CN108865934B - A kind of Bacillus saphoe HMD9204 and its bacterial agent and application - Google Patents

Abstract

The invention discloses a bacillus safensis HMD9204, a microbial inoculum and an application thereof, wherein the strain is preserved in Guangdong province microbial strain preservation center in 2017, 12 and 18 months, and the preservation number is as follows: GDMCC No. 60302. The bacterial strain is matched with an organic fertilizer or a farmyard manure for use, has very obvious control effect on eggplant wilt and banana wilt, and can reduce the morbidity of the eggplant wilt to 2-5% and the banana wilt to 6.7-9.6%.

Description

A kind of Bacillus saphoe HMD9204 and its bacterial agent and application

technical field

The invention belongs to the technical field of microorganisms, and in particular relates to a kind of Bacillus saphobia HMD9204 and its bacterial agent and application.

Background technique

Planting a certain crop for a long period of time has caused serious secondary salinization of soil and aggravated soil-borne diseases, resulting in reduced crop quality and yield, which has become an important obstacle factor restricting the sustainable development of facility agriculture. Plant soil-borne diseases are a kind of important plant diseases. There are many kinds of pathogens (fungi, bacteria, nematodes, viruses) that cause soil-borne diseases. They usually infect the roots of plants, causing diseases of plant roots and even the whole plant, causing major Economic losses. Among them, soil-borne pathogenic fungi have become one of the main constraints for stable and high yield of crops and vegetables.

Banana wilt is a soil-borne disease caused by a fungus that infects the vascular bundles of banana plants. It is a devastating disease, an international phytosanitary object, caused by Fusarium oxysporum Cuban-specialized infestation. It has occurred in banana producing areas in several provinces in southern China. The pathogenic bacteria can survive in the soil for several years, generally reducing the yield by more than 20%, and even the harvest of serious fields is completely lost. Therefore, in order to effectively prevent and control soil-borne diseases and ensure the stable growth of crop and vegetable yields in my country, comprehensive control is necessary. Among them, because the biological control is green, harmless and efficient, it has been widely concerned by the government, farms and farmers, and the research on biocontrol microbial resources is imminent.

SUMMARY OF THE INVENTION

The object of the present invention is to provide Bacillus safensis (Bacillussafensis) capable of preventing and controlling soil-borne diseases, and the bacterial strain can effectively prevent and control soil-borne diseases to crops, improve crop yield, and particularly has obvious control effect on fusarium wilt. At the same time, the invention also provides the preparation and application of the bacterial agent of the strain.

The present invention is specifically realized through the following technical solutions:

The present invention provides Bacillus safensis HMD9204 capable of preventing and controlling soil-borne diseases. The strain has been preserved, and the preservation unit is: Guangdong Microbial Culture Collection Center (GDMCC), address: Xianlie Middle Road, Guangzhou, China The fifth floor of the laboratory building of No. 100 Provincial Institute of Microbiology, preservation date: December 18, 2017, preservation number: GDMCCNo.60302.

The main biological characteristics of the Bacillus saffordii HMD9204 of the present invention are Gram-positive (G+), the cell shape is rod-shaped, the cell size is 1.0-1.2 μm in length and 0.5-0.7 μm in width, aerobic, spore-producing and movement. The colony was 3-7mm in diameter when grown in TSA medium for 2 days, and the colony was gray-white, round, and wavy at the edge. The strain can grow under the conditions of 0-10% (w/v) NaCl, 10-50°C, and the optimum growth conditions are 35°C, pH 7 and 0.5% (w/v) NaCl. Catalase, oxidase, alkaline phosphatase and lipase were positive, and could hydrolyze gelatin, casein, pyridoxine and RNA, but could not hydrolyze indole and starch.

The 16S rRNA sequence of Bacillus suffolk HMD9204 of the present invention is: SEQ ID NO: 1.

Another object of the present invention is to provide the above-mentioned Bacillus safflower HMD9204 or its bacterial suspension or its culture solution or its fermentation product or its inoculum.

A bacterial seed preparation for preventing and treating soil-borne diseases, the bacterial seed preparation comprises Bacillus saphobia HMD9204 or its bacterial suspension or its culture solution or the powder of its fermentation product.

The strain preparation of the present invention is usually obtained by conventional liquid or solid culture, including bacterial culture, produced by conventional liquid fermentation, and then added with surfactants such as dispersing agents, stabilizers, wetting agents, binders, disintegrating agents, etc. One or more of the decomposers, etc., or the adsorption carrier is mixed in a certain proportion, and then prepared into a powder.

