CN112522144A - Bumper-fragrant endogenous Bacillus belgii and application thereof - Google Patents

Abstract

The invention discloses Bumper-gum endophytic Bacillus belgii and application thereof. The strain is named as Bacillus velezensis B1 with the preservation number of GDMCC No: 61296, the strain was deposited in Guangdong provincial collection of microorganisms in building 59 of Tokyo large institute 100, Michelia furiosa, Guangzhou, 11.19.2020. The bacterial strain of the invention belongs to the endophytic of the pelargonium odoratissimum, and has no pathogenicity; the bacterial strain has good inhibition effect on the bacterial wilt of the tobacco, can obviously enhance the resistance of the tobacco to the bacterial wilt and delay the occurrence of diseases, and has good persistence; has obvious promotion effect on the growth of tobacco; the strain has obvious inhibition effect on phytophthora parasitica of cucumber, phytophthora capsici, late blight of tomato, colletotrichum gloeosporioides, guava pycnidium fruit rot, mangosteen fruit rot, luffa wilt, colletotrichum gloeosporioides and banana wilt.

Description

Bumper-fragrant endogenous Bacillus belgii and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to Bumper-gum endophytic Bacillus belgii and application thereof.

Background

Tobacco bacterial wilt is one of the main diseases of tobacco in tropical and subtropical regions, has extremely wide distribution range, and poses a great threat to the production of high-quality tobacco (which outlook et al, 1995). The ralstonia solanacearum survives in the soil for a long time, which results in the soil becoming the main primary source of infection, and this pathogen can invade the vascular bundle system directly from the wound at the root stem of a healthy tobacco plant, or from the root cap of a secondary root, and when ralstonia solanacearum successfully colonizes in the xylem, forms a pus on the surface of the root, returns to the soil and re-infects (Araud et al, 1998; McGarvey et al, 1999; Huang et al, 2000; Pantaimen et al, 2010). Tobacco bacterial wilt is often mixed with root-knot nematode, tobacco black shank, tobacco hollow stem disease and the like in production, so that the prevention and the control are very difficult, and the tobacco industry economy of China is seriously influenced.

At present, the tobacco bacterial wilt prevention and treatment measures mainly comprise agricultural prevention and treatment, chemical prevention and treatment and the like. Wherein, the continuous cropping of disease-resistant varieties can cause the resistance decline of crops (Fangzhimin et al, 2002), the control effect is poor, and the chemical pesticide residue brings serious environmental pollution. Compared with the traditional control method, biological control becomes a research hotspot for controlling plant diseases in recent years due to the characteristics of environmental friendliness, remarkable control effect and the like.

The microbial biocontrol mechanisms mainly include competition for ecological loci and nutrients, production of antibacterial substances, induction of resistance, promotion of plant growth, and the like (Verma et al, 2002). In recent decades, scholars at home and abroad develop a great deal of research work aiming at the biological control of tobacco bacterial wilt and try to utilize a plurality of beneficial microorganisms and metabolites thereof to control diseases. The poplar and the like (2014) screen 36 antagonistic bacteria from the rhizosphere soil and tobacco tissues of the tobacco field, wherein the strain YH-22 has stronger antagonistic activity, is identified as bacillus, has certain colonization ability in the rhizosphere soil and the tobacco tissues of the tobacco, can occupy good ecological sites, and secretes partial extracellular enzymes, thereby achieving the effect of effectively inhibiting the tobacco bacterial wilt; the method is characterized in that an XQ biocontrol bacterium agent consisting of a Bacillus pumilus flora completely nontoxic to human and livestock is utilized to inoculate tobacco, and the results show that the cured tobacco has good growth vigor after the XQ biocontrol bacterium is applied, the incidence rate of bacterial wilt of the cured tobacco is reduced to 10.2%, the disease index is reduced to 3.85%, the relative prevention effect is increased to 81.87%, the physical properties of the cured tobacco are good, and the production quality of the cured tobacco is remarkably improved.

