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Biocontrol strain of Aspergillus flavus resistant to fungicides and not producing aflatoxin and its application

A technology of aflatoxin and biocontrol strains, applied in the application, biocide, microorganism and other directions, can solve the problems of biocide sensitivity, affecting biocontrol effect and control effect stability, etc. Colonization ability, effectiveness in overcoming biocide susceptibility issues

Active Publication Date: 2018-08-24
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Common non-toxin-producing Aspergillus flavus biocontrol strains are sensitive to fungicides, and the use of fungicides will inhibit the proliferation of these biocontrol bacteria populations in farmland systems, thereby affecting the biocontrol effect and stability of control efficacy

Method used

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  • Biocontrol strain of Aspergillus flavus resistant to fungicides and not producing aflatoxin and its application
  • Biocontrol strain of Aspergillus flavus resistant to fungicides and not producing aflatoxin and its application
  • Biocontrol strain of Aspergillus flavus resistant to fungicides and not producing aflatoxin and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Isolation and identification of 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 colony is p...

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-Kuang Chang (Chang, P.K., Horn, B.W. and Dorner, J.W. (2005) Sequence breakpoints in the aflatoxin biosynthesis Gene cluster and flanking regions in nonaflatoxigenic Aspergillus flavus isolates. Fungal Genet Bio 42,914–923.) The identification primers for toxin synthesis genes were designed 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 to...

Embodiment 3

[0059] Example 3 Indoor test to evaluate the resistance of the non-aflatoxin-producing Aspergillus flavus strain AF054 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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Abstract

The invention discloses an aspergillus flavus biocontrol strain realizing resistance on bactericides without producing aflatoxin and application of the aspergillus flavus biocontrol strain. The aspergillus flavus biocontrol strain is named as aspergillus flavus AF054; the preservation number is CGMCC NO.9860; and the preservation date is November 20, 2014. The aspergillus flavus obtained through separation does not generate the aflatoxin; the formation of the aflatoxin in agricultural products can be effectively inhibited; the aflatoxin pollution in the agricultural products is reduced; the agricultural product quality is improved; the resistance on common-use bactericides such as carbendazol, tebuconazole and amistar is realized; the problem of sensitivity of the existing biocontrol strain on the bactericides in a farmland system is solved; and the colonizing capacity and the adaptability of the biocontrol strain in the field are effectively improved, so that the biocontrol effect of biocontrol bacteria is improved.

Description

technical field [0001] The invention relates to the field of agricultural product safety, in particular to a biocontrol strain of Aspergillus flavus which is resistant to fungicides and does not produce aflatoxin and an application thereof. Background technique [0002] Aflatoxins are mainly composed of metabolites of Aspergillus flavus and Aspergillus parasiticus, and are the most toxic type of biotoxins found so far in contaminated agricultural products. Among them, aflatoxin B1 is the most toxic and harmful, and its toxicity is 10 times that of potassium cyanide and 68 times that of arsenic. It is listed as a highly toxic substance under strict control. Aflatoxin contamination of agricultural products has become an important pollutant affecting food safety and endangering people's health. [0003] According to the World Food and Agriculture Organization estimates, currently 25% of the world's crop products are contaminated with mycotoxins, mainly aflatoxins. Aflatoxin p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/14A01N63/04A01P21/00C12R1/67
Inventor 陈云马忠华张承启高启讯
Owner ZHEJIANG UNIV
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