Bacillus aquimaris 2G-3 as well as fermentation product, fungicide and application of bacillus aquimaris 2G-3

Abstract

The invention discloses bacillus aquimaris 2G-3 with the preservation number of CGMCC No.32837, and relates to the technical field of microorganisms. The bacillus aquimaris 2G-3 has extremely strong prevention and treatment effects on root-knot nematodes regardless of fermentation bacteria liquid, fermentation supernate or volatile components generated by fermentation, and the fatality rate of the bacillus aquimaris 2G-3 on second-stage larvae can reach 100%; and the strain has a good growth promoting effect on the reticulated melons. The strain is applied to prevention and treatment of root knot nematode disease in agricultural production, and has the advantages of high efficiency, environmental protection and cost saving.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and more specifically, to a strain of Bacillus aquimaris 2G-3, its fermentation product, microbial agent and application. Background Art

[0002] Root-knot nematodes (Meloidogyne spp.) are obligate endoparasitic nematodes that can infect more than 3,000 plants including vegetables, fruit trees, grain and oil crops. The losses caused by them account for almost half of all agricultural losses. Root-knot nematode disease is the most important biological limiting factor in the production of protected vegetables. Under the cultivation conditions of protected vegetables, it only takes about 20 days for root-knot nematodes to complete one generation. Because of their huge reproductive quantity and rapid population growth, they are extremely likely to become rampant again, bringing great pressure to prevention and control. Root-knot nematodes damage the underground parts of plants, causing the roots of plants to swell and deform; they damage the vascular tissue of plants, resulting in abnormal water absorption by plants, and thus causing water loss, wilting and withering of the above-ground parts, and ultimately resulting in a decrease in yield.

[0003] At present, the application of chemical nematicides in production is still the main method for controlling nematodes. However, there are many disadvantages in chemical control of root-knot nematodes, such as the residues of chemical pesticides, the generation of nematode drug resistance and the re-rampancy of nematodes after use. With the enhancement of people's environmental protection awareness and the pursuit of a healthy life, some highly toxic and high-residue nematicides have been phased out and prohibited. As an important part of the sustainable management strategy of plant nematodes, biological control has attracted more and more attention due to its safety and environmental friendliness. Exploring plant endophytic bacteria with good control effects on root-knot nematodes is of great significance for the control of root-knot nematode disease in protected vegetables and the healthy development of the vegetable industry. Summary of the Invention

[0004] In view of this, the present invention provides a strain of Bacillus aquimaris 2G-3, its fermentation product, microbial agent and application.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] A strain of Bacillus aquimaris 2G-3, and the preservation number of the 2G-3 is CGMCC No. 32837.

[0007] Another object of the present invention is to provide a fermentation product obtained by fermenting the above-mentioned Bacillus aquimaris 2G-3.

[0008] Another object of the present invention is to provide a microbial agent including the above-mentioned Bacillus aquimaris 2G-3.

[0009] Another object of the present invention is to provide the application of the above-mentioned Bacillus marinus 2G-3, or the above-mentioned fermentation product, or the above-mentioned microbial inoculum in the control of root-knot nematodes.

[0010] Another object of the present invention is to provide the application of the above-mentioned Bacillus marinus 2G-3, or the above-mentioned fermentation product, or the above-mentioned microbial inoculum in the preparation of a product for controlling root-knot nematodes.

[0011] Another object of the present invention is to provide the application of the above-mentioned Bacillus marinus 2G-3, or the above-mentioned fermentation product, or the above-mentioned microbial inoculum in plant growth promotion.

[0012] Beneficial effects: The present invention discloses a strain of Bacillus marinus 2G-3, which has extremely strong control effect on root-knot nematodes. Whether it is the fermented bacterial liquid, the fermented supernatant or the volatile components produced by fermentation, the mortality rate of the second-stage larvae of root-knot nematodes can reach 100%; and this strain shows good growth promotion effect on netted melons. Applying this strain to agricultural production for controlling root-knot nematode disease has the advantages of high efficiency, environmental protection and cost saving. Description of the Drawings

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0014] Figure 1 It is the colony morphology of Bacillus marinus 2G-3.

[0015] Figure 2 It is the Gram staining result of Bacillus marinus 2G-3.

[0016] Figure 3 It is the phylogenetic tree of Bacillus marinus 2G-3. Detailed Embodiments

[0017] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of 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 belong to the scope of protection of the present invention.