Many researches have found that the bacillus can control plant diseases by competing with pathogenic bacteria for nutrition and ecological sites, secreting antibacterial substances to inhibit the growth of pathogenic bacteria and exciting plant system disease resistance (

et al, 2013; lugtenberg et al,2009) and has some growth promoting effect on plants. In addition, the bacillus can generate endogenic spores, can resist various adverse environments such as high temperature, drought, ultraviolet irradiation and the like, and has strong viability. Therefore, bacillus is one of the ideal strains of biocontrol strains, and the development value and theoretical research of the bacillus are more and more valued by people. At present, no endophytic bacillus which has a good effect of promoting tobacco growth and can inhibit tobacco bacterial wilt is reported.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide the endogenous Bacillus belgii for the pelargonium odoratissimum.

The invention also aims to provide application of the pelargonium odoratissimum endophytic Bacillus beijerinckii.

The purpose of the invention is realized by the following technical scheme: a Bumper incense endogenous Bacillus belgii, which is named as Bacillus velezensis B1 and has the collection number of GDMCC No: 61296, the strain was deposited in Guangdong provincial collection of microorganisms in building 59 of Tokyo large institute 100, Michelia furiosa, Guangzhou, 11.19.2020.

The nucleotide sequence of 16S rRNA of the banana endophytic Bacillus belgii is shown as SEQ ID No.1, and the sequence length is 1434 bp; the nucleotide sequence of rpoA is shown as SEQ ID No.2, and the sequence length is 226 bp; the nucleotide sequence of rpoB is shown as SEQ ID No.3, and the sequence length is 590 bp; the nucleotide sequence of gyrA is shown as SEQ ID No.4, and the sequence length is 716 bp; the nucleotide sequence of gyrB is shown as SEQ ID No.5, and the sequence length is 1160 bp.

The application of the Bulbophyllum odoratissimum endophytic Bellis bacillus in preventing and treating diseases such as tobacco bacterial wilt, cucumber phytophthora blight, phytophthora capsici, tomato late blight, papaya anthracnose, guava fruit rot, mangosteen fruit rot, cucumber fusarium wilt, cabbage anthracnose and banana fusarium wilt; preferably tobacco bacterial wilt.

The pathogenic bacteria of the disease are Ralstonia solanacearum, Phytophthora melonis (Phytophthora melonis), Phytophthora capsici (Phytophthora capsici), tomato late blight (Phytophthora infestans), papaya anthracnose (Colletotrichum gloeosporioides), guava pyva (Pestalotiopsis micropora), mangosteen (Gliocephalum bulbophyllum), towel gourd Fusarium oxysporum (Fusarium oxysporum), cabbage anthracnose (Colletotrichum higginsianum), and banana Fusarium oxysporum (Fusarium oxysporum f.sp.cubense).

The application of the pelargonium odoratissimum endogenous belief bacillus in promoting the growth of tobacco.

The application method comprises the step of preparing the Bacillus belgii into a fermentation solution and a composite biological agent.

A composite biological agent comprises the Bacillus belgii and a plant immunity inducer.

The compound biological agent is used for enhancing the resistance of tobacco to tobacco bacterial wilt.

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

1. the strain B1 has obvious inhibition effect on phytophthora parasitica of cucumber, phytophthora capsici, late blight of tomato, colletotrichum gloeosporioides, guava pythium, mangosteen pythium, luffa fusarium oxysporum, colletotrichum gloeosporioides and banana fusarium oxysporum.

2. The strain B1 belongs to the endophytic of the pelargonium odoratissimum, and has no pathogenicity; the compound pesticide has good effect of inhibiting the bacterial wilt of the tobacco, can obviously enhance the resistance of the tobacco to the bacterial wilt and delay the occurrence of diseases, and has good persistence.

3. The strain B1 has obvious promotion effect on the growth of tobacco.

Drawings

FIG. 1 is a map showing morphological identification of Bacillus belgii B1; wherein, a is a colony characteristic diagram on an LB culture medium, b is a gram staining diagram, and c is a spore staining diagram.

FIG. 2 is a phylogenetic tree of strain B1 constructed based on the 16S rDNA partial sequence.

FIG. 3 is a phylogenetic tree of strain B1 constructed based on the partial sequence of rpoA.

FIG. 4 is a phylogenetic tree of strain B1 constructed based on the partial sequence of rpoB.

Fig. 5 is a phylogenetic tree of strain B1 constructed based on the gyrA partial sequence.

Fig. 6 is a phylogenetic tree of strain B1 constructed based on the gyrB partial sequence.