On the basis of the preparation of the present invention, the present invention also provides a method for preventing and controlling soil-borne diseases, specifically mixing the powder of Bacillus saphobia HMD9204 with organic fertilizer or farmyard manure in a mass ratio of 1.5 to 3:1000, 3 ～10kg/mu bottom application or top dressing, avoid using together with pesticides during application.

The number of effective viable bacteria after the Bacillus saphobia HMD9204 powder is mixed with organic fertilizer or farmyard manure is not less than 100 million cfu/g.

The present invention also provides the application of the described Bacillus saphobia HMD9204 or its bacterial suspension or its culture solution or its fermentation product in preventing and treating crop soil-borne diseases, wherein the soil-borne diseases are fusarium wilt, and the crops Choose from banana or eggplant.

At the same time, the application of the above-mentioned Bacillus saphobia HMD9204 powder in the prevention and control of crop soil-borne diseases is also within the protection scope of the present invention.

The beneficial effects of the present invention are:

Fusarium oxysporum causes fusarium wilt of many crops and spreads through soil. It is difficult to control with general chemical agents, and it is easy to bring chemical pollution. Biological control is the most feasible and environmentally friendly means. The invention isolates a strain of bacillus saphobia HMD9204 with antagonistic effect on fusarium wilt, and is used in combination with organic fertilizer or farmyard manure, and has obvious control effect on fusarium wilt of eggplant and fusarium wilt of banana, and can reduce the incidence of fusarium wilt of eggplant. To 2-5%, the incidence of banana fusarium wilt drops to 6.7-9.6%.

Detailed ways

The actual technical solutions of the present invention will be clearly and completely described below with reference to the specific embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1 Isolation and identification of Bacillus suffolk HMD9204

1) Isolation of Bacillus saphobia HMD9204

The soil separated in this example was collected from the saline soil (37°34'N 116°49'E) of the experimental base of the Chinese Academy of Agricultural Sciences in Ling County, Dezhou, Shandong. The five-point sampling method is adopted, that is, firstly determine the midpoint of the diagonal as the central sampling point, and then select four points on the diagonal with the same distance from the central sampling point as the sampling points. ^The improved farming soil plot of 1000m2 of the plot was sampled, and the surface soil 15-20cm from the ground was selected for testing. The collected soil samples were divided into sample bags and recorded, and returned to the laboratory in time.

Weigh 5 g of soil, add it to a triangular flask (containing glass beads) containing 45 ml of sterile physiological saline, shake at 30° C., 180 rpm in a constant temperature shaker for 20 min to disperse the soil evenly. Soil solutions of different dilution concentrations, such as 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 and 10 ^-7 g/ml, were sequentially prepared in a sterile bench. Pipette 0.1ml of soil solution with a dilution of 10 ^-3 -10 ^-7 , spread it evenly on the NA solid medium plate with a coating rod, set 3-5 replicates for each sample, and invert in a constant temperature incubator to cultivate, NA The culture is based on 30 °C, observe the growth of bacteria on the plate in 2-5 days, record the number and growth of colonies on the surface of the plate, and inoculate the single colony growing on the plate on a new plate by the four-zone streak method until the strain is pure. bacteria.

2) Identification of Bacillus saphobia HMD9204

The isolated and purified strains were subjected to 16S rRNA gene amplification and PCR amplification, and were sequenced and spliced by a sequencing company. The results were compared and identified on the Ezbiocloud website. The isolated strains were observed under a microscope, Gram staining was positive (G+), the cell shape was rod-shaped, and the cell size was 1.0-1.2 μm in length and 0.5-0.7 μm in width. Sporulation. The colony was 3-7mm in diameter when grown in TSA medium for 2 days, and the colony was gray-white, round, and wavy at the edge. It can grow under the conditions of 10-50℃, and the optimum growth condition is 35℃.

The contact enzyme test was carried out on the strain according to the "Berger Bacterial Identification Manual", and the results were positive for catalase, oxidase, alkaline phosphatase and lipase, and it could hydrolyze gelatin, casein, pyridoxine and RNA, but could not hydrolyze indole and starch.

Analysis of cell wall composition, cells were harvested, inactivated in a water bath, peptidoglycan was isolated, and fatty acid composition was determined. The results showed that the main fatty acid components were iso-C _15:0 , anteiso-C _15:0 , iso-C _17:0 and anteiso-C _17:0 .

Stress tolerance test, inoculated into beef extract peptone medium with different pH values and different concentrations of NaCl. The strain can grow in 0-10% (w/v) NaCl conditions, with an optimum pH of 7 and 0.5% (w/v) NaCl.

