CN109988727B

CN109988727B - Verticillium dahliae endophyte and application thereof

Info

Publication number: CN109988727B
Application number: CN201811591585.3A
Authority: CN
Inventors: 张�林; 陶冶; 陈俊梅; 李文鹏; 黄冰纷; 牛秋红
Original assignee: Nanyang Normal University
Current assignee: Nanyang Normal University
Priority date: 2018-12-25
Filing date: 2018-12-25
Publication date: 2022-07-15
Anticipated expiration: 2038-12-25
Also published as: CN109988727A

Abstract

The invention provides an endophyte of Verticillium dahliae, which is Enterobacter maritimus V1 strain with the preservation number of CCTCC M2018715. The invention adopts Enterobacter (Enterobacter xiangfangensis) V1 strain, which has the function of inhibiting Verticillium dahliae. The total length of the peroxidase gene in the enterobacter is 603bp, 200 amino acids are coded, and after the gene is expressed in a heterologous way, the expression product also has the function of inhibiting verticillium dahliae. The invention provides microbial resources for preventing and treating cotton verticillium wilt by using a biocontrol technology, and simultaneously avoids the problems of environmental pollution and the like compared with chemical prevention and treatment.

Description

Verticillium dahliae endophyte and application thereof

Technical Field

The invention belongs to the technical field of biological control of plant diseases, and particularly relates to verticillium dahliae endophyte and application thereof.

Background

Cotton is an important fiber plant, and verticillium wilt caused by infection of verticillium dahliae is one of the main diseases of cotton, which is called cotton cancer. The pathogenic bacteria have strong viability, the sclerotium can survive for more than 10 years under the condition of no host in the soil, and the pathogenic bacteria have rich genetic diversity, easy variation and the like. In addition, the host range of the verticillium dahliae is extremely wide, and 660 plants of 38 families can be killed by foreign reports, wherein 184 crops and 153 weeds are planted. According to the identification of China, at least 20 host plants are 80, and field crops comprise sunflower, eggplant, peppery seeds, tomatoes, tobacco, potatoes, melons, watermelons, cucumbers, peanuts, kidney beans, mung beans, soybeans, sesame, beet and other crops. The cotton is one of economic crops which are distributed most widely and have the largest planting area in the world and is also one of crops which are most seriously damaged, the cotton verticillium wilt is a worldwide dangerous disease and is also a serious disease in cotton production in China, and the verticillium dahliae can form microsclerotia with a dormant structure, has stronger resistance to adverse environments and can resist high temperature of 80 ℃ and low temperature of-30 ℃, so the microsclerotia with stronger protection effect can be formed under extreme conditions, and a fungus state with stronger susceptibility can be formed when the external environment is suitable for germination. The disease area of cotton verticillium wilt in China is nearly 15% -20% of the planting area of China every year, the disease area can even reach as much as 50% in severe years, and some planting areas with severe diseases can be harvested absolutely. The verticillium wilt not only reduces the yield of cotton, but also reduces the fiber quality, seed yield, oil yield of seeds and the like of the cotton, thereby causing severe economic loss.

At present, the prevention and treatment of cotton verticillium wilt at home and abroad mainly adopt the traditional means of crop rotation, breeding disease-resistant varieties, chemical prevention and the like, but the disease prevention effect is not ideal, and the methods have certain limitations and disadvantages, poor operability and adaptability or unsatisfactory effect. Biological control of plant diseases is increasingly attracting attention because of its safety, effectiveness, low toxicity and few residues, and is considered to be the most promising method for control. As a biological control resource, endogenous bacteria become a hot spot for developing biological control microbial agents in recent years, and therefore, biological control research on verticillium wilt naturally becomes a key and focus problem.

Disclosure of Invention

The present invention has been made to overcome the above problems, and an object of the present invention is to provide an endophyte of Verticillium dahliae having a high control effect on Verticillium dahliae and little environmental pollution, and a peroxidase contained in the endophyte.

In order to solve the problems, the invention adopts the technical scheme that:

the invention provides an endophyte of Verticillium dahliae, which is Enterobacter maritimus V1 strain with the preservation number of CCTCC M2018715.

The invention provides an application of verticillium dahliae endophyte in inhibiting activity of verticillium dahliae.

The invention provides application of verticillium dahliae endophytes in cotton verticillium wilt prevention and treatment or preparation of microbial inoculum for cotton verticillium wilt prevention and treatment.

The invention provides application of peroxidase in verticillium dahliae to inhibition of activity of verticillium dahliae, prevention and treatment of cotton verticillium wilt or preparation of a microbial inoculum for prevention and treatment of cotton verticillium wilt.

Preferably, the cDNA nucleotide sequence of the peroxidase gene is SEQ ID NO.1 in a sequence table.

Preferably, the amino acid sequence of the peroxidase gene is SEQ ID NO.2 in the sequence table.

Preferably, after the peroxidase is expressed in a heterologous way, the expression product has the effect of inhibiting the activity of Verticillium dahliae or preventing and treating cotton verticillium wilt.