FIG. 7 is a graph showing the effect of Bacillus belgii B1 and its metabolites on bacterial inhibition of Ralstonia solanacearum; wherein a is the direct inhibition effect of Bacillus belgii B1 on Ralstonia solanacearum; c is the inhibition effect of metabolite of the strain B1 on ralstonia solanacearum measured by a cellophane method; b. d is the control of a and c respectively.

FIG. 8 is a plate inhibition of a portion of phytopathogenic fungi by Bacillus belgii B1; wherein a is the inhibiting effect of the strain B1 on the fusarium wilt of cucurbita moschata; b is the inhibiting effect of the bacterial strain B1 on banana fusarium wilt.

FIG. 9 is a plate inhibition of B1 metabolite of Bacillus belgii on a part of plant pathogenic fungi; wherein a is papaya colletotrichum; b is mangosteen crown rot; c is phytophthora cucumeri; d is colletotrichum gloeosporioides; e is guava pythium aphanidermatum; number 1 is control group, and number 2 is treatment group.

FIG. 10 is a graph of the growth promoting effect of Bacillus beleisi B1 on tobacco; wherein, A is CK, B is the intelligent clever group of plant immunity inducer, C is the group of improved Hoagland nutrient solution, D is the group of Bacillus belgii B1, E is the group of Bacillus belgii B1+ intelligent clever group.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto. 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.

Example 1 isolation and identification of Bacillus belgii B1

(1) Isolation and purification of the Strain

Healthy and well-grown tabasheer plants are selected, and the vigorously-growing leaves, stems and roots are cut and used for separating endophytes. The specific separation steps are as follows: cleaning the separation material with clear water, soaking in 70% (v/v) ethanol for 30sec, quickly transferring the separated tissue into 5% (v/v) sodium hypochlorite, soaking for 3min, sterilizing the surface, and killing bacteria on the tissue surface; it was then transferred to sterile water and washed 3 times to remove residual tissue. The separated material was cut into small pieces with scissors and transferred to petri dishes with NA medium, 4 tissues placed evenly per dish. After the separation is completed, a label is attached, the tissue separation and the separation period are indicated, and then the culture is carried out at the temperature of 25-28 ℃. After 2d, judging the separated colonies according to the characteristics of colony morphology, color, edge state, transparency and the like and counting. And (3) coating 0.1mL of sterile water for the last time of cleaning after tissue disinfection on the surface of a culture medium, and checking whether the surface of the sample is disinfected completely. And culturing the obtained bacteria by adopting a plate marking method to obtain pure colonies, and then transferring the pure colonies to an inclined plane for low-temperature storage for later use.

(2) Identification of endogenic Bacillus belgii

Morphological characteristics of the Strain

The bacterial colony of the strain B1 is white after being cultured for 20 hours at 37 ℃ on an LB culture medium; the surface is rough, the shape is irregular, and the wrinkles are not obvious; the cells are rod-shaped, gram-positive, have spores, and are located in the center or slightly off-center of the cells (FIG. 1).

Physiological and biochemical characteristics of strain

The strain B1 can hydrolyze starch and gelatin, and glucose, starch, citrate, catalase and phenylalanine deaminase are positive, and the methyl red test is negative, and the V-P test is positive. The results of measurement of the specific physiological index are shown in Table 1.

TABLE 1 physiological and biochemical assays for Strain B1

Note: "+" represents > 90% of the strains as positive, "-" represents > 90% of the strains as negative, and "d" represents 11% -89% of the strains as positive.

Molecular characterization of strains

A single colony of the strain B1 is picked up and inoculated in 5mL LB culture medium, cultured at 37 ℃ and 170rpm for 20h, and 1 microliter of bacterial liquid is taken as a template to perform PCR amplification on partial sequences of 16S rDNA, rpoA, rpoB, gyrA and gyrB respectively. The amplification product is detected to be clear and single-banded by agarose electrophoresis, and is sent to Shanghai biological products GmbH for sequencing. The partial sequences of 16S rDNA, rpoA, rpoB, gyrA and gyrB of the strain are shown as SEQ ID No. 1-SEQ ID No.5 in sequence. Selecting other strains of Bacillus belgii and other species of Bacillus, and constructing the evolutionary tree by using partial sequences of 16S rDNA, rpoA, rpoB, gyrA and gyrB. The results showed that strain B1 is highly homologous to Bacillus belgii (Bacillus velezensis) and has the closest genetic distance (fig. 2, fig. 3, fig. 4, fig. 5, fig. 6). The phylogenetic tree cluster analysis of gyrB shows that the sequence of the strain B1 is greatly different from that of a previously reported Bacillus belgii R-9 strain (CN107779420B, two endogenous Bacillus belgii antagonistic to the tobacco bacterial wilt and application thereof) for preventing and treating the tobacco bacterial wilt, and the two strains have large difference in colony morphology and belong to the same species and different strains.

