microorganisms, etc., can solve the problems of fungicide sensitivity, affecting the effect of bio-control and the stability of control effect, so as to reduce the inhibitory effect, improve the fitness and Colonization ability, the effect of maintaining the effect of biological control
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Embodiment 1
[0027] Example 1 The isolation and identification of the Aspergillus flavus strain AF054 that does not produce aflatoxin
[0028] 1. Isolation of Aspergillus flavus strains from soil, corn, peanut and other habitats
[0029] Soil was collected from corn and peanut fields in Jiangsu, Zhejiang, Shandong, Fujian, Anhui and other places, and the soil was cultured with YES medium to isolate Aspergillus flavus strains. Ingredients per liter of YES medium: Yeast extract 1g, sucrose 10g, NaCl 60g, agar 20g, add CuSO after sterilization 4 .ZnSO 4 1 mL, 0.4% clonamide 5 ml, streptomycin 100 μg / mL.
[0030] 2. Use bioassay, thin-layer chromatography and enzyme-linked immunoassay to analyze the aflatoxin production of the strain.
[0031] details as follows:
[0032] Bioassay method: Inoculate the isolated Aspergillus flavus strain on YES medium containing 0.3% cyclodextrin, culture it at 30°C for 7 days, fumigate the colony with 25% ammonia water for 3 minutes, if the back of the col...
Embodiment 2
[0051] Example 2 Deletion Identification of Aflatoxin Synthetic Gene in Aspergillus flavus Strain AF054 That Does Not Produce Aflatoxin
[0052] (1) Primer synthesis
[0053] In order to clarify the deletion of the aflatoxin synthesis gene in the non-aflatoxin-producing Aspergillus flavus strain AF054, the test refers to the literature Perng-KuangChang (Chang, P.K., Horn, B.W. and Dorner, J.W. (2005) Sequence break points intheaflatoxinbiosynthesisgeneclusterandflankingregionsinnononaflatoxigenicAspergillusflavusisolates49Bioisolates49Bioisolates42. The identification primers about toxin synthesis genes designed in ) were used to identify the deletion of related genes in strain AF054.
[0054] (2) Gene deletion identification
[0055] Using the extracted DNA of Aspergillus flavus strain AF054 as a template, conventional PCR reactions were carried out with the above primers, and each reaction had a DNA control and a negative control (use sterile water as a template) of the tox...
Embodiment 3
[0059] Example 3 Indoor test evaluates the resistance of Aspergillus flavus strain AF054 that does not produce aflatoxin to amida, carbendazim and tebuconazole
[0060] Inoculate AF054 or toxin-producing Aspergillus flavus standard strain NRRL3357 on PDA plates containing 10 μg / mL carbendazim or 25 μg / mL tebuconazole or 100 μg / mL amida, respectively, and observe the effect of the strains on fungicides after culturing at 25 degrees for 3 days. resistance. The experiment took PDA without fungicide as the control. from figure 2 It can be seen that the non-toxin-producing Aspergillus flavus strain AF054 of the present invention can resist carbendazim, tebuconazole and amida. And AF054 on the plate containing fungicide will not affect its ability to produce conidia.
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