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Endogenous bacillus subtilis positive mutant strain, preparation method thereof, prepared biocontrol agent and application of biocontrol agent in preventing and controlling pomegranate dry rot

A technology for dry rot of Bacillus subtilis and pomegranate, which is applied in biochemical equipment and methods, botanical equipment and methods, microorganism-based methods, etc., and can solve the problems of difficulty in popularization and low bacteriostatic effect.

Inactive Publication Date: 2014-09-03
ZAOZHUANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, endophytic bacteria screened from inside plant tissues generally have low antibacterial effect. Mutation breeding is an important direction of industrial microorganism breeding, and among them, ultraviolet radiation mutagenesis is the most commonly used in physical mutagenesis methods. Other physical mutagenesis methods Factors are limited by equipment conditions and are difficult to popularize. Therefore, the method of ultraviolet mutagenesis can be used to breed positive mutants with high resistance activity, so that endogenous biocontrol bacteria have greater application potential in the prevention and treatment of plant diseases.
At present, the research on the endophytic bacteria of pomegranate dry rot is still blank at home and abroad

Method used

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  • Endogenous bacillus subtilis positive mutant strain, preparation method thereof, prepared biocontrol agent and application of biocontrol agent in preventing and controlling pomegranate dry rot
  • Endogenous bacillus subtilis positive mutant strain, preparation method thereof, prepared biocontrol agent and application of biocontrol agent in preventing and controlling pomegranate dry rot
  • Endogenous bacillus subtilis positive mutant strain, preparation method thereof, prepared biocontrol agent and application of biocontrol agent in preventing and controlling pomegranate dry rot

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Screening of wild-type endophytic strain NS04

[0053] 1. Isolation and Purification of Endophytic Strains

[0054] Collect healthy pomegranate leaves and weigh 3.0 g, wash them with tap water, rinse them with sterile water three times, cut them into small pieces of 4-5 mm square with sterile scissors, and wash them with 75% alcohol in turn on the ultra-clean workbench. Soak for 5 s, soak in 0.1% mercuric chloride for 50 s, rinse with sterile water for 3 times, then add 30 mL of sterile water to grind in a sterile mortar; after grinding, keep the layers, and the supernatant is used as the leaching solution for later use. 0.1 g / mL, take 1 mL of the supernatant for serial dilution to 10 -5 , from 10 -3 、10 -4 、10 -5 Take 0.2 mL of each coated plate from the diluted suspension, and carry out sterility test with 0.2 mL of the last rinsing solution on the coated plate, with 3 replicates for each treatment. Inverted culture in a constant temperature incubator at...

Embodiment 2

[0070] Embodiment 2: the preparation method of positive mutant strain NS04307

[0071] 2.1 Determination of the growth curve of wild-type strain NS04

[0072] Make the starting strain after activation into a concentration of 1×10 8 For cfu / ml bacterial suspension, take 20 μl and put it into a test tube containing 10 ml beef extract peptone liquid medium, connect 18 tubes in total, place it at 37°C, and shake it at 150 r / min for culture. After 2 hours, At the beginning, take out a test tube every 2 hours and put it in a refrigerator at 4 °C for storage. After all the tubes are taken out, use a spectrophotometer to measure their OD values ​​at a wavelength of 625 nm. For the control, 3 repetitions were performed for each treatment. The growth curve of the strain was drawn with the culture time as the abscissa and the OD value as the ordinate to determine its logarithmic growth phase. The results showed that the growth curve of the NS04 strain was S-shaped, with a lag phase of...

Embodiment 3

[0087] Embodiment 3: the preparation method of NS04307 bacterial strain biocontrol agent

[0088] 1. The effect of ventilation on the growth and antibacterial performance of NS04307 strain

[0089] Use a 250ml Erlenmeyer flask to fill 50ml, 75ml, 100ml, 125ml, 150ml of beef extract peptone liquid medium, inoculate with 1% of the liquid volume, and inoculate the NS04307 strain at 37°C and 180 r / min Cultured on a shaking table respectively, and the OD of the fermentation broth was measured after 24 h 625 value. Then adopt the zone of inhibition method described in Example 2 to do antagonism experiment between fermented liquid and pomegranate dry rot pathogen, measure the bacteriostatic situation of fermented liquid under each ventilation rate, and measure the size of the zone of inhibition. The results showed that the growth of the strain NS04307 was the best when 50 ml of NS04307 was added, and the filling volume was 20%, but there was no significant difference in the antibac...

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Abstract

The invention relates to an endogenous bacillus subtilis positive mutant strain, a preparation method thereof, a prepared biocontrol agent and an application of the biocontrol agent in preventing and controlling pomegranate dry rot, including (1) screening of a Bacillus subtilis wild-type strain NS04 and ultraviolet mutation and breeding of a positive mutant biocontrol strain NS04307; (2) antibacterial stability of the positive mutant strain NS04307; (3) a preparation method of the positive mutant strain NS04307 biocontrol agent; and (4) application of the positive mutant strain NS04307 in preventing and controlling plant diseases. The biocontrol agent disclosed by the invention has the characteristics of wide bactericidal spectrum, good effect, difficulty in disease drug resistance generation, safety on human, livestock and crops, environmental friendliness and the like and has a better effect on the field control of the pomegranate dry rot especially. In addition, the biocontrol agent disclosed by the invention achieves outstanding inhibition activity on the growth of 15 different pathogenic bacteria, such as Sclerotiumrolfsii, Trichotheciumroseum, Exserohilumrostratum, Fusariumoxysporum, Alternariaalternate and the like.

Description

technical field [0001] The invention belongs to the technical field of biological pesticides, and in particular relates to an endophytic Bacillus subtilis positive mutant strain NS04307 with high activity against pomegranate dry rot, its preparation and application for preventing and treating pomegranate dry rot. Background technique [0002] Pomegranate dry rot is an important disease affecting the pomegranate industry, and its pathogen is Ascospora pomegranate ( Coniella granati (Sacc.)Petr.&Syd) Synonyms: Zythia Versoniana Sacc., belonging to the subphylum Agnostica ( Deuteromycotina ), Spheroides ( Sphaeropsidales ). It mainly infects fruits and trunks, but can also infect pomegranate flower organs, fruit branches and new shoots. The mycelium or conidia of the pathogen can overwinter on the diseased fruit, diseased branch, and diseased fruit table, causing the diseased fruit peel and diseased branch to become the source of primary infection. When there is a lot ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N13/00A01N63/00A01P3/00C12R1/125
Inventor 谭小艳马耀华黄思良刘晶张盼盼牛小瑞罗伟
Owner ZAOZHUANG UNIV
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