Example 2 bacteriostatic Effect of Bacillus belgii B1 on plates

1. Bacterial strain B1 bacteriostatic effect on ralstonia solanacearum

The activated B1 strain was cultured on an LB medium plate for 48 hours, and the cake was punched out with a punch having a diameter of 5mm and inoculated into a medium to which Ralstonia solanacearum was added. Culturing and observing in an incubator at 30 ℃, and recording whether the inhibition zone exists or not. After culturing for 3d, measuring the size of the inhibition zone by a cross method. Each treatment was repeated 3 times, with no addition of strain B1 as a control.

The result shows that after 3d of culture, the bacterial strain B1 has good inhibition effect on tobacco ralstonia solanacearum (figures 7a and B), and the inhibition zone can reach 25.7 mm. After 6 days, the size of the inhibition zone of the B1 strain is basically unchanged compared with that of the B1 strain cultured for 3 days, which indicates that the persistent effect is better.

In order to determine the inhibition effect of metabolites of the endogenous antagonistic strain B1 on ralstonia solanacearum, strain B1 was coated on an NA medium paved with cellophane, the medium was cultured at 30 ℃ for 72 hours, the cellophane was removed, and ralstonia solanacearum was coated on the medium, with 3 replicates per treatment. The control was made by inoculating Ralstonia solanacearum only to the NA medium. The results showed that the metabolite produced by strain B1 completely inhibited the growth of the test tobacco ralstonia solanacearum (FIGS. 7c, d).

2. Bacteriostatic effect of strain B1 on other plant pathogenic fungi

The bacterial strain B1 has a wide antibacterial spectrum and has obvious inhibitory effect on various plant pathogenic fungi. Carrying out a flat plate confronting experiment: inoculating the lawn of the endophyte to be detected in the center of a PDA (personal digital assistant) plate, inoculating 4 plant pathogen target bacteria at the distance of 3cm around the endophyte to be detected, repeating the step for 3 times for each bacterium to be detected, culturing for one week at 25 ℃, and observing the bacteriostatic activity. Pathogenic bacteria having inhibitory activity were identified, and these were inoculated into a PDA plate together with the strain B1 again to culture the same, and the inhibitory activity of the strain B1 was confirmed. The results show that the strain B1 has obvious inhibiting effects on Phytophthora cucumeri (Phytophthora melonis), Phytophthora capsici (Phytophthora capsici), Phytophthora infestans (Phytophthora infestans), Colletotrichum gloeosporioides (Colletotrichum gloeosporioides), psidium guajava fruit rot (Phytophthora micropora), mangosteen fruit rot (Gliocephalum bulbophyllum), luffa Fusarium oxysporum (Fusarium oxysporum), Colletotrichum anthracnose (Colletotrichum higginsnum) and Fusarium oxysporum (Fusarium oxysporum f.sp. culense) (all the pathogenic bacteria can be purchased from the market). Fig. 8 shows the inhibitory effect of the strain B1 on Fusarium oxysporum (Fusarium oxysporum) and Fusarium oxysporum f.sp.cubense.

On the basis of a confrontation experiment, an endogenous antagonistic strain B1 is coated on a PDA culture medium paved with cellophane, the PDA culture medium is cultured for 72 hours at the temperature of 30 ℃ (B1 bacterial colony fully grows over the culture medium, but does not exceed the range of the cellophane), the cellophane is removed, each test plant pathogenic bacterium is inoculated on the culture medium, the bacteriostatic action of a growth metabolite of the test plant pathogenic bacterium is tested, and 3 times of treatment are set for each treatment. The control was the treatment without inoculating the strain B1 and with inoculating only the corresponding pathogenic bacteria on PDA medium. The results show that the metabolite produced by the strain B1 has better inhibitory effect on various pathogenic bacteria (Table 2, FIG. 9).

