A broad-spectrum antifungal plant endophytic Bacillus subtilis and its application

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

Active Publication Date: 2012-02-01
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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  • A broad-spectrum antifungal plant endophytic Bacillus subtilis and its application
  • A broad-spectrum antifungal plant endophytic Bacillus subtilis and its application
  • A broad-spectrum antifungal plant endophytic Bacillus subtilis and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0028] In the greenhouse of the orchid production base of Zhuhai Agricultural Science Research Center, healthy leaves of Dendrobium bicolor were collected, rinsed with tap water, dried and treated with 75% alcohol for 1 min, then treated with 5% NaOCl for 5 min, and rinsed 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, spread them on NA (beef extract peptone medium) plates containing 50 μg / mL cycloheximide; culture at a constant temperature of 28°C , observe every day, pick out once bacteria grow out of the incision, purify and culture them on a new NA plate, and store them in the form of bacterial liquid with 20% glycerol at -20°C and -80°C. Then take 200 μl of sterile water rinsed for the fourth time and apply it on the NA plate, do 3 repetitions...

Embodiment 2

[0095] Embodiment 2: Antagonism test of R31 to different Fusarium pathogenic bacteria

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

[0097] Source of isolation: Collect typical banana fusarium wilt, celery verticillium wilt, and dendrobium leaf spot disease tissues in Zhuhai and surrounding cities to isolate pathogenic bacteria and test the antagonistic activity of R31 against different Fusarium pathogenic bacteria.

[0098] Separation and purification method: clean the diseased tissue with clean water, dry it, cut the diseased tissue at the junction of diseased and healthy tissue with a sterile blade and cut it into an appropriate size, disinfect the surface with 70% alcohol for 30 seconds under sterile conditions, and then use available chlorine Treat with 5% NaOCl for 5 minutes, rinse with sterile water for 4 times, cut the surface-sterilized diseased tissue into tissue pieces of about 0.2 cm × 0.5 cm with sterile scissors and tweezers, ...

Embodiment 3

[0104] Embodiment 3: The antagonistic activity of R31 to other phytopathogenic fungi, the results are shown in Table 5

[0105] The method during bioassay antagonistic activity is with embodiment 2, and wherein Alternaria, Curvularia lunata, mango stalk rot, watermelon wilt etc. are respectively the phytopathogenic fungi that Zhuhai City True Green Technology Co., Ltd. presents, and D. solani Rhizoctonia is a plant pathogenic fungus donated by Dr. Yi Runhua of Guangdong Ocean University. The larger the inhibition zone, the stronger the antibacterial effect.

[0106] The antagonistic activity of table 4R31 to different host source pathogenic fungi Fusarium spp.

[0107]

[0108] Table 5R31 plate antagonistic activity to several plant pathogenic fungi

[0109]

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Abstract

The present invention relates to a strain of Bacillus subtilis R31 with broad-spectrum antagonistic ability to phytopathogenic fungi, in particular to provide Bacillus subtilis R31 (Bacillus subtilis R31) bacterial strain (CCTCC NO: M 209261) and its growth to phytopathogenic fungi Inhibition, especially in the biological control of banana wilt. The plant endophytic Bacillus subtilis provided by the present invention is proved to be a new Bacillus subtilis through the obtained 16S rDNA, gyrA and gyrB gene fragments, and through BLAST search and comparison on NCBI, capable of treating various plant pathogens The fungus has good plate antagonistic activity, especially for various soil-borne vascular diseases such as Fusarium oxysporum, Rhizoctonia solani, Alternaria and other soil-borne vascular diseases, and has shown good activity in greenhouses and fields. Control effect.

Description

technical field [0001] The present invention relates to a strain of Bacillus subtilis R31 with broad-spectrum antagonistic ability to phytopathogenic fungi, in particular to provide Bacillus subtilis R31 (Bacillus subtilis R31) bacterial strain (CCTCC NO: M 209261) and its growth to phytopathogenic fungi Inhibitory effect, especially the ability in the biological control of banana wilt, it belongs to the category of biological pesticides and disease-resistant microbial fertilizers. Background technique [0002] The losses of horticultural crops such as fruits and vegetables in our country due to diseases are very serious. According to the results of the 1984-1986 national census of garden plant diseases and insect pests, 5508 kinds of plant diseases were found on 1256 kinds of garden plants, with an average of 4.4 kinds on each plant. Vegetables, flowers The occurrence of diseases of horticultural crops such as , fruit trees, etc. is far more serious than that of bulk cereal...

Claims

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Application Information

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Patent Type & Authority Patents(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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