Preferably, the heterologous expression of the peroxidase is induced by ligating the peroxidase to the vector PET32a, introducing the ligated product into competent cells, and expressing the product.

Preferably, the induction of expression is at 25 ℃ for 8h with 0.5mM IPTG.

Preferably, a protein purification step is further included after said inducing expression.

The invention adopts Enterobacter (Enterobacter xiangfangensis) V1 strain, which has the function of inhibiting Verticillium dahliae. The total length of the peroxidase gene in the enterobacter is 603bp, 200 amino acids are coded, and after the gene is expressed in a heterologous way, the expression product also has the function of inhibiting verticillium dahliae. The antagonism function of the gene is mainly the increase of the activity of peroxidase, and can further increase the synthesis of resistance components such as phenol, lignin and the like in cotton bodies, thicken cell walls and play a role in protecting plant cells. And the peroxidase is associated with disease resistance reaction generated in plants. The invention provides microbial resources for the prevention and treatment of cotton verticillium wilt by using a biocontrol technology, and simultaneously avoids the problems of environmental pollution and the like compared with chemical prevention and treatment.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a plate confrontation experiment of Enterobacter xiangfangensis V1 strain.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.

Example 1

Step 1) screening a V1 strain from resistant cotton leaves in a cotton test field of Nanyang faculty academy of faculty, wherein the strain has stronger antagonistic action on Verticillium dahliae, and after the antagonistic activity is determined through a plurality of times of plate confrontation experiments, the strain is preserved in China center for type culture Collection (CCTCC, preservation address: in Wuhan university school of Wuhan 229 eight-way in Wuchang district, Wuhan city, Hubei province, the preservation number is: CCTCC M2018715, and identifying the strain as Enterobacter xiangfangensis V1 strain.

The results of the plate confrontation experiment are shown in FIG. 1.

Step 2), inoculating the V1 bacterial cells into LB liquid medium test tubes (each tube is filled with 5ml), and carrying out shake culture at 37 ℃ and 220rpm for 12h to obtain test tube species. Then inoculating the strain into a 500ml triangular flask LB liquid culture medium, and carrying out shake cultivation at 37 ℃ and 220rpm for 56h to obtain a fermentation culture.

Centrifuging the fermentation culture at 10000rpm for 20min, taking supernatant, adding solid ammonium sulfate respectively until the saturation is 20%, 30%, 40%, 50%, 60%, 70% and 80%, stirring at 4 ℃ for 2-3 h, standing at 4 ℃ for 2-3 h, centrifuging at 8000r/min for 20min, and dissolving the precipitate in PBS buffer solution to obtain a crude extract. The crude extract is put into a dialysis bag for dialysis for 24 hours, and SDS-PAGE electrophoresis is carried out after dialysis. The activity of each component is respectively measured by adopting a filter paper method, and the optimal saturation degree of the ammonium sulfate is determined to be 40 percent, and the molecular weight is 35 KDa. The band was sent to Shanghai Biopharmaceutical science and technology Limited to perform mass spectrometric identification and identified as peroxidase.

The nucleotide sequence of the peroxidase in Enterobacter xiangfangensisV1 strain is as follows:

ATGGTTCTGGCAACTCGTCCGGCTCCGGATTTGACAGCTGCCGCCGTTCTGGGCAACGGTGACATCGTTGAAAACTTCAACTCCAAACAGCACAACACCGGTAAAGCGACCGTTCTGTCTTTCTGGCCAATGGACTTCACTTTCGCTTGCCCCTCTGAGCTGATCGCGTTCGCCAAACGTTACGAAGAATTCCAGAAACGTGGCGAGGAAGTGGTTGGCGTCTCCTTCGACTCTGAATTTGTACACAAAGCATGGCGTATCACCCCTGTCGAAAACGGCGACATCGATGCGGTGAAATACGCGATGGTTGCGGACATCAAACGTGGAACCCAGCAGGCTTACGGTATCGAACATCCGGACGCTGGCGTTGCGCTGCGTGGTTCTTTCCTGATCGACGCGAACGACATCGTTTGTCACCAGGCTGTGAACGATCTGCTGCTGGATCGTAACATTGACGAAATGCGGCGCATGGTTGACGCGCTCCTGTTCCACGAAGCGCACGGTGAAGTGTGCCTGGCTCAGTGGGAAAAAGGTATAGAAGGTATGGCGGCTTCCCTAGACGGCGTAGCTAAACACCTGTCTGAGAACGTATACAGcCTGTAA

the coded amino acid sequence is as follows:

MVLATRPAPDLTAAAVLGNGDIVENFNSKQHNTGKATVLSFWPMDFTFACPSELIAFAKRYEEFQKRGEEVVGVSFDSEFVHKAWRITPVENGDIDAVKYAMVADIKRGTQQAYGIEHPDAGVALRGSFLIDANDIVCHQAVNDLLLDRNIDEMRRMVDALLFHEAHGEVCLAQWEKGIEGMAASLDGVAKHLSENVYSL

and 3) constructing a protein expression vector PET32a of the peroxidase gene, carrying out double enzyme digestion on the V1 quality-improved grains, introducing the quality-improved grains into BL21 competent cells after the double enzyme digestion is successfully verified, adding an inducer IPTG with a certain concentration for induction expression, and improving the expression level of the peroxidase by optimizing three factors of induction time, induction temperature and the inducer IPTG concentration, wherein the expression condition optimization parameters are shown in Table 1.