TABLE 2 inhibitory Effect of metabolites of Strain B1 on test plant pathogenic fungi

Note: the same column indicates that different letters represent significant differences (P <0.05)

Example 3 potting experiment of Bacillus belgii B1 for controlling tobacco bacterial wilt

Selecting tobacco seedlings with consistent sizes for transplanting, and after 15d of transplanting, (1) irrigating B1 bacterial suspension (OD)₆₀₀1.3-1.4)20mL, and pouring again after 7 d; (2) 20mL of plant immune inducer (intelligent agent) is poured, and the plant immune inducer is poured for another time after 7 days; (3) 20mL of plant immunity inducer (intelligent agent) is poured, and B1 bacterial suspension (OD) is poured after 7 days₆₀₀1.3-1.4)20mL, treating for 24h, and then adopting a root irrigation method to treat ralstonia solanacearum suspension (OD)₆₀₀0.3-0.4)25mL of the solution was poured into tobacco plant soil. The treatment of irrigating 15mL of the pseudomonas solanacearum suspension after normal irrigation of the nutrient solution is taken as a positive control; in normal irrigationAnd (3) irrigating 15mL of sterilized water after the nutrient solution is used as a negative control, irrigating 15mL of tobacco ralstonia solanacearum suspension after the nutrient solution is normally irrigated, and irrigating 15mL of streptomycin after 12h is used as a medicament control. 30 tobacco plants were used for each treatment.

The results show (Table 3), the resistance of tobacco to bacterial wilt can be obviously enhanced after the tobacco is irrigated with the strain B1, and the occurrence of diseases is delayed. The prevention and treatment effect of the strain B1 on tobacco is not obvious from the effect of spraying agricultural streptomycin, and when the strain B1 is used together with a plant immunity inducer, the occurrence date of diseases can be delayed, and the prevention and treatment effect of the strain B1 is improved.

TABLE 3 disease index after inoculation of ralstonia solanacearum with different treatments

Note: the same column indicates that different letters represent significant differences (P <0.05)

Example 4 potting experiment of Bacillus belgii B1 for promoting tobacco growth

Activating the bacillus B1 strain stored at low temperature, dipping a single colony in 5mL of NA liquid culture medium by using an inoculating ring, performing shake culture for 12h at 37 ℃ and 170r/min, and then transferring the single colony into 100mL of NA liquid culture medium according to the proportion of 1: 50. Shake culturing at 37 deg.C and 170r/min for 24 h.

Transplanting 1 tobacco seedling with almost the same size in each planting bag, and after 15d, irrigating B1 bacterial suspension (OD) by root irrigation method (1)₆₀₀1.3-1.4)20mL, and pouring into tobacco plant soil; (2) 20mL of sterile water (CK) is poured; (3) 20mL of improved Hoagland nutrient solution is irrigated, and irrigation is carried out once after 7 days; (4) intelligently and intelligently mixing soil, sowing tobacco seeds, and filling 20mL of sterile water into the tobacco plant soil after seedlings are transplanted for 15 days; (5) intelligently mixing soil, sowing tobacco seeds, transplanting seedlings for 15d, and irrigating B1 bacterial suspension (OD)₆₀₀1.3-1.4)20 mL. 30 tobacco plants were used for each treatment. After treatment of each experimental groupAnd 15d, measuring the leaf length, leaf width, plant height, stem thickness, leaf number, total root length and maximum leaf area of the tobacco.

The result shows that the strain B1 is non-pathogenic to tobacco and has obvious promotion effect on the growth of the tobacco. After the tobacco is irrigated for 15d by B1, compared with the tobacco irrigated with sterile water and the tobacco irrigated with the improved Hoagland nutrient solution for 2 times, each index of the tobacco is obviously improved; the intelligent agent of the plant immunity inducer can promote the growth of tobacco, but the effect is not obvious, and the effect of the intelligent agent of the plant immunity inducer is not obviously different from that of the intelligent agent of the plant immunity inducer used in combination with the strain B1 compared with the effect of the strain B1 used alone (table 4, figure 10).

