CN114015618B

CN114015618B - Bacillus belgii and application thereof in plant disease control

Info

Publication number: CN114015618B
Application number: CN202111499658.8A
Authority: CN
Inventors: 张康; 邢继红; 杜月; 郭浩; 曹宏哲; 司贺龙; 董金皋
Original assignee: Hebei Agricultural University
Current assignee: Hebei Agricultural University
Priority date: 2021-12-09
Filing date: 2021-12-09
Publication date: 2023-04-14
Anticipated expiration: 2041-12-09
Also published as: CN114015618A

Abstract

The invention discloses a Bacillus subtilis GL-03 with the preservation number of CGMCCNo.16931. The microbial agent has better control effect on plant fungal diseases caused by fusarium graminearum, layered fusarium, pseudoverticillium dahliae, northern leaf blight, corn microsporum, trichoderma harzianum and curvularia lunata after being applied. The strain can play a role in agricultural production in various forms, bacterial liquid can be used for root irrigation, powder can be used for seed dressing, and the strain can also be prepared into a biological compound fertilizer for application, so that the strain has the effects of obviously reducing the diseases of the corn stalk rot, increasing the crop yield and improving the quality.

Description

Bacillus belgii and application thereof in plant disease control

Technical Field

The invention relates to the technical field of microbiology and plant disease biocontrol, in particular to a bacillus belgii strain, a microbial agent and application thereof.

Background

Corn stalk rot is a major disease that impairs corn yield and quality, and it can cause lodging of corn plants and rot of ears. The main pathogenic fungi are fusarium graminearum, which is a soil-borne fungus and can also produce vomitoxin, so that the human body health can be harmed when the fusarium graminearum is eaten. At present, chemical pesticide methods are mostly adopted for treating the corn stalk rot, and the methods have quick response and simple operation, but cause harm to the environment.

Plant rhizosphere bacteria are beneficial bacteria which colonize in plant rhizosphere, can prevent and control fungal diseases to a certain extent, arouse the attention of many researchers, and are mostly used for researching biological prevention and control.

Bacillus velezensis is a kind of spore-producing gram-positive bacteria, and has the characteristics of perigenesis flagella, most rod-shaped forms, strong adaptability, high growth speed, strong bacteriostatic ability and the like. It is a bacillus amyloliquefaciens late isoform capable of producing a variety of secondary metabolites such as: enzymes, antibacterial substances, etc. The bacillus subtilis has antagonistic action on various plant pathogenic fungi and becomes a research hotspot of biocontrol bacillus in recent years. It can also secrete IAA (indoleacetic acid) and iron ionophore to promote plant growth, and has obvious growth promoting effect.

However, currently known bacillus belgii still has some drawbacks, such as: most of the methods have the defects of poor prevention and control effect, single action object and the like. These problems result in a great reduction in the control effect of the strain on plant diseases.

Disclosure of Invention

In order to solve the technical problems, the invention provides a bacillus beiLeisi, a microbial agent and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

1. the strain GL-03 is obtained by separating and purifying rhizosphere soil of a corn plant with serious stem rot disease in Dongling farmland in Shenyang city, liaoning province. It is identified as Bacillus velezensis.

The identification process of strain GL-03 is as follows:

when GL-03 strain is cultured on NA culture medium plate, its colony is milk white, circular, its edge is irregular, and its centre is raised. The thalli is observed by a microscope to be unicellular, rod-shaped and gram-negative. After the physiological and biochemical indexes are used for identifying the protein, the protein is found to be positive in starch hydrolysis, gelatin hydrolysis, V-P determination and the like; m.r. reaction, nitrite reduction test, malonate test, phenylalanine deaminase test, H2S production test, etc. were negative. And then, amplifying the specific fragment of the strain by using a 16S rDNA gene specific primer by using a molecular biology means, recovering and sequencing an amplification product, and performing Blast analysis on a sequencing result to find that the strain highly homologous with the strain GL-03 is Bacillus velezensis (Bacillus velezensis) and is identified as the Bacillus velezensis by combining physiological and biochemical characteristics.

2. As a second aspect of the present invention, the present invention provides a microbial agent prepared from Bacillus belgii GL-03 strain.

The microbial agent is prepared by the following method:

culturing the Bacillus belgii GL-03 strain in LB liquid culture medium at 37 deg.C for 48 hr at 220rpm, centrifuging the fermentation liquid, suspending the strain in physiological saline, and regulating the cell number to 1-1.5 × 10 ⁸ cfu/m L, and the ratio of the bacterial suspension of the Bacillus belgii to the sterilized starch is V: m starch = 1-2: 1-2, airing in the shade at room temperature to obtain the Bacillus belief inoculum.

