Endogenous Bacillus Velezii of Suaeda salsa and application thereof

Abstract

The present invention discloses an endophytic Bacillus Velez subtilis of Suaeda salsa and its application, and belongs to the field of microbial technology. The strain of the present invention was deposited in the China Center for Type Culture Collection on August 15, 2022, with a deposit number of CCTCC NO: M20221277. The Bacillus Velez subtilis of the present invention has good tolerance to chemical fungicides and salts, can significantly reduce the salt-alkali content in the soil, and has a preventive and therapeutic effect on pathogens such as gray mold, peanut white rot, tomato wilt, cucumber wilt, and strawberry root rot. Therefore, the Bacillus Velez subtilis can be prepared into a microbial preparation, which can be used in the fields of pathogen control, saline-alkali land improvement, and plant growth promotion, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to an endophytic Velez subspore of Suaeda salsa and application thereof. Background Art

[0002] The pathogen of grape gray mold is Botrytis cinerea, which can infect grape inflorescence, leaves and young fruits, and even endanger mature fruits, thereby endangering the entire industrial chain of fruits and vegetables such as grapes from planting to fruit storage, causing significant economic losses. Peanut is the second largest oil crop in my country except rapeseed. Peanut white rot caused by Sclerotium rolfiiSacc. is one of the main diseases in my country's peanut industry, which can endanger the base of peanut stems, fruit stalks, pods, seeds and roots, thereby causing large-scale peanut production reduction and quality degradation. The sclerotia produced by the fungus can survive in the soil for many years, causing soil-borne pollution and prevention and control difficulties, seriously restricting the increase in production and income of oil crops such as peanuts. Chemical agents are used for prevention and control, such as cypermethrin, oxadiazine and thiophanate-methyl, which have caused the average frequency of resistance to gray mold and peanut white rot to exceed 70%. Compared with chemical fungicides, finding potential strains for preventing and controlling plant diseases from microorganisms has become one of the important directions for the development of green pesticides in the future. Moreover, microbial fungicides have the advantages of good degradability, high safety, and low resistance to drug resistance, which are of great significance to the development of environmentally friendly ecological agriculture. Summary of the invention

[0003] The technical solution of the present invention is as follows:

[0004] The present invention provides an endophytic Bacillus Velezii G-3-1 of Suaeda salsa, which is isolated from the root tissue of Suaeda salsa collected from the Yellow River estuary wetland in Dongying City, and was deposited in the China Center for Type Culture Collection on August 15, 2022, with a preservation number of CCTCC NