CN114134064A

CN114134064A - Bacillus subtilis XX and application thereof in prevention and treatment of soil-borne fungal diseases

Info

Publication number: CN114134064A
Application number: CN202111087271.1A
Authority: CN
Inventors: 李敏慧; 曾敬; 魏玉清; 谷亚南; 钟家祺; 习平根; 卓侃; 姜子德; 孔广辉
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-09-16
Filing date: 2021-09-16
Publication date: 2022-03-04
Anticipated expiration: 2041-09-16
Also published as: CN114134064B

Abstract

The invention discloses a bacillus subtilis XX and application thereof in preventing and treating soil-borne fungal diseases, and belongs to the technical field of biological prevention and treatment of plant diseases. The strain is preserved in the Guangdong province microorganism strain preservation center of No. 59 building of the Mieli Zhonglu No. 100 college of Vibrio prefecture in Guangdong province, Guangzhou city, 6.7 days 2021, with the preservation number being GDMCC No: 61713. the bacillus subtilis strain XX and the fermented supernatant thereof have good inhibition effects on fusarium oxysporum, fusarium solani and rhizoctonia solani, have excellent inhibition effects on guava naeslunella, and have important practical significance on preventing and treating banana wilt, pachyrhizus angulatus root rot, guava blight, rice sheath blight and the like. The strain culture condition is simple, the propagation speed is high, the supernatant fluid source is environment-friendly and nontoxic, and the influence on the ecological environment is small. The thallus and the bacterial liquid can be used as main bodies for prevention and control, are easy to store and transport, and have good development and application prospects.

Description

Bacillus subtilis XX and application thereof in prevention and treatment of soil-borne fungal diseases

Technical Field

The invention belongs to the technical field of biological control of plant diseases, and particularly relates to a bacillus subtilis XX and application thereof in control of soil-borne fungal diseases.

Background

The soil-borne disease refers to a disease that pathogenic bacteria live in soil, propagate in large quantities under proper conditions, invade into the plant body from the wound at the root, stem base or rhizome base of the plant, propagate in large quantities at the catheter or sieve tube part of the plant, and block the normal transportation of water and nutrient substances, thereby causing wilting of the whole plant. Examples of the plant pathogenic fungi causing soil-borne diseases include fungi of the genera Fusarium (Fusarium), Nalanthamla (Nalanthamala), Rhizoctonia (Rhizoctonia), and Sclerotium (Sclerotium). Wherein Fusarium oxysporum (F. oxysporum) can cause blight of more than 150 plants such as banana, tomato, etc. (Li Shihui et al, 2019); fusarium solani (f. solani) is also the main cause of root rot of plants such as pachyrhizua angulatus (linnaeus et al, 2003); in recent years, guava blight caused by Psidium guajava (N.psidii) in Guangdong, has become more serious, and researches have found that the pathogen can be transmitted by soil, invades from the root of the guava, firstly causes branches to die and finally causes the whole plant to die (Pekun, 2000; Hong et al, 2015). In addition, rice sheath blight disease caused by rhizoctonia solani (r.solani), and flower southern blight caused by sclerotinia sclerotiorum (s.rolfsii) can be spread as a pest through soil.

After the soil-borne fungal diseases occur, pathogenic bacteria can live through the winter in the soil to resist the external adverse environment and are difficult to be eradicated completely. When the temperature condition is proper, the plants are continuously infected and harmful to healthy plants, so that the disease condition is aggravated circularly, and the crop production is seriously influenced. Currently, the occurrence of soil-borne diseases is accelerated due to single planting mode and artificial factors such as improper use of pesticide and fertilizer. In production, chemical agents are commonly used for fumigating soil or rotation with other non-host crops and other methods are used for preventing and controlling the diseases, but the prevention and control effect is poor. Although the pesticide has a good bacteriostatic effect on soil-borne pathogenic fungi under laboratory conditions, the control effect is greatly reduced once the pesticide is applied to soil, which is related to microorganisms which contain a large amount of degradable pesticide in the soil. Moreover, the use of a large amount of bactericide can cause the risk of pesticide residue in agricultural products and accelerate the generation of drug resistance of pathogenic bacteria to different degrees. At present, the biological control method is applied to the production to control the soil-borne fungal diseases most effectively. With the popularization and the use of the microbial agent, the use amount of pesticides is greatly reduced, and the microbial agent has important practical significance for repairing a soil ecosystem, maintaining the soil fertility and improving the soil structure.