TABLE 1 peroxidase expression conditions optimization parameters

The SDS-PAGE electrophoresis running results show that the expression level of the peroxidase is highest under the expression condition of inducing for 8h at 25 ℃ by 0.5mM IPTG, and the optimal expression condition of inducing for 8h at 25 ℃ by 0.5mM IPTG is determined. Induced expression was performed with 0.5mM IPTG and protein purification was performed using a one-stop His-tagged protein miniprep kit.

Step four, testing the antagonistic capacity of the peroxidase gene expression product by using a confronting culture method, measuring the diameter of an inhibition zone, and calculating the inhibition rate, wherein the inhibition rate (%) is (the diameter of a control group colony-the diameter of a treatment group colony)/(the diameter of a control group colony-the diameter of a test pathogenic bacterium cake) multiplied by 100, and the inhibition rate can reach 40% through a three-time plate confronting test. The expression product of the gene has good bacteriostatic activity.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> south Yang college of learning

<120> Verticillium dahliae endophyte and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 603

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggttctgg caactcgtcc ggctccggat ttgacagctg ccgccgttct gggcaacggt 60

gacatcgttg aaaacttcaa ctccaaacag cacaacaccg gtaaagcgac cgttctgtct 120

ttctggccaa tggacttcac tttcgcttgc ccctctgagc tgatcgcgtt cgccaaacgt 180

tacgaagaat tccagaaacg tggcgaggaa gtggttggcg tctccttcga ctctgaattt 240

gtacacaaag catggcgtat cacccctgtc gaaaacggcg acatcgatgc ggtgaaatac 300

gcgatggttg cggacatcaa acgtggaacc cagcaggctt acggtatcga acatccggac 360

gctggcgttg cgctgcgtgg ttctttcctg atcgacgcga acgacatcgt ttgtcaccag 420

gctgtgaacg atctgctgct ggatcgtaac attgacgaaa tgcggcgcat ggttgacgcg 480

ctcctgttcc acgaagcgca cggtgaagtg tgcctggctc agtgggaaaa aggtatagaa 540

ggtatggcgg cttccctaga cggcgtagct aaacacctgt ctgagaacgt atacagcctg 600

taa 603

<210> 2

<211> 200

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Val Leu Ala Thr Arg Pro Ala Pro Asp Leu Thr Ala Ala Ala Val

1 5 10 15

Leu Gly Asn Gly Asp Ile Val Glu Asn Phe Asn Ser Lys Gln His Asn

20 25 30

Thr Gly Lys Ala Thr Val Leu Ser Phe Trp Pro Met Asp Phe Thr Phe

35 40 45

Ala Cys Pro Ser Glu Leu Ile Ala Phe Ala Lys Arg Tyr Glu Glu Phe

50 55 60

Gln Lys Arg Gly Glu Glu Val Val Gly Val Ser Phe Asp Ser Glu Phe

65 70 75 80

Val His Lys Ala Trp Arg Ile Thr Pro Val Glu Asn Gly Asp Ile Asp

85 90 95

Ala Val Lys Tyr Ala Met Val Ala Asp Ile Lys Arg Gly Thr Gln Gln

100 105 110

Ala Tyr Gly Ile Glu His Pro Asp Ala Gly Val Ala Leu Arg Gly Ser

115 120 125

Phe Leu Ile Asp Ala Asn Asp Ile Val Cys His Gln Ala Val Asn Asp

130 135 140

Leu Leu Leu Asp Arg Asn Ile Asp Glu Met Arg Arg Met Val Asp Ala

145 150 155 160

Leu Leu Phe His Glu Ala His Gly Glu Val Cys Leu Ala Gln Trp Glu

165 170 175

Lys Gly Ile Glu Gly Met Ala Ala Ser Leu Asp Gly Val Ala Lys His

180 185 190

Leu Ser Glu Asn Val Tyr Ser Leu

195 200

Claims

1. Verticillium dahliae endophyte is characterized in that: the endophyte is Enterobacter xiangfangensis V1

The strain has a preservation number of CCTCC M2018715.

2. The use of the verticillium dahliae endophyte of claim 1 to inhibit activity of verticillium dahliae.

3. The use of the verticillium dahliae endophyte of claim 1 in the prevention and treatment of cotton verticillium wilt or in the preparation of microbial inoculum for the prevention and treatment of cotton verticillium wilt.