Biocontrol strain hw17 for controlling cucumber green mottle mosaic virus and its application

A technology for green mottled flowers and biocontrol strains, applied in the field of agricultural microorganisms, can solve the problems of difficult prevention and control, complex genetic mechanism and background, and difficulty in cultivating varieties with stable resistance, etc., and achieves the effect of reducing residues and saving expenses.

Active Publication Date: 2018-03-09
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although cultivating disease-resistant varieties is the fastest and most effective method, due to the complexity of its genetic mechanism and background, it is difficult to breed varieties with stable resistance. At the same time, due to the high price of disease-resistant varieties, ordinary farmers do not have the ability to purchase them. The promotion and application of disease-resistant varieties
On the other hand, cucumber green mottle mosaic virus disease is mainly transmitted by seed transmission and contact infection. The virus can enter the tissue through the host's tiny wounds, and it can also infect healthy plants with the virus through grafting operations. The virus can survive the winter in the soil , and the activity can be maintained for more than 1 year, becoming the source of infection in the next year, making it difficult to prevent and control

Method used

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  • Biocontrol strain hw17 for controlling cucumber green mottle mosaic virus and its application
  • Biocontrol strain hw17 for controlling cucumber green mottle mosaic virus and its application
  • Biocontrol strain hw17 for controlling cucumber green mottle mosaic virus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Screening method: Bacillus subtilis HW17 (CGMCC NO.11025) was isolated from healthy watermelon rhizosphere in Dingji Town, Huaian City, Jiangsu Province. Cut the root tissue of watermelon into small pieces of 1 cm, soak it in 1% sodium hypochlorite for 5 minutes, soak it in 70% alcohol for 1-2 minutes, and wash it with sterile water for 3 times (take 100 μl of the last cleaning solution and apply it on R 2 A solid culture medium, if sterile growth after 48h, it is considered that the surface is clean and sterile). Take 3g of the sterilized sample and place it in a sterile mortar, add 27ml of sterile 0.85% NaCl, macerate the tissue, grind it, and dilute with sterile 0.85% NaCl gradient. take 10 -1 、10 -2 、10 -3 100 μl each of the three gradient dilutions were applied to R 2 On A, culture at 28°C for 48h. Pick the largest single colony to inoculate into fresh R 2 A solid medium, through repeated transfer to obtain purified strains. The purified strains were stored ...

Embodiment 2

[0046] The preparation of embodiment 2 HW17 biocontrol agent

[0047] Inoculate the Bacillus subtilis HW17 (CGMCC NO.11025) strain into the LB culture medium for 16 hours at 28°C with shaking at 200rpm, then take a sample every 2 hours to measure its OD value at 600nm at 600nm, when the OD value is 0.8 When the culture is over, this bacterial solution is used as the seed bacterial solution. Inoculate the seed bacteria solution into LB fermentation medium at a volume ratio of 1:500 for fermentation, culture at 28°C and 200 rpm for 48 hours, then centrifuge at 6000 rpm for 10 minutes, and dilute the precipitate to obtain the biocontrol agent HW17. The total concentration of viable bacteria in the resulting biocontrol agent was 1×10 9 ~1×10 10 CFU / mL.

Embodiment 3

[0048] Example 3 Greenhouse Test Determination of the Control Effect of HW17 on Cucumber Green Mottled Mosaic Virus

[0049] The cucumbers are transplanted at the 2-3 leaf stage, and the roots are irrigated and sprayed at the same time. Example 2 The preparation of HW17 (CGMCCNO.11025) bacterial agent irrigation root concentration is 1 × 10 8 CFU / mL, the dosage for each seedling is 30ml. The spray concentration is 1×10 7 CFU / mL, the spray standard is that there is a mist-like droplet distribution on each leaf, subject to not falling, spray evenly. After 7 days of biocontrol agent treatment, inoculate cucumber green mottle mosaic virus (the pathogenic bacteria used is the virus CGMMV, donated by Zhejiang Academy of Agricultural Sciences, it is an infectious cloned Agrobacterium, the strain name is called EHA105 in the experiment, and the transformed plasmid is pCB-CGMMV Prepare EHA105 in LB culture solution containing rifampicin and kanamycin (the final concentration is 50mg...

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Abstract

The invention belongs to the field of agricultural microorganisms, and relates to a biocontrol bacterial strain HW17 for preventing and treating cucumber green mottle mosaic virus disease and an application thereof. The biocontrol strain HW17, which was identified as belonging to the genus Bacillus subtilis, was deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on June 29, 2015, and the strain preservation number is CGMCC NO.11025. At the same time, the greenhouse test results showed that the biocontrol agent HW17 can significantly reduce the disease severity of cucumber green mottle mosaic virus disease, and has a significant effect on the control of cucumber green mottle mosaic virus disease, with a control effect between 49.63% and 53.76%. It has a very good growth-promoting effect on cucumbers, the biomass can be increased by 87.54%, and the chlorophyll content of cucumber leaves can be significantly increased. Therefore, the bacterial strain and the microbial agent prepared therefrom can be used in the prevention and treatment of cucumber green mottled mosaic virus disease and growth promotion.

Description

technical field [0001] The invention belongs to the field of agricultural microorganisms, and relates to a biocontrol bacterial strain HW17 for preventing and treating cucumber green mottle mosaic virus disease and an application thereof. Background technique [0002] Cucumber green mottle mosaic virus (CGMMV) is an important quarantine virus on Cucurbitaceae plants in many countries and regions in the world, seriously threatening the production of watermelon, muskmelon, cucumber and other crops. In 1935, Ainsworth of the United Kingdom first reported the damage of CGMMV on cucumbers, and it was later spread to more than 20 countries in Europe, Asia, and South America. The disease was first discovered in Gaizhou, Liaoning, my country in 2005. Because of its serious damage, the Ministry of Agriculture issued Announcement No. 788 on December 21, 2006, identifying CGMMV as a national agricultural plant quarantine pest. [0003] The natural host of cucumber green mottle mosaic ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20A01N63/02A01P1/00A01P21/00C12R1/125
Inventor 刘红霞李宏伟
Owner NANJING AGRICULTURAL UNIVERSITY
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