In biological control of plant diseases, Bacillus spp is a bio-control bacterium widely used. Among them, bacillus subtilis has a broad-spectrum effect of inhibiting plant pathogenic fungi by producing substances such as bacteriostatic proteins or antimicrobial peptides, and is an ideal biocontrol bacterium widely existing in soil, air, water and other environments.

In summary, screening effective biocontrol bacteria, developing broad-spectrum, safe and effective microbial bactericides and controlling the occurrence of soil-borne fungal diseases are urgent needs for improving the yield of economic crops, reducing loss and promoting sustainable development of agricultural economy.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a strain of bacillus subtilis.

Another object of the present invention is to provide a biological agent.

Still another object of the present invention is to provide the use of the above-mentioned Bacillus subtilis and/or biological agents.

The purpose of the invention is realized by the following technical scheme:

a strain of Bacillus subtilis, named as Bacillus subtilis XX, is preserved in Guangdong province microbial strain preservation center (GDMCC) of No. 59 building of Zhou Lu 100, Pieli Zhonglu, Calif., Guangdong province, Guangzhou city, 6.7 days 2021, with the preservation number of GDMCC No: 61713.

the bacillus subtilis XX is separated from rhizosphere soil of healthy bananas and has the following characteristics:

1. morphology, culture characteristics: bacillus subtilis XX is gram-positive short rod-shaped bacteria, has spores, is mesogenic or subterminal, and is oval; the surface of the bacterial colony is rough and opaque, and is dry and irregular in edge;

2. physiological and biochemical characteristics: as shown in table 1 below:

TABLE 1 physiological and biochemical characteristics of Bacillus subtilis XX

("+" indicates a positive reaction and "-" indicates a negative reaction)

3. 16S rDNA gene sequence analysis: a phylogenetic tree is constructed by adopting MEGA-X software according to 16S rDNA sequences of the bacillus subtilis XX and other bacillus strains by an adjacent method, repeated calculation is carried out for 1000 times, the nodes show Bootstrap values larger than 50%, and superscript T represents a model strain. The result shows that the 16S rDNA sequence obtained by XX sequencing of the strain is 1386bp, as shown in SEQ ID No.1, and the 16S rDNA sequence submitted to Genebank is compared with the registered sequence by nucleotide homology, the 16S rDNA sequence of the strain has 100 percent of homology with bacillus subtilis (B.subtilis) and 99.93 percent of homology with bacillus tequilensis (B.tegilensis).

According to the morphological, culture and physiological and biochemical characteristics of the strain and the analysis result of the 16S rDNA sequence, the strain XX is identified to be the bacillus subtilis.

A biological agent comprising a fermentation supernatant of the above Bacillus subtilis XX and/or Bacillus subtilis XX.

The fermentation supernatant of the bacillus subtilis XX is prepared by the following method:

inoculating the bacillus subtilis XX into a fermentation culture medium, and culturing in a shaker at 25-30 ℃ at 150-200 rpm for 10-15 h, preferably in a shaker at 28 ℃ at 180rpm for 12 h; centrifuging the bacterial liquid at 2500-3500 rpm for 8-12 min, preferably at 3000rpm for 10min, and filtering and removing thallus with a bacterial filter with a filter membrane pore size of 0.2 μm to obtain a fermentation supernatant.

The fermentation medium is preferably LB liquid medium.

The preparation method of each 1000mL system of the LB liquid culture medium is preferably as follows: weighing 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, adding water to 1000mL, uniformly stirring, adding 15g of agar, and performing moist heat sterilization at 121 ℃ for 20 min.