3. As a third aspect of the invention, the invention provides an application of a Bacillus belgii GL-03 strain and a biological agent containing the strain in the field of plant fungal disease control.

The fungal pathogens of the plant fungal diseases are Fusarium graminearum (Fusarium graminearum), fusarium laminarinum (f.proliferatum), fusarium verticillium (f.verticillium), northern leaf spot corn (Exserohilum turcum), southern leaf spot corn (Helminthosporium maydis), trichoderma harzianum (Trichoderma harzianum), curvularia lunata (Curvularia lunata) and the like.

Compared with the prior art, the invention has the following technical effects:

(1) In the plate confronting method, the Bacillus beijerinckii GL-03 strain has good inhibition effect on plant pathogenic fungi such as fusarium graminearum, fusarium laminarinum, fusarium verticillioides, northern leaf blight, southern leaf blight, trichoderma harzianum, curvularia lunata and the like, and the strain has a wider antibacterial spectrum.

(2) In field experiments, the Bacillus belgii microbial agent has better control effect on fusarium graminearum fungal diseases after being applied.

(3) The Bacillus belgii GL-03 strain can play a role in agricultural production in various forms, a bacterial liquid can be used for root irrigation, a powder can be used for seed dressing, and a biological compound fertilizer can be prepared for application, so that the Bacillus belgii GL-03 strain has the effects of obviously reducing corn stalk rot diseases, increasing crop yield and improving quality.

Description of the deposit certificate:

the preservation organization: china general microbiological culture Collection center;

the preservation number is: CGMCC No.16931;

the preservation date is as follows: 12 months and 20 days 2018;

and (4) storage address: xilu No.1 Hospital No.3, beijing, chaoyang, north;

taxonomic nomenclature: bacillus velezensis (Bacillus velezensis).

Drawings

FIG. 1 is a graph showing the effect of a Bacillus beijerinckii strain on inhibiting Fusarium graminearum in example 1 of the present invention;

FIG. 2 is a graph showing the cluster analysis of 16S r DNA of Bacillus belgii strain in example 1 of the present invention;

FIG. 3 is a graph showing the bacteriostatic effect of Bacillus belgii on phytopathogenic fungi in example 2 of the present invention;

FIG. 4 is a schematic view showing the inhibition of the growth of Fusarium graminearum hyphae by Bacillus belgii in example 2 of the present invention;

FIG. 5 is a graph showing the effect of the field test on the disease resistance of Bacillus belgii microbial agents against Fusarium graminearum in example 4 of the present invention.

Detailed Description

The invention discloses bacillus beleisi with high antifungal activity, which can be realized by appropriately improving process parameters by the technical personnel in the field by referring to the content. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be included within the invention. While the Bacillus belgii strains of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and appropriate modifications and combinations of the methods and applications described herein may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

The technical solution of the present invention is further illustrated in detail by the following examples.

Example 1

Isolation of GL-03 Strain

Rhizosphere soil is taken from rhizosphere of a corn plant with serious stem rot disease in Dongling farmland in Shenyang city of Liaoning, and the rhizosphere soil is collected and sealed by a clean sample bag. Taking 10g of rhizosphere soil, fully and uniformly mixing in 200mL of sterile physiological saline, carrying out gradient dilution, and taking 50 mu L of 10 ^-6 、10 ^-7 、10 ^-8 The mixed liquid with 3 dilution gradients is evenly coated on an LB bacterial culture plate (each test is repeated for 3 times), the coated plate is placed in an incubator at 37 ℃ for culture, and a new single colony is picked up and cultured on the bacterial culture plate for preservation and standby.

2. Screening of antagonistic Strain

Fusarium graminearum (f.graminearum) is used as a pathogen, and a bacterial disc is inoculated in the center of a PDA plate. And (3) adopting a plate confronting method to streak and inoculate the separated and purified Bacillus belgii GL-03 strain at a position 3cm away from the disc. The blank was inoculated with Fusarium graminearum alone. Three control groups were inoculated according to the above inoculation method, respectively, and cultured in a dark environment at 27 ℃ for 7 days to observe antagonistic effects. Determining the bacteriostatic effect according to the size of the bacteriostatic zone, and selecting the strain with the best bacteriostatic effect (figure 1) to obtain the Bacillus beiLeisi GL-03.

3. Sequence determination and analysis of 16S r DNA of strain and physiological and biochemical test identification

The DNA extraction of the Bacillus beleisi GL-03 adopts a bacterial genome DNA rapid extraction kit (DL 111-01), and 16S rDNA sequences are amplified by using bacterial universal primers, wherein the primers are 27F and 1492R, and the primer sequences are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.

