Broad-spectrum antifungal plant endophytic bacillus subtillis and application thereof

A technology of Bacillus subtilis and plants, applied in the field of Bacillus subtilis R31, can solve the problems of failure, high similarity between sequences, and inability to effectively distinguish Bacillus subtilis groups, and achieve good antagonistic ability, good control effect, and good plate antagonism active effect

Active Publication Date: 2010-06-30
GUANGDONG GEOLONG BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The species of the Bacillus subtilis group include Bacillus subtilis subsp.subtilis (Smith et al., 1964; Nakamura et al., 1999), Bacillus lichenifromis (Skerman et al., 1980), Bacillus atrophaeus (Nakamura, 1989), Bacillus mojavensis (Robert et al., 1994), Bacillus vallismortis (Roberts et al., 1996), Bacillus subtilis subsp. spizizenii (Nakamura et al., 1999), Bacillus sonorensis (Palmisano et al., 2001), Bacillus subtilis subsp. et al., 2009), these species are closely related and can be distinguished by fatty acid composition analysis, DNA-DNA hybridization analysis and restriction enzyme analysis, but it is difficult to distinguish them by phenotypic characteristic analysis (Fritze, 2004, Wang, et al., 2007), although the 16S rDNA / RNA gene sequence is widely used in bacterial identification or to construct the phylogenetic relationship of bacteria, but among taxonomic groups with close kinship, due to the high similarity between the sequences Failed (Christense et al., 1998), 16S rDNA sequence cannot effectively distinguish the species of Bacillus subtilis group

Method used

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  • Broad-spectrum antifungal plant endophytic bacillus subtillis and application thereof
  • Broad-spectrum antifungal plant endophytic bacillus subtillis and application thereof
  • Broad-spectrum antifungal plant endophytic bacillus subtillis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Separation, purification and physical and chemical properties of R31

[0027] (1) Separation, purification and preservation of R31

[0028] Collect healthy two-color dendrobium leaves in the greenhouse of the orchid production base of the Zhuhai Agricultural Science Research Center, rinse them with tap water, dry them and treat them with 75% alcohol for 1 min, then treat them with 5% NaOCl for 5 min, and rinse them with sterile water 4 times; After drying, use sterile scissors and tweezers to cut the leaves into tissue pieces of about 0.2cm×0.5cm, and spread them on NA (beef extract peptone medium) plates containing 50μg / mL cycloheximide; culture at 28°C Observe every day. Once bacteria are found to grow from the incision, they are picked out, purified and cultured on a new NA plate, and stored in the form of bacteria solution with 20% glycerol at -20℃ and -80℃. Then take 200μl of sterile water from the fourth rinse and spread it on the NA plate, do 3 repetitions...

Embodiment 2

[0095] Example 2: Antagonistic test of R31 against different pathogens of Fusarium

[0096] (1) Isolation and purification of different Fusarium pathogens

[0097] Isolation source: Collect typical banana wilt, celery verticillium wilt and dendrobium leaf spot disease tissues in Zhuhai City and surrounding cities, and isolate the pathogen to test the antagonistic activity of R31 against different Fusarium pathogens.

[0098] Separation and purification method: clean the diseased tissue with clean water, blow dry, use a sterile blade to cut the diseased tissue at the junction of disease and health and cut into suitable size, sterilize the surface with 70% alcohol for 30s under aseptic conditions, and then use effective chlorine Treat the 5% NaOCl for 5 minutes, rinse with sterile water 4 times, cut the diseased tissue after surface disinfection with sterile scissors and tweezers into a tissue block of about 0.2cm×0.5cm, dry the surface of the diseased tissue block and place it on Add...

Embodiment 3

[0104] Example 3: The antagonistic activity of R31 against other plant pathogenic fungi, the results are shown in Table 5

[0105] The method for bioassay of antagonistic activity is the same as that of Example 2. Among them, Alternaria, Curvularia lunata, Mango stalk rot, Watermelon wilt, etc. are plant pathogenic fungi donated by Zhuhai True Green Technology Co., Ltd. Rhizoctonia is a plant pathogenic fungus presented by Dr. Yi Runhua from Guangdong Ocean University. The larger the antibacterial band, the stronger the antibacterial effect.

[0106] Table 4 The antagonistic activity of R31 against the pathogenic fungus Fusarium spp. from different hosts

[0107]

[0108] Table 5 Plate antagonistic activity of R31 against several plant pathogenic fungi

[0109]

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Abstract

The invention relates to bacillus subtillis R31 with broad-spectrum antagonism capacity on plant pathogenic fungi, and in particular provides a bacterial strain (CCTCC NO: M209261) of the Bacillus subtillis R31 as well as growth inhibition action thereof on the plant pathogenic fungi and especially capacity thereof on the biological prevention and control of banana vasicular wilt. The plant endophytic bacillus subtillis is proved to be new bacillus subtillis through obtained 16S rDNA and gyrA and gyrB gene segments as well as BLAST search and comparison on NCBI (National Center of Biotechnology Information) and has better flat antagonism activity on multiple plant pathogenic fungi, better antagonism capacity especially on various soil-borne and vascular bundle diseases, i.e. fusarium oxysporum, rhizoctonia solani, alternaria, and the like, and better prevention and control effect in greenhouses and fields.

Description

Technical field [0001] The present invention relates to a Bacillus subtilis R31 strain with broad-spectrum antagonistic ability against plant pathogenic fungi, and specifically provides Bacillus subtilis R31 (Bacillus subtilis R31) strain (CCTCC NO: M 209261) and its growth against plant pathogenic fungi Inhibition, especially in the biological control of banana wilt, belongs to the category of biological pesticides and disease-resistant microbial fertilizers. Background technique [0002] The losses caused by diseases of horticultural crops such as fruits and vegetables in our country are very serious. According to the results of the national census of pests and diseases of garden plants from 1984 to 1986, 5508 kinds of plant diseases were found on 1256 kinds of garden plants, with an average of 4.4 on each plant, vegetables and flowers Diseases of horticultural crops such as fruit trees and fruit trees are far more serious than those of bulk cereal crops. According to statisti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A01N63/00A01P3/00C12R1/125
Inventor 喻国辉黎永坚陈燕红杨紫红陈远凤程萍
Owner GUANGDONG GEOLONG BIOTECH
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