The results of agarose gel electrophoresis showed that the size of the amplified fragment of the 16S rRNA gene of the strain was about 1446, and the measured sequence was shown in SEQ ID NO: 1. The phylogenetic analysis showed that the similarity between the strain HMD9204 and Bacillus safensis was 99.65%. Based on the physicochemical properties and 16S rRNA gene phylogenetic tree of the bacteria, it can be determined that the bacteria is Bacillus saphobia.

Based on the above characteristics, the strain HMD9204 was identified as Bacillus safensis. The strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on December 18, 2017, and the strain collection number is GDMCC No.60302.

The inhibitory effect of embodiment 2HMD9204 bacteria on banana fusarium wilt and eggplant Fusarium wilt

Confrontation test: The test consists of 3 groups: 1) Banana Fusarium wilt-Bacillus suffolk group; 2) Eggplant Fusarium wilt-Bacillus saffron group; 3) Control group. 3 repetitions per set. The center of the nutrient broth medium was punched with a sterile 5mm diameter hole punch, and the pathogenic bacteria cake was inoculated in the center. After culturing for 24 hours, a sterile 5mm diameter punch was used on both sides at a distance of 3 cm from the edge of the central pathogenic bacteria cake. The hole was punched and inoculated with Bacillus suffolk. After the negative control group was inoculated with pathogenic bacteria, the Bacillus saffron cake was replaced by agar block and placed in a 28°C incubator for 48 hours. The inhibition zone was measured and the inhibition was calculated. Rate. The results are shown in Table 1.

Table 1 Inhibitory effect of HMD9204 on banana Fusarium wilt and Eggplant Fusarium wilt

Inhibition zone diameter (mm) Bacteriostatic rate (%) Fusarium wilt of banana 27.1 ± 1.2 63.8 ± 2.1 Fusarium wilt of eggplant 23.3±1.5 59.0±3.7

It can be seen from Table 1 that the strain HMD9204 has a good inhibitory effect on banana fusarium wilt and eggplant fusarium wilt, and the bacteriostatic rate reaches more than 60%.

Example 3

After the Bacillus saphobia HMD9204 powder provided by the invention is mixed with organic fertilizer or farmyard manure in proportion, the control effect on eggplant fusarium wilt and banana fusarium wilt is very obvious. It measures the number of effective viable bacteria. The results are shown in Table 2.

Table 2HMD9204 powder and organic fertilizer mixed effective viable count detection

According to the test results of the effective viable count of HMD9204 inoculum mixed with organic fertilizer, the effective viable count of powder/organic fertilizer can reach 100 million cfu/g when the ratio of powder/organic fertilizer is above 1.5:1000.

The influence of embodiment 3HMD9204 powder and organic fertilizer mixed use on banana fusarium wilt etc.

This example was set in the Hainan Brazil banana field that had been continuously cropped for 2 years (the incidence rate in the previous year was about 40%).

The specific method is:

Control group: 50kg of base fertilizer compound fertilizer per mu, 10kg of topdressing compound fertilizer in flowering period, 10kg of urea, 10kg of topdressing compound fertilizer in fruiting period, and 20kg of potassium chloride;

Experimental group 1: 1.5 kg of powder + 0.5 ton of organic fertilizer per mu, 2 times, bottom application and top dressing at flowering period. 40kg of compound fertilizer at the bottom, 10kg of compound fertilizer in flowering period, 10kg of urea, 10kg of compound fertilizer in fruit period, and 20kg of potassium chloride;

Experimental group 2: 2.5 kg of powder + 0.75 tons of organic fertilizer per mu, 2 times, once at the bottom and once at the flowering stage. 40kg of compound fertilizer at the bottom, 10kg of compound fertilizer in flowering period, 10kg of urea, 10kg of compound fertilizer in fruit period, and 20kg of potassium chloride;

Experimental group 3: 5 kg of powder + 1.25 tons of organic fertilizer per mu, 2 times, bottom application and top dressing at flowering period. Apply 40kg of compound fertilizer at the bottom, 10kg of compound fertilizer in flowering period, 10kg of urea, 10kg of compound fertilizer in fruit period, and 20kg of potassium chloride.

Table 3 Influence of mixed use of HMD9204 powder and organic fertilizer on the incidence of bananas

The results show that: HMD9204 powder mixed with organic fertilizer can be applied to banana fusarium wilt and has good control and effect, improves soil acidity, increases soil organic matter content, and the incidence of banana fusarium wilt is reduced to 6.7-9.6%. Showed obvious effect, and the yield at least more than doubled.

The influence of embodiment 4HMD9204 powder and organic fertilizer mixed use on eggplant fusarium wilt etc.