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

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

And (3) PCR reaction system: 25 μ L of 1 × EcoTaqPCR Supermix, 0.4 μmol/L forward and reverse primers, 2 μ L genomic DNA, in a total volume of 50 μ L. And (3) PCR reaction conditions: pre-denaturation at 95 deg.C for 4min, denaturation at 94 deg.C for 1min, annealing at 1min, extension at 70 deg.C for 1min, and final extension at 70 deg.C for 10min.

The PCR amplification products were subjected to electrophoresis (1% agarose gel). Sequencing the purified PCR product by Huada Gene Co., ltd to obtain a 16srDNA sequence of the GL-03 strain shown as SEQ ID NO. 1.

The sequencing result is submitted to NCBI, similarity comparison is carried out between Blast function options in NCBI databases and known sequences, the maximum likelihood method in Mega 7.0 software is utilized to analyze the evolutionary relationship of 16S rDNA genes of bacteria, and the result is shown in figure 2, and the Bootstrap value is set as 1000. According to the comparison result, GL-03 is concluded to be Bacillus beleisis.

4. Performing primary physiological and biochemical identification on the separated and screened antagonistic strain

The method comprises gram determination, catalase reaction, VP test, starch hydrolysis test and the like, and the test method adopts the conventional test method for determining physiological and biochemical indexes in the field, is not the essential point of the invention, and is not described herein again. The results are shown in Table 1.

TABLE 1 determination of physiological and biochemical indices of the strains

Measurement index	Characteristics of the strains	Measurement index	Characterization of the strains
				Starch hydrolysis	+	Glucose sugar	+
VP assay	+	Sucrose	+
				Indole production	+	Maltose	+
Ammonia production test	+	Lactose	+
				Phenylalanine deaminase assay	-	Sorbitol	+
Hydrogen peroxide reductase	+	Fructose	+
				Gram stain	-	Glycerol	+
M.r test	-	Citrate test	+
				Nitrate reduction test	+	Dynamic experiment	+
Malonate test	-	H ₂ S Generation test	-

The results show that the screened antagonistic bacteria have positive effects on starch hydrolysis, gelatin hydrolysis, V-P determination and the like; M.R. reaction, nitrite reduction test, malonate test, phenylalanine deaminase test, H2S production test and the like are negative, and the physiological and biochemical characteristics of the Bacillus belgii are met.

Example 2

1. Growth inhibition test of GL-03 isolated in example 1 on northern leaf blight, fusarium laminarinum, fusarium graminearum, fusarium verticillium, curvularia lunata, microsporum cerealis and Trichoderma harzianum

A single colony of the Bacillus beleisi GL-03 is picked and cultured overnight in a shaking table with the temperature of 37 ℃ at 220r/min in three bottles with 20mL of LB liquid culture medium and 50mL, and the OD600=0.5 is adjusted. The plant pathogenic fungi with the diameter of 8mm and the GL-03 fermentation broth of the invention are subjected to opposite culture by adopting a plate opposite method, each treatment is repeated for 3 times, the culture is carried out in a constant-temperature incubator at 27 ℃ for 7 days, and the antibacterial effect is observed, and the result is shown in figure 3.

According to the bacteriostatic effect, the strain GL-03 disclosed by the invention has a good inhibitory effect on plant pathogenic fungi such as corn northern leaf blight, layered fusarium, fusarium graminearum, pseudoverticillium dahliae, curvularia lunata, microspora parvus and trichoderma harzianum, and the strain disclosed by the invention has a wide bacteriostatic spectrum.

2. EXAMPLE 1 growth inhibition test of isolated GL-03 Strain on Fusarium graminearum hyphae

The method adopts a plate mutual confrontation method, the Belgium glaucum GL-03 and the fusarium graminearum are oppositely cultured in a constant temperature incubator at the temperature of 27 ℃, a plate without point-contact antagonistic strains is used as a blank control group, after 7d, the plate is placed under a microscope for observation, and the growth conditions of fusarium graminearum hyphae of an experimental group and the control group are observed. The experiment was set up in 3 replicates. The results are shown in FIG. 4.

As can be seen from the growth condition of hyphae, the strain GL-03 can obviously inhibit the growth of the hyphae of fusarium graminearum, and has a good control effect on fungal diseases caused by fusarium graminearum.

Example 3

Preparation of microbial inoculum comprising GL-03 Strain as described in examples 1 to 2

Culturing the GL-03 strain of example 1-2 in LB liquid medium at 37 deg.C for 48h at 220rpm to obtain fermentation broth, centrifuging the fermentation broth, suspending the strain in physiological saline, and adjusting cell number to 10 ⁸ cfu/m L, mixed with sterilized starch. Mixing the bacterial suspension with starch according to the ratio of V: and (3) mixing the components according to the proportion of M =1, and airing at a cool place at room temperature to obtain the microbial inoculum.