The application of the bacillus subtilis and/or the biological agent in preventing and treating soil-borne fungal diseases;

the soil-borne fungi include banana wilt pathogen, pachyrhizus angulatus root rot pathogen, guava blight pathogen and rice sheath blight pathogen.

The Fusarium oxysporum f.sp.cubense Tropical race 4 is a Fusarium oxysporum cubense special Tropical No. 4 physiological race which is a main pathogen causing the current banana Fusarium oxysporum f.sp.cubense and is also a physiological race with the most serious harm; the root rot of kudzu vine is Fusarium solani; the guava dry blight bacteria is guava naesli (Nalanthamala psidii); the Rhizoctonia solani is Rhizoctonia solani.

Compared with the prior art, the invention has the following advantages and effects:

1. the bacillus subtilis strain XX has a good inhibition effect on banana fusarium wilt, and has an important practical significance for preventing and treating banana fusarium wilt.

2. The bacillus subtilis strain XX has a good inhibition effect on the pueraria thomsonii and has an important practical significance for preventing and treating pueraria thomsonii rot.

3. The bacillus subtilis strain XX has a good inhibition effect on psidium guajava dry blight bacteria, and has important practical significance for preventing and treating psidium guajava dry blight.

4. The bacillus subtilis strain XX has a good inhibition effect on rhizoctonia solani and has an important practical significance for preventing and treating rice sheath blight.

5. The biological preparation of the bacillus subtilis strain XX has a good inhibition effect on banana fusarium oxysporum, pachyrhizua angulatus and rice sheath blight, has an excellent inhibition effect on guava blight, is environment-friendly and nontoxic in source, and has little influence on ecological environment.

6. The Bacillus subtilis XX strain has simple culture condition and high propagation speed. The thallus and the bacterial liquid can be used as main bodies for prevention and control, are easy to store and transport, and have good development and application prospects.

Drawings

FIG. 1 is a graph showing the inhibitory effect of Bacillus subtilis XX strain on Fusarium oxysporum F sp.

FIG. 2 is a diagram showing the inhibitory effect of Bacillus subtilis XX strain on Rhizopus pachyrhizus.

FIG. 3 is a graph showing the inhibitory effect of Bacillus subtilis XX strain on Psidium guajava L.blight.

FIG. 4 is a graph showing the inhibitory effect of Bacillus subtilis XX strain on Rhizoctonia solani.

FIG. 5 is a colony and microscopic morphology of Bacillus subtilis strain XX; wherein Panel A is a colony morphology of strain XX; panel B is a microscopic morphology of strain XX.

FIG. 6 is a phylogenetic tree diagram of Bacillus subtilis XX strain based on 16S rDNA sequence analysis.

FIG. 7 is a graph showing the inhibitory effect of a biological agent of Bacillus subtilis XX strain on banana vascular wilt; wherein, panel A is the colony growth of banana fusarium oxysporum on the PDA culture medium without biological agent; panel B shows the growth of colonies of Fusarium oxysporum f.banana on PDA medium containing a biological preparation of Bacillus subtilis XX strain.

FIG. 8 is a graph showing the inhibitory effect of a biological agent of Bacillus subtilis XX strain on Rhizopus pachyrhizus; wherein, the graph A shows the growth condition of the pueraria rot fungus colonies which are powdered on the PDA culture medium without biological agents; panel B shows the growth of colonies of Rhizopus pachyrhizus on PDA medium containing a biological preparation of Bacillus subtilis XX strain.

FIG. 9 is a graph showing the inhibitory effect of a biological agent of Bacillus subtilis XX strain on Psidium guajava L.blight; wherein panel A is the growth of colonies of Psidium guajava L.on PFA medium without biological agents; panel B shows the growth of colonies of Psidium guajava L.on a PFA medium containing a biological agent of strain XX of Bacillus subtilis.