TABLE 4 determination of agronomic performance index of tobacco treated differently

Note: the same column indicates that different letters indicate significant difference (P <0.05)

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

<110> southern China university of agriculture

<120> Bumper-top endophytic Bacillus belgii and application thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1434

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of 16S rRNA

<400> 1

cctaatacat gcaagtcgag cggacagatg ggagcttgct ccctgatgtt agcggcggac 60

gggtgagtaa cacgtgggta acctgcctgt aagactggga taactccggg aaaccggggc 120

taataccgga tggttgtctg aaccgcatgg ttcagacata aaaggtggct tcggctacca 180

cttacagatg gacccgcggc gcattagcta gttggtgagg taacggctca ccaaggcgac 240

gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact 300

cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc 360

cgcgtgagtg atgaaggttt tcggatcgta aagctctgtt gttagggaag aacaagtgcc 420

gttcaaatag ggcggcacct tgacggtacc taaccagaaa gccacggcta actacgtgcc 480

agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc gtaaagggct 540

cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct caaccgggga gggtcattgg 600

aaactgggga acttgagtgc agaagaggag agtggaattc cacgtgtagc ggtgaaatgc 660

gtagagatgt ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa ctgacgctga 720

ggagcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacga 780

tgagtgctaa gtgttagggg gtttccgccc cttagtgctg cagctaacgc attaagcact 840

ccgcctgggg agtacggtcg caagactgaa actcaaagaa ttgacggggg cccgcacaag 900

cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc 960

tctgacaatc ctagagatag gacgtcccct tcgggggcag agtgacaggt ggtgcatggt 1020

tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgat 1080

cttagttgcc agcattcagt tgggcactct aaggtgactg ccggtgacaa accggaggaa 1140

ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat 1200

ggacagaaca aagggcagcg aaaccgcgag gttaagccaa tcccacaaat ctgttctcag 1260

ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa tcgcggatca 1320

gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccacgagagt 1380

ttgtaacacc cgaagtcggt gaggtaacct ttttaggaag cccaagctcc gccg 1434

<210> 2

<211> 226

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of rpoA

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ttctggggta ctccttacgt cgtatcctct tatcctcact ccctggtgcc gctgtaacat 60