This example was set up in the Hainan eggplant field experiment with continuous cropping for 2 years (the incidence rate in the previous year was about 50%).

The specific method is:

Control group: 40kg of compound fertilizer was applied at the bottom of each mu; 10kg of compound fertilizer and 5kg of urea were applied in the mid-term;

Experimental group 1: 3 kg of powder + 0.5 tons of organic fertilizer per mu at the bottom, 30 kg of compound fertilizer at the bottom; 10 kg of compound fertilizer and 5 kg of urea applied in the mid-term;

Experimental group 2: 5 kg of powder + 1.25 tons of organic fertilizer per mu, 30 kg of compound fertilizer at the bottom; 10 kg of compound fertilizer and 5 kg of urea applied in the mid-term;

Experimental group 3: 10 kg of powder + 2.5 tons of organic fertilizer per mu at the bottom, 30 kg of compound fertilizer at the bottom; 10 kg of compound fertilizer and 5 kg of urea applied in the mid-term.

Table 4 Effects of mixed use of HMD9204 powder and organic fertilizer on the incidence of eggplant

The results show that: HMD9204 powder mixed with organic fertilizer can be applied to eggplant fusarium wilt and has good control and effect, improve soil acidity, increase soil organic matter content, banana fusarium wilt incidence rate is reduced to 2-5%, The obvious effect is shown, and the yield is increased by about two times.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, and substitutions can be made in these embodiments without departing from the principle and spirit of the invention and modifications, the scope of the present invention is defined by the appended claims and their equivalents.

sequence listing

<110> Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences

<120> A kind of Bacillus saphobia HMD9204 and its bacterial agent and application

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<170> SIPOSequenceListing 1.0

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<212> DNA

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gggggggggg ctatacatgc agtcgagcgg acagaaggga gcttgctccc ggatgttagc 60

ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag actgggataa ctccgggaaa 120

ccggagctaa taccggatag ttccttgaac cgcatggttc aaggatgaaa gacggtttcg 180

gctgtcactt acagatggac ccgcggcgca ttagctagtt ggtggggtaa tggctcacca 240

aggcgacgat gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc 300

ccagactcct acgggaggca gcagtaggga atcttccgca atggacgaaa gtctgacgga 360

gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaag ctctgttgtt agggaagaac 420

aagtgcgaga gtaactgctc gcaccttgac ggtacctaac cagaaagcca cggctaacta 480

cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa 540

agggctcgca ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt 600

cattggaaac tgggaaactt gagtgcagaa gaggagagtg gaattccacg tgtagcggtg 660

aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg actctctggt ctgtaactga 720

cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt 780

aaacgatgag tgctaagtgt tagggggttt ccgcccctta gtgctgcagc taacgcatta 840

agcactccgc ctggggagta cggtcgcaag actgaaactc aaaggaattg acgggggccc 900

gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt 960

gacatcctct gacaacccta gagatagggc tttcccttcg gggacagagt gacaggtggt 1020

gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080

ccttgatctt agttgccagc attcagttgg gcactctaag gtgactgccg gtgacaaacc 1140

ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg 1200

ctacaatgga cagaacaaag ggctgcaaga ccgcaaggtt tagccaatcc cataaatctg 1260

ttctcagttc ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg ctagtaatcg 1320

cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1380

cgagagtttg caacacccga agtcggtgag gtaaccttta ggagccagcc gccggaaggg 1440

tggggc 1446

Claims (6)

1. a kind of Bacillus safensis ( Bacillus safensis ) HMD9204 for preventing and controlling soil-borne diseases, is characterized in that, this bacterial strain has been preserved in Guangdong Province Microorganism Culture Collection Center on December 18, 2017, preservation number: GDMCCNo .60302. 2 . A bacterial seed preparation for preventing and treating soil-borne diseases, characterized in that, the bacterial seed preparation comprises the powder of Bacillus safophila HMD9204 or its bacterial suspension or its culture solution according to claim 1 . 3 . 3 . The preparation according to claim 2 , wherein the preparation is mixed with organic fertilizer or farmyard manure at a mass ratio of 1.5 to 3:1000 when in use. 4 . 4. The preparation according to claim 3, characterized in that, after the preparation is mixed with organic fertilizer or farmyard manure, the number of effective viable bacteria is not less than 100 million cfu/g. 5. the application of bacillus saffordii HMD9204 according to claim 1 or its bacterial suspension or its culture solution in the prevention and treatment of fusarium wilt of banana and eggplant. 6. the application of the bacterial strain preparation described in claim 2 in the control of banana and eggplant fusarium wilt.