FIG. 10 is a graph showing the inhibitory effect of a biological agent of Bacillus subtilis XX strain on Rhizoctonia solani; wherein, the graph A shows the colony growth of Rhizoctonia solani on the PDA culture medium without biological agent; panel B shows the growth of a colony of Rhizoctonia solani on PDA medium containing a biological preparation of strain XX of Bacillus subtilis.

FIG. 11 is a comparison graph of colony diameters of banana fusarium oxysporum, pachyrhizus angulatus, guava fusarium solani and rice sheath blight bacteria before and after treatment of a biological agent of Bacillus subtilis XX strain.

FIG. 12 shows the growth inhibition rate of Bacillus subtilis XX treated with the biological agent on banana wilt, arrowroot rot, guava blight and rice sheath blight.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to embodiments, but it will be understood by those skilled in the art that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The following examples refer to biological materials:

banana wilt bacterium FOC4 strain XJZ2 has been disclosed in the document "Li Yi Hui, Du Xian Tao, Wang hong Fei, etc.. quick detection and identification of No.1 and No. 4 physiological races of banana wilt bacterium [ J ]. Chinese agricultural science, 2012,45(19): 3971-3979.";

the root rot of Pueraria thomsonii has been disclosed in the literature, "stable in Linlan, Mega male, Heagainst Wenwei, Huangsaihua, identification of the etiology of root rot of Pueraria thomsonii [ J ]. ecological environment, 2003(04): 516-;

psidium guajava L.is disclosed in "C, F, Hong, et al. Import of root infection in guava with used by Nalantha psidii [ J ]. Plant Pathology,2014,64(2): 450-;

the Rhizoctonia solani strain GD-118 has been disclosed in the literature "Yangying, Li Ming sea, Yang Mei, et al. optimization of protoplast preparation and regeneration conditions for Rhizoctonia solani". J. proceedings of university of Huazhong agriculture, 2010,29(5):546 551 ".

Example 1 isolation screening and identification of Bacillus subtilis Strain XX

(1) Preparing a PDA culture medium: firstly, washing and peeling potatoes, weighing 200g of potatoes, cutting into small pieces, adding water, boiling for 20-30 min, and filtering with eight layers of gauze. Adding 20g of glucose and 15g of agar powder, fully heating to dissolve, fixing the volume to 1000mL, subpackaging into 250mL triangular bottles, performing damp-heat sterilization at 121 ℃ for 20min, and storing for later use.

(2) Preparation of PFA culture medium: firstly, washing and peeling potatoes, weighing 200g of potatoes, cutting into small pieces, adding water, boiling for 20-30 min, and filtering with eight layers of gauze. Adding 20g of fructose and 15g of agar powder, fully heating to dissolve, fixing the volume to 1000mL, subpackaging into 250mL triangular bottles, performing damp-heat sterilization at 121 ℃ for 20min, and storing for later use.

(3) Preparing an LB culture medium: weighing 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, adding 1000mL of water, uniformly stirring, adding 15g of agar powder, fully heating to dissolve, fixing the volume to 1000mL, subpackaging into 250mL triangular bottles, performing moist heat sterilization at 121 ℃ for 20min, and preparing into plates for storage.

(4) Separation and purification of the strains: the strains were isolated from healthy banana rhizosphere soil and soil samples were collected in the Guangzhou city, Guangdong province, floribunda. The method comprises the following specific steps: taking 10g of healthy banana rhizosphere soil sample, placing the sample into a 250mL triangular flask filled with 90mL of sterile water, carrying out shaking culture at 180rpm for 30min at 28 ℃, standing for 10min, sucking 1mL of supernatant, and diluting the supernatant into 10 times by using a concentration gradient method^-1To 10^-5The concentration of (2) was measured by pipetting 150. mu.L of 10^-3，10^-4And 10^-5Uniformly coating the diluted solution with concentration on an LB flat plate, placing the flat plate in an incubator at 28 ℃ for culturing for 12 hours, picking growing single colony bacteria on the LB flat plate, and carrying out single colony separation and purification again by using a plate marking method for later use.