cgatccagat agatggtgta ctgcacgaat tctcgacaat cgaaggcgtt gtggaagatg 120

ttacaacgat aatcttgcac attaaaaagc ttgcattgaa aatctactct gatgaagaga 180

agacgcttga aattgatgta cagggcgaag gaactgtaac ggcaga 226

<210> 3

<211> 590

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of rpoB

<400> 3

tttgaggtca aactgttcag tatggacgac accgccagcg cagaagctak ggtcgcatta 60

gcgaagtgtt agaattacca aatctcattg aaattcaaac ctcttcttat cagtggtttc 120

ttgatgaggg tcttagagag atgtttcaag acatatcacc aattgaggat ttcactggta 180

acctctctct agagttcatt gactacagtt taggagatcc taagtatccc gttgaagagt 240

caaaagaacg tgatgtgact tactcagctc cgctgagagt gaaggttcgt ttaattaaca 300

aagaaactgg agaggtaaaa gaccaggatg tcttcatggg tgatttccct attatgacag 360

ataccggtac ttttatcatc aacggtgcag aacgtgttat cgtatctcag cttgttcggt 420

ctccaagtgt atatttcagt ggtaaagtag acaagaacgg taaaaaaggt tttaccgcga 480

ctgtcattcc aaaccgtggc gcatggttag aatacgaaac tgatgcgaaa gatgttgkgy 540

attccgcatt gatcgcacac gtaagttgcc ggttaacggt tctaaaaaac 590

<210> 4

<211> 716

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of gyrA

<400> 4

actgggtgta ccacccgcac ggtgactcag cggtttacga atcaatggtc agaatggcgc 60

aggattttaa ctaccgctac atgcttgttg acggacacgg caacttcggt tcggttgacg 120

gcgactcagc ggccgcgatg cgttacacag aagcgagaat gtcaaaaatc gcaatggaaa 180

tccttcggga cattacgaaa gatacgattg attatcaaga taactatgac ggcgcagaaa 240

gagaacctgt cgtcatgcct tcgagatttc cgaatctgct cgtcaacgga gctgccggta 300

ttgcggtcgg aatggcgaca aatattcctc cgcatcagct tggggaagtc attgaaggcg 360

tacttgccgt aagtgagaat cctgagatta caaaccagga gctgatggaa tacatcccgg 420

gcccggattt tccgactgca ggtcagattt taggccggag cggcatccgc aaggcatatg 480

aatccggacg gggatccatt acgatccggg ctaaggctga aatcgaagag acatcatcgg 540

gaaaagaaag aattattgtc acggaacttc cttatcaggt gaacaaagcg agattaattg 600

aaaaaatcgc agatcttgtc cgggacaaaa aaatcgaagg aattacggat ctgcgtgacg 660

aatccgaccg taacggaatg agaatcgtca ttgagatccg ccggtgacgc aaagaa 716

<210> 5

<211> 1160

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of gyrB

<400> 5

ccccttgtgc aaaagccgga tatatagtat ccggcggtct tcacggtgta ggggcatctg 60

tcgtaaacgc cttgtcgacc actcttgacg ttacggttca tcgtgacgga aaaatccact 120

atcaggcgta cgagcgcggt gtacctgtgg ccgatcttga agtgatcggt gatactgata 180

agaccggaac gattacgcac ttcgttccgg atccggaaat cttcaaagaa acaaccgtat 240

acgactatga tctgctttca aaccgtgtcc gggaattggc cttcctgaca aaaggcgtaa 300

acatcacgat tgaagacaaa cgtgaaggac aagaacggaa aaacgagtac cactacgaag 360

gcggaatcaa aagctatgtt gagtacttaa accgttccaa agaagtcgtt catgaagagc 420

cgatttatat cgaaggcgag aaagacggca taacggttga agttgcattg caatacaacg 480

acagctatac aagcaatatt tattctttca cgaataatat caacacatac gaaggcggga 540

cgcacgaagc cggatttaaa accggtctga cccgtgtcat aaacgactat gcaagaagaa 600

aagggatttt caaagaaaat gatccgaatt taagcgggga tgatgtgaga gaagggctga 660

ctgccattat ttcaattaag caccctgatc cgcaattcga agggcagacg aaaaccaagc 720

tcggcaactc cgaagcgaga acgatcactg atacgctgtt ttcttctgcg ctggaaacat 780

tccttcttga aaatccggac tcagcccgca aaatcgttga aaaaggttta atggccgcaa 840

gagcgcggat ggcagcgaaa aaagcacggg aattgacccg gcgcaaaagt gcgcttgaga 900

tttccaatct gccgggcaaa ctggcggact gttcttctaa gatccgagca tttccgagct 960

gtatatcgta gagggtgact ctgcgggcgg atcagcgaaa caggacggga ccgtcatttc 1020

cagctattct gccgctgcgc gtagattctg aacgttgaga aagccagact gataagattc 1080

tctcaaacaa tgaagttcag atcaatgatc acgtccctcg gacagaaatc gagaagaatt 1140

ttaatcttga aaaagcgccg 1160

Claims (7)

1. The Bacillus licheniformis for endophytic Collybia robusta is characterized in that the Bacillus subtilis B1 is named as Bacillus velezensis B1, and the preservation number is GDMCC No: 61296, the strain was deposited in Guangdong provincial collection of microorganisms in building 59 of Tokyo large institute 100, Michelia furiosa, Guangzhou, 11.19.2020.

2. The use of the Bacillus belgii endophytic bacterium of claim 1 for the control of diseases tobacco bacterial wilt, cucumber phytophthora blight, phytophthora capsici, tomato late blight, papaya anthracnose, guava fruit rot, mangosteen fruit rot, cucumber fusarium wilt, cabbage anthracnose, and banana fusarium wilt.

3. The use according to claim 2, wherein the pathogenic bacteria of the disease are Ralstonia solanacearum (Ralstonia solanacearum), Phytophthora cucumeri (Phytophthora melonis), Phytophthora capsici (Phytophthora capsici), tomato late blight (Phytophthora infestans), papaya anthracnose (colletotrichoides), guava pythium (petiolositum), mangosteen pythium (glochytrium bulbophyllum), luffa Fusarium oxysporum (Fusarium oxysporum), Colletotrichum anthracnose (Colletotrichum higginsianum), banana Fusarium oxysporum (Fusarium oxysporum f.

4. Use of the brevibacillus bayperi endophytic bacterium of claim 1 for promoting the growth of tobacco.

5. The use of claim 4, wherein the method of use comprises preparing the Bacillus belgii into a fermentation broth, a complex biological agent.

6. A complex biological agent comprising the Bacillus belgii of claim 1 and a plant immunity inducer.

7. The composite biological agent as claimed in claim 6, wherein the composite biological agent is used for enhancing the resistance of tobacco to tobacco bacterial wilt.

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