Example 4

Example 3 field test of disease resistance of the microbial Agents against Fusarium graminearum

The experiment is carried out in a test field of the university of agriculture in Hebei, wherein seeds with full grains and consistent sizes are selected for sowing, and a control group and an experimental group are respectively arranged. After the corn seedlings emerge, 3g of microbial inoculum is applied to the corn roots in the experimental group, and the control group is not treated. At the 10-leaf stage, the second stem part of the corn, which is separated from the ground, is punctured in parallel by using an injector, 2mL of fusarium graminearum suspension is injected into the second stem part, and the control group and the experimental group are injected. Randomly sampling 3 corns in the two groups at 7d, 14d, 21d and 28d after inoculation respectively, and cutting stalks to observe the morbidity. As shown in table 2, table 3 and fig. 5.

The level of resistance of the test maize to maize stalk rot is divided into 5 grades depending on how many diseased plants are in the population.

Stage 1: the proportion of the diseased corn plants is 0 to 5.0 percent;

and 3, stage: the proportion of diseased corn plants is 5.1-10.0%;

and 5, stage: the proportion of the diseased corn plants is 10.1-30.0%;

and 7, stage: the proportion of diseased corn plants is 30.1-40.0%;

and 9, stage: the proportion of the plants with diseases is 40.1-100.0%.

Disease index = [ ∑ (number of diseased stem segments at each level × number of relative diseased stem segments)/(number of stem segments in investigation × 9) ] × 100%;

control effect (%) = [ (CK-Pt)/CK ] × 100%;

CK represents the disease index of the control group, and Pt represents the disease index of the treatment group.

TABLE 2 field control data (1)

Group of

Disease index (%)

Control effect (%)

Ear row number

Number of lines and grains

Ear thickness (cm)

Ear length (cm)

Control group

51.74±3.89

—

15.52±0.08

27.87±0.65

4.34±0.38

16.33±0.36

GL-03

26.44±2.78

48.90

17.22±0.13

32.91±0.23

4.44±0.27

17.84±0.31

TABLE 3 field control data (2)

Group of	Long bald tip (cm)	Water content (%)	Thousand Kernel weight (g)	Percentage of seed production (%)	Yield (kg/hm) ² )
						Control group	2.15±0.51	12.5±0.51	279±11.22	83.93±0.31	636.02±14.12
GL-03	1.40±0.08	12.5±0.23	335±7.99	89.67±0.15	699.97±15.21

Through measurement and analysis of the growth condition and disease index of the corn, the following results are found: after the microbial inoculum is used for treating the seedlings, the disease resistance of the seedlings is obviously enhanced, and the highest control effect reaches 48.90 percent; after the microbial inoculum is used for treating seedlings, the corn yield is remarkably improved by more than 10 percent.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

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Claims

1. A composition containing Bacillus belgiiBacillus velezensis) The application of GL-03 microbial inoculum in preventing and treating plant fungal diseases is characterized in that,

the preservation number of the Bacillus belgii GL-03 is CGMCC No.16931;

the microbial agent is formed by mixing Bacillus belgii GL-03 bacterial suspension and sterilized starch, wherein the Bacillus belgii GL-03 bacterial suspension and the starch are mixed according to the ratio of V: m =1 to 2, and the ratio is 1 to 2, and the viable count in the Bacillus beilesensis GL-03 suspension is 1 to 1.5 multiplied by 10 ⁸ cfu/m L；

The plant fungal disease is corn stalk rot, and the pathogenic bacteria is fusarium graminearum.

2. The use of claim 1, wherein the suspension of bacillus beijerinckii GL-03 is mixed with starch according to a V: m =1 and mixing.

3. The use of claim 2, wherein the suspension of Bacillus belgii GL-03 has a viable count of 1 x 10 ⁸ cfu/m L。

4. A composition containing Bacillus belgiiBacillus velezensis) The application of GL-03 microbial inoculum in preventing and treating plant fungal diseases is characterized in that,

the preservation number of the Bacillus belgii GL-03 is CGMCC No.16931;

the microbial agent is prepared by mixing a bacillus belgii GL-03 bacterial suspension and sterilized starch, wherein the bacillus belgii GL-03 bacterial suspension and the starch are mixed according to the ratio of V: m =1 to 2, and the ratio is 1 to 2, and the viable count in the Bacillus beilesensis GL-03 suspension is 1 to 1.5 multiplied by 10 ⁸ cfu/m L；

The pathogenic bacteria of the plant fungal diseases are one or more of Fusarium stratification, fusarium verticillioides, northern leaf blight of corn, southern leaf blight of corn, trichoderma harzianum and Curvularia lunata.