(5) Screening of high-efficiency antagonistic strain for soil-borne fungal diseases

A bacterial cake with the diameter of 7mm is placed on a PDA plate culture medium by taking the tropical No. 4 physiological race XJZ2 strain of the fusarium oxysporum f.sp.cubense as a target, a little bacterial colony of the separated and purified strain in the step (3) of the example 1 is dipped by a sterilizing toothpick, streaked at the position which is about 5cm away from the bacterial cake, and inversely cultured in an incubator at the temperature of 28 ℃ for 4-5 days. A strain with obvious bacteriostasis to the target bacteria is screened out and named as XX. The result shows that the strain XX has better inhibition effect on the fusarium oxysporum XJZ2 strain of banana (figure 1). According to the method, the pachyrhizua angulatus rhizoctonia and the rice sheath blight GD-118 are cultured by a plate confronting method respectively, and the guava rhizoctonia solani plate confronting test is carried out on a PFA plate culture medium. The results show that the strain XX also has a good inhibition effect on the root rot of pachyrhizua angulatus, the guava dry rot and the rice sheath blight GD-118 (fig. 2, fig. 3 and fig. 4).

(6) Identification of Strain XX

A. Morphological identification

Selecting single colony, streaking on LB plate culture medium by plate streaking method, and culturing at 28 deg.C in dark for 2 d. As shown in FIG. 5, the strain XX is a gram-positive, short rod-shaped bacterium, having spores, mid-spore or partial-terminal growth, oval. The colony surface is rough and opaque, dry and has irregular edges.

B. Molecular identification

A single colony is picked up and put in 3mL LB liquid medium, and is put at 28 ℃ and cultured on a shaking table at 180rpm for 12h, and the DNA of the strain XX is extracted according to the method of a bacterial DNA extraction kit OMEGA D3350-1 Bacteria DNAkit, and is put at-20 ℃ for storage and standby. PCR amplification was performed with 16S rDNA universal primer 27F/1492R, and after cloning of the PCR product into a T vector, sequencing was performed by Compton Biotechnology engineering (Shanghai) Ltd. The 16S rDNA sequence of the strain XX is shown in SEQ ID No.1, and the length is 1386 bp.

27F:5’-AGAGTTTGATCCTGGCTCAG-3’(SEQ ID No.2)；

1492R:5’-GGTTACCTTGTTACGACTT-3’(SEQ ID No.3)。

And (3) submitting the obtained sequence to Genebank for nucleotide homology comparison with the registered sequence, downloading the sequence with higher homology, constructing a phylogenetic tree by adopting a neighbor method for 16S rDNA sequences of the strain and other bacillus strains by adopting MEGA-X software, repeating for 1000 times, wherein the nodes show Bootstrap values of more than 50 percent, and the superscript 'T' represents a model strain. The result shows (figure 6), 16S rDNA sequence obtained by strain sequencing is 1386bp, and the sequence is submitted to Genebank to be compared with the registered sequence for nucleotide homology, the 16S rDNA sequence of the strain has 100 percent of homology with Bacillus subtilis and 99.93 percent of homology with Bacillus teguilens (Bacillus terkesii).

According to the morphology and the molecular identification result, the strain XX is determined to be bacillus subtilis (B.subtilis). The strain is preserved in Guangdong province microbial strain preservation center (GDMCC) of No. 59 building of Mieli 100 college of the Pieli-Mieli, California, Guangdong province in No. 6 and 7 days of 2021, with the preservation number being GDMCC No: 61713, named Bacillus subtilis XX.

Example 2 preparation of Bacillus subtilis XX biologics

Dipping a small amount of bacillus subtilis XX into a 250mL conical flask containing 200mL liquid LB culture medium, and placing the conical flask in a shaking table at 28 ℃ and shaking at 180rpm for 12 h. Centrifuging the bacterial liquid at 3000rpm for 10min, and filtering and removing bacterial thallus with a bacterial filter (the aperture of the filter membrane is 0.2 μm) to obtain the biological agent of the strain XX.

