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Method for obtaining endophytic bacteria for degrading verticillium dahlia toxin protein and application in preventing and treating plant verticillium wilt

A technology of plant endophytic bacteria, Verticillium dahliae, applied in the fields of botanical equipment and methods, plant growth regulators, microorganism-based methods, etc., can solve problems such as uninquiry

Inactive Publication Date: 2013-02-06
NANJING NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the published materials, there is no query about the method of screening endophytic bacteria for degrading toxic proteins in vitro and the application of plant endophytic bacteria to prevent and treat Verticillium dahliae by degrading Verticillium dahliae toxins in plants

Method used

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  • Method for obtaining endophytic bacteria for degrading verticillium dahlia toxin protein and application in preventing and treating plant verticillium wilt
  • Method for obtaining endophytic bacteria for degrading verticillium dahlia toxin protein and application in preventing and treating plant verticillium wilt
  • Method for obtaining endophytic bacteria for degrading verticillium dahlia toxin protein and application in preventing and treating plant verticillium wilt

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Isolation and Endophytic Determination of Plant Endophytic Bacteria

[0044] (1) Isolation of strains: Collect cotton, tomato, strawberry, cucumber and other plants from different planting places, wash and dry the roots, stems and leaves with tap water, weigh 1-2g each, soak in 1% NaClO for 5min , washed 5 times with sterile water, and then surface-sterilized with 0.1% mercuric chloride. Leaves were washed for 1 minute, roots and stems for 3 minutes, washed 5 times with sterile water, and blotted dry with sterile filter paper. Put the leaves into a sterile tissue homogenizer, weigh the roots and stems, put them into a sterile mortar and grind them, take 1g sample and add 9mL sterile water to make 10 -1 , diluted with sterile water to 10 -5 , After resting for 15 minutes, take 200μl each and spread on the plate, and repeat 3 times for each treatment. Take 200 μL of sterile water from the last rinse and smear it on the plate to verify whether the disinfection...

Embodiment 2

[0047] Example 2: Identification of Plant Endophytic Bacteria

[0048] Example 1 The isolated plant endophytic bacterial strains take DP10 as an example: DP10 was streaked and inoculated on NA medium, cultured at 28°C for 24 hours, firstly, 30 μL of sterile water was added to a 1.5 mL centrifuge tube, and picked with an inoculation needle Take a small amount of bacteria to make a suspension in water, boil for 10 minutes, quickly ice-bath for 5 minutes, centrifuge at 12000r / min at 4°C for 5 minutes, and the supernatant is used as a PCR template.

[0049] Reaction system (50 μL): 10×PCR buffer (5 μL), dNTP mixture (10 mmol / L) 1 μL, MgCl2 (25 mmol / L) 4 μL, primer P0 (5 μmol / L) 1 μL, primer P5 (5 μmol / L) 1 μL, Taq DNA polymerase (5U / L) 0.5μL, 5μL supernatant as Template, add ddH 2 O 32.5 μL to a final volume of 50 μL.

[0050] Design 16SrDNA primers: forward primer: 5'-GAGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1)

[0051] Reverse primer: 5'-CTACGGCTACCTTGTTACGA-3' (S...

Embodiment 3

[0056] Embodiment 3: Preparation of Verticillium dahliae toxin VD solution and plant endophytic bacteria suspension

[0057] The used Verticillium dahliae can be published standard strains such as ACCC36120, ACCC36129, ACCC36217, ACCC36211 of China Agricultural Microorganism Collection Center, or can be isolated as in Example 4.

[0058] Put the Verticillium dahliae fermentation broth in a water bath at 100°C for 1 hour, vacuum filter with a 6-layer filter paper Buchner funnel, discard the mycelium, centrifuge the filtrate at 8000r / min for 10 minutes, and take the supernatant and pass it through 0.45μm and 0.22μm microporous membranes in turn Vacuum filtration, the filtrate is crude toxin liquid, namely VD liquid.

[0059] Dip a circle of activated endophytic bacterial strains (SZ5, CCM9 and DP10 strains) with an inoculation needle, inoculate them into a 250 mL Erlenmeyer flask containing 30 mL of NB culture solution, and culture them on a shaker at 28 °C at 150 r / min for 48 h...

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Abstract

The invention relates to a method for obtaining endophytic bacteria for degrading verticillium dahlia toxin protein and an application in preventing and treating plant verticillium wilt, and belongs to the biotechnological field. The method for obtaining endophytic bacteria for degrading verticillium dahlia toxin protein comprises the steps of culturing endophytic bacteria separated from the plant and verticillium dahlia toxin; using polyacrylamide gel electrophoresis to detect protein band after culture, comparing with protein band of the original toxin; and taking mixed culture system without 26KD protein band as a target strain. The method has clear target and mechanism. The method overcomes the shortcomings that the bio-control microorganism in biological control soil borne disease is easily influenced by soil environment and has unstable effect. The method uses pathogenic character of the verticillium dahlia and conduction characteristic of the endophytic bacteria in the plant to directly screen endophytic bacteria for grading verticillium wilt toxin result in plant disease so as to achieve the aim of effectively preventing and treating plant verticillium wilt.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for effectively preventing and controlling plant Verticillium wilt damage by degrading Verticillium dahlia toxins through plant endophytic bacteria. Background technique [0002] Verticillium dahliae can cause plant Verticillium wilt, which is a very harmful disease of the vascular system. The disease is characterized by yellowing of leaves of susceptible plants, sudden wilting of leaves after prolonged drought and rain, so it is called " Verticillium wilt". Verticillium dahliae has a wide range of hosts, with as many as 660 species of host plants. Among them, there are 184 kinds of crops, 323 kinds of ornamental plants and 153 kinds of weeds. In crops, it is infective to cotton, potato, eggplant, tomato, pepper, etc., and can transfect each other. Grass crops such as rice, wheat, corn, etc. and some weeds are rarely infringed. In recent years, Verticillium w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A01N63/02A01P3/00C12R1/125
Inventor 闫淑珍陈双林许芳
Owner NANJING NORMAL UNIVERSITY
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