[0018] Example 1 Isolation, Identification and Preservation of Strain 2G-3

[0019] 1. Isolation

[0020] Weigh 1 g of Orychophragmus violaceus leaves, disinfect them with 1% sodium hypochlorite for 5 min, rinse them five times with sterile water, place them in a sterile mortar and grind them thoroughly. Add 100 ml of sterile water and mix well. Use a spreading rod to evenly spread the dilution on the LB medium for the isolation of biocontrol bacteria. Seal the plate with a sealing film and then invert the plate and culture it in an incubator at 30 °C. Observe the growth of bacteria after culturing for 2 - 3 d, pick single colonies and purify them on the LB medium to obtain strain 2G-3.

[0021] 2. Identification

[0022] The plate streaking method was used to observe the morphological characteristics of strain 2G-3 and perform Gram staining on it. Preliminary identification was carried out with reference to "Bergey's Manual of Determinative Bacteriology" and others. Finally, the total DNA of the biocontrol bacteria was extracted, and gene fragment amplification was carried out using the 16S rRNA identification primers 27F / 1492R. After purification and recovery, it was sent to the company for sequencing. After the sequence was returned, the sequence identity comparison was carried out using the BLAST method to determine the bacterial species.

[0023] (1) Morphological observation and physiological and biochemical identification: Strain 2G-3 grew orange-yellow round colonies on the LB medium, which were sticky, with raised surfaces, opaque, and the peripheries of the colonies were neat, and there was no obvious change in the color of the medium (see Appendix Figure 1 ). After Gram staining, it showed purple and was a Gram-positive bacterium (see Appendix Figure 2 ). The catalase and citrate utilization reactions were positive, the nitrate reduction was positive, and it could hydrolyze starch and gelatin.

[0024] (2) Molecular identification: The total DNA of strain 2G-3 was extracted using the bacterial genomic DNA extraction kit TIANamp Bacteria DNA Kit. The 16S rDNA universal primers for bacteria 27F / 1492R (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′; 1492R: 5′-GGTTACCTTGTTACGACTT-3′) were used for PCR amplification. After the PCR amplification was completed, 1% agarose gel electrophoresis was used for detection. After passing the detection, bidirectional sequencing was carried out, and the 16S rDNA sequence of strain 2G-3 was obtained. High-matching sequences and species were obtained through the NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) database. A phylogenetic analysis was constructed based on the obtained sequences using MEGA7. The phylogenetic results showed (see Appendix Figure 3 ) that strain 2G-3 belonged to the same branch as Bacillus aquimaris. Therefore, it was identified as Bacillus aquimaris by combining the morphological and physiological and biochemical characteristics of the colonies and cells.

[0025] 3. Preservation

[0026] The strain 2G-3 was deposited on November 28, 2024 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101), classified and named as Bacillus aquimaris, and the deposit number is CGMCC No. 32837.

[0027] Example 2 Contact toxicity test of strain 2G-3 against second-stage larvae of Meloidogyne incognita

[0028] 1. Preparation of strain fermentation broth and fermentation supernatant

[0029] The preserved strain 2G-3 was streaked on an LB plate, sealed with a sealing film, and inverted in an incubator at 37°C for activation culture. After 1 day, a single colony was picked and transferred to a sterile Erlenmeyer flask containing LB liquid medium, and cultured at 37°C and 160 r until OD 600 reached 2.0 to obtain the fermentation broth. The fermentation broth was centrifuged at 9000 rmp for 5 min to collect the supernatant to obtain the fermentation supernatant. The obtained fermentation broth and fermentation supernatant were used for the subsequent contact toxicity test of second-stage larvae.

[0030] 2. Contact toxicity test against second-stage larvae of Meloidogyne incognita

[0031] 1 mL of the fermentation supernatant and fermentation broth were respectively added to a 24-well plate, and an equal amount of sterile water was used as a control. Approximately 100 to 200 second-stage root-knot nematodes were picked and placed into each well, and incubated at a constant temperature of 28°C. After culturing for 24 h respectively, the number of dead nematodes was counted and the corrected mortality rate of the nematodes was calculated. Each treatment was repeated biologically three times, and the experimental results were averaged.

[0032] Mortality rate = (number of dead nematodes / total number of test nematodes) × 100%

[0033] Corrected mortality rate = (mortality rate of treated nematodes - mortality rate of control nematodes) / (1 - mortality rate of control nematodes) × 100%

[0034] Table 1 Determination of contact toxicity effect of strain 2G-3 against second-stage larvae of Meloidogyne incognita

[0035]

[0036]

[0037] The test results are shown in Table 1. After the contact treatment with the fermentation broth and supernatant of strain 2G-3, the corrected mortality rate and mortality rate of nematodes were both 100%, which were significantly higher than those of the untreated control group. Therefore, strain 2G-3 has a good contact killing effect on Meloidogyne incognita.

