Pseudomonas aeruginosa for preventing and treating vertieillium wilt in crops and application of pseudomonas aeruginosa

A technology for Pseudomonas aeruginosa and crops, applied in the direction of chemicals for biological control, methods based on microorganisms, applications, etc., can solve the problems of large differences in antagonism effects, and achieve high-efficiency antagonism effects

Active Publication Date: 2016-04-06
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the antagonistic effects of different species, even strains from different sources of the same species, vary greatly

Method used

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  • Pseudomonas aeruginosa for preventing and treating vertieillium wilt in crops and application of pseudomonas aeruginosa
  • Pseudomonas aeruginosa for preventing and treating vertieillium wilt in crops and application of pseudomonas aeruginosa
  • Pseudomonas aeruginosa for preventing and treating vertieillium wilt in crops and application of pseudomonas aeruginosa

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 strain isolation and screening

[0027] The tested strains were isolated from the cotton experimental field of Zhejiang University Huajiachi Campus in Hangzhou City, Zhejiang Province.

[0028] Screening of biocontrol bacteria in soil by double-layer plate method. Put the Verticillium dahliae cake that has been cultured for 4-5 days in sterile water, and prepare a concentration of 10 6 per mL of spore suspension. Take an appropriate amount (1g) of soil sample to prepare a soil suspension in sterile water, and serially dilute to 10 -2 -10 -3 . Double-layer plate method: evenly coat the prepared spore suspension of Verticillium dahliae on PDA, grow at 28°C for 48 hours, pour LB on the upper layer of PDA, and evenly coat the soil suspension. Place the plate under dark conditions at 28°C for culture. After the colonies grow out, select the colonies with inhibition zones for purification and culture, and then carry out re-screening.

[0029] A total of 1 stra...

Embodiment 2

[0036] The identification of embodiment 2 bacterial strains

[0037] Morphological identification

[0038] The edge of the colony of the strain on LB medium was irregular, and a transparent halo was formed around the colony. Stain test determined Gram-negative bacteria. Under the scanning electron microscope, it is rod-shaped bacteria with a size of 0.7-0.9μm×1.8-3μm( figure 1 B).

[0039] 16SrDNA sequence analysis

[0040] The strain was inoculated in NA culture medium (yeast powder 1g / L, beef extract 3g / L, peptone 5g / L, sucrose 10g / L, pH=7.0), and after culturing at 30°C and 180rpm for 120 hours, DNA extraction was performed; 16SrDNA general primers were used for PCR amplification to obtain an electrophoretic band of about 877bp, which was recovered and sequenced by Sangon Bioengineering Co., Ltd. (Shanghai). The determined 16SrDNA sequence was compared with the sequence in the GenBank database to determine its taxonomic status.

[0041]16SrDNA sequencing, after compar...

Embodiment 3

[0049] The fermentation culture of embodiment 3 bacterial strain KK9a and the detection of antagonistic component

[0050] The culture medium can be LB liquid medium, its formula is: tryptone 10g / L, yeast extract powder 5g / L, sodium chloride 10g / L, adjust the pH to 7.0-7.2.

[0051] 50L automatic fermenter culture: inoculate Pseudomonas aeruginosa in LB culture medium and cultivate to culture medium OD 600 0.6 to 0.8, to obtain the seed solution; inoculate the seed solution into a 50L fermenter, the expansion culture temperature should be between 35 and 37°C, the culture time should be between 48 and 60h, the aeration ratio should be 0.4 to 2:1, and the rotation speed 300~600rpm, pH should be between 7~7.5. The fermentation medium is: tryptone 10g / L, sucrose 3g / L, sodium chloride 2.5g / L, adjust the pH to 7.0-7.2, and sterilize at 121°C for 30 minutes. The final bacterial concentration can usually reach 1×10 10 ~1×10 11 CFU / mL.

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Abstract

The invention discloses pseudomonas aeruginosa for preventing and treating vertieillium wilt in crops and application of the pseudomonas aeruginosa. The strain is named as pseudomonas aeruginosa KK9a, with preservation number as CGMCC 11637. The invention provides an inoculant containing the pseudomonas aeruginosa. The strain of the invention has an inhibitory effect on both spore germination and mycelial growth of verticillium dahlia and the strain has an efficient antagonistic action. The strain of the invention is capable of preventing and treating crop verticillium wilt caused by the verticillium dahlia, the strain has a significant effect on preventing and treating cotton verticillium wilt and control efficacy of pot experiments reaches 86% or above; therefore, the strain has a great potential in the application to bio-pesticides.

Description

technical field [0001] The invention belongs to the field of development and utilization of microbial germplasm resources, and specifically relates to Pseudomonas aeruginosa used for preventing and treating verticillium wilt of crops caused by Verticillium dahliae and application thereof. Background technique [0002] Verticillium dahliae (Verticilliumdahliae) belongs to Ascomycota fungus, and it mainly overwinters with microsclerotia on soil, diseased tissues, infected seeds and unripe soil miscellaneous fertilizers, and can harm 660 species of plants in 38 families, of which Daejeon Crops include cotton, sunflower, eggplant, pepper, tomato, tobacco, potato, etc., and the disease caused is called Verticillium wilt. After overwintering, the microsclerotium germinates and produces a large number of conidia. The conidia germinate and invade root hairs, root epidermal cells or root wounds, enter the vessel through the cortex, destroy the vascular system of the plant, and produc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A01N63/00A01P3/00C12R1/385
CPCA01N63/00C12N1/205C12R2001/385
Inventor 张敬泽李晓林祝水金
Owner ZHEJIANG UNIV
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