Example 3 growth Rate assay for Activity of Bacillus subtilis XX biologics

The biological preparation of strain XX and PDA medium were mixed in a 1: 9 to prepare a plate, and LB liquid medium was used in place of the biological agent of the strain XX and PDA medium in the same ratio to prepare a plate as a Control (CK). Respectively connecting bacterial cakes of banana wilt pathogen XJZ2, pachyrhizus angulatus and rice sheath blight pathogen GD-118 with the diameters of 7mm in the centers of the treated and control plates, culturing at 28 ℃, stopping culturing until the target fungi grow over the whole culture dish on the Control (CK) plate, respectively measuring the bacterial colony diameters of the target fungi on the treated and control plates, repeating each treatment for three times, and calculating the bacteriostasis rate of the bacterial strain XX. PFA medium was used for both the guava-dried blight and Control (CK) tests. The calculation formula is as follows: the inhibition rate (%) × (control colony diameter-treatment colony diameter)/(control colony diameter-cake diameter) × 100%.

The results show that the biological preparation of the bacillus subtilis XX has better inhibition effects on banana fusarium oxysporum, pachyrhizus angulatus and rice sheath blight GD-118, has excellent inhibition effects on guava blight (figures 7-10), has an inhibition rate of 37.24% on banana fusarium oxysporum XJZ2, 13.67% on pachyrhizus angulatus, 70.33% on guava blight and 42.59% on rice sheath blight GD-118 (figures 11-12).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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Claims

1. A bacillus subtilis strain is characterized in that: designated as Bacillus subtilis XX, which is preserved in No. 59 great institute of Guangdong province microbial culture collection center (GDMCC) of No. 59 great institute of Pielie Zhonglu, Vibrio, Guangdong province on 7 th 6 th 2021, with the preservation number being GDMCC No: 61713.

2. a biological agent characterized by: a fermentation supernatant comprising bacillus subtilis XX and/or bacillus subtilis XX of claim 1.

3. The biological agent according to claim 2, characterized in that: the fermentation supernatant of the bacillus subtilis XX is prepared by the following method:

inoculating the bacillus subtilis XX into a fermentation culture medium, and culturing for 10-15 h in a shaking table at 25-30 ℃ and at 150-200 rpm; centrifuging the bacterial liquid at 2500-3500 rpm for 8-12 min, and filtering and removing thalli by using a bacterial filter to obtain a fermentation supernatant.

4. The biological agent according to claim 2, characterized in that: the fermentation supernatant of the bacillus subtilis XX is prepared by the following method:

inoculating the bacillus subtilis XX into a fermentation medium, and culturing for 12h in a shaking table at 28 ℃ and at 180 rpm; centrifuging the bacterial liquid at 3000rpm for 10min, and filtering and removing thallus with bacterial filter with filter membrane pore diameter of 0.2 μm to obtain fermentation supernatant.

5. A biological agent according to claim 3 or 4, characterized in that:

the fermentation medium is LB liquid medium.

6. The biological agent according to claim 5, characterized in that:

the preparation method of each 1000mL system of the LB liquid culture medium comprises the following steps: weighing 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, adding water to 1000mL, uniformly stirring, adding 15g of agar, and performing moist heat sterilization at 121 ℃ for 20 min.

7. Use of a bacillus subtilis according to claim 1 and/or a biological agent according to any one of claims 2 to 6 for the control of soil-borne fungal diseases, characterized in that: the soil-borne fungi include banana wilt pathogen, pachyrhizus angulatus root rot pathogen, guava blight pathogen and rice sheath blight pathogen.

8. Use according to claim 7, characterized in that:

the Fusarium oxysporum f.sp.cubenseTropical race 4 is Fusarium oxysporum cubense special tropical race 4; the root rot of kudzu vine is Fusarium solani; the guava dry blight bacteria is guava naesli (Nalanthamala psidii); the Rhizoctonia solani is Rhizoctonia solani.