[0038] 3. Fumigation effect on the second-stage larvae of Meloidogyne incognita

[0039] Add 1 mL of the fermentation broth and 1 mL of the second-stage larvae of Meloidogyne incognita (about 100) to both sides of the two-compartment plate respectively. An equal amount of LB is used as a control. Seal the gap of the two-compartment plate with a sealing film to prevent the volatilization of organic substances and affect the experimental results. Incubate the two-compartment plate at a constant temperature of 28 °C, and record the experimental results after 12 hours and 24 hours respectively to observe the toxic effect of the volatile organic substances of strain 2G-3 on Meloidogyne incognita. Each treatment is repeated biologically three times, and the results are averaged.

[0040] Table 2 Determination of the fumigation effect of strain 2G-3 on the second-stage larvae of Meloidogyne incognita

[0041]

[0042] The test results are shown in Table 2. After treatment with the volatile organic substances of the fermentation broth of strain 2G-3 for 12 h and 24 h, the corrected mortality rates of Meloidogyne incognita were 71.58% and 100% respectively. The experimental results show that the volatile organic substances produced by the fermentation of strain 2G-3 have an obvious fumigation effect on Meloidogyne incognita without direct contact with nematodes.

[0043] Example 3 Pot experiment to determine the effect of strain 2G-3 on netted melons

[0044] 1. Control effect on root-knot nematodes

[0045] Soak the netted melon seeds (Zhongkemi No. 6) in normal temperature water for 3 h, and then place them on a moist gauze for germination for 3 days. After the seeds germinate, transplant them into small flower pots. Conduct the control effect test after 4 true leaves grow. Prepare a suspension of Meloidogyne incognita at a concentration of 600 nematodes / mL, and evenly inoculate 1 mL of the nematode suspension into each flower pot. The experimental group (2G-3 treatment group) was irrigated with the 2G-3 bacterial fermentation broth with the final adjusted cell concentration of 10 9 CFU / mL, and 10 mL was irrigated into each flower pot in the treatment group. The negative control group (control treatment group) was irrigated with 10 mL of clear water at the roots of the netted melons, and the positive control group (abamectin treatment group) was irrigated with 5 mL of 1.8% abamectin (1:5000). There were 18 netted melons in each treatment. The experiment was set with 3 biological replicates.

[0046] Forty-two days after inoculation, the number of root knots in the roots of the treated group and the control group of netted melons was detected respectively, and the control effect of 2G-3 on root-knot nematodes was calculated.

[0047] Control effect (%) = (number of root knots per gram of roots in the negative control - number of root knots per gram of roots in the treatment group) × 100 / number of root knots per gram of roots in the negative control.

[0048] Table 3 Pot experiment on the control effect of root-knot nematodes

[0049]

[0050] The results are shown in Table 3. After treatment with the fermentation broth of strain 2G-3, the number of root knots in the roots of netted melons decreased significantly, and the control effect was 76.83%. There was no significant difference from the result of abamectin treatment, indicating that Bacillus aquimaris 2G-3 can effectively control root-knot nematodes in netted melons and can be used as a substitute for abamectin. Since strain 2G-3 is an endophytic bacterium of plants, it can quickly colonize in the plant roots after application, and has the advantages of high efficiency, environmental protection and cost saving in the control of root-knot nematode disease in agricultural production.

[0051] 2. Growth promotion effect

[0052] On the 35th day after inoculation with root-knot nematodes and the fermentation broth of 2G-3, the plant height, above-ground fresh weight, underground root length and underground fresh weight of the above-ground part of the netted melons were measured respectively. The results are shown in Table 4. It was found during the investigation that the netted melons treated with the fermentation broth of strain 2G-3 had significant increases in both plant height, above-ground fresh weight, underground root length and underground fresh weight compared with the control (clean water). This indicates that Bacillus aquimaris 2G-3 strain has an obvious growth promotion effect on netted melons.

[0053] Table 4 Growth promotion effect of strain 2G-3 on netted melons

[0054]

[0055] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. For the same or similar parts among the embodiments, reference can be made to each other.

[0056] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Bacillus aquimaris 2G-3, characterized in that: The deposit number of 2G-3 is CGMCC No.32837.

2. A fermentation product, characterized in that Obtained by fermentation of the marine Bacillus 2G-3 described in claim 1.

3. A microbial agent, characterized in that: Including the marine Bacillus 2G-3 described in claim 1.

4. Use of the marine Bacillus 2G-3 according to claim 1, the fermentation product according to claim 2, or the microbial agent according to claim 3 in controlling root-knot nematodes.

5. Use of the marine Bacillus 2G-3 according to claim 1, the fermentation product according to claim 2, or the microbial agent according to claim 3 in the preparation of products for controlling root-knot nematodes.

6. Use of the marine Bacillus 2G-3 according to claim 1, the fermentation product according to claim 2, or the microbial agent according to claim 3 in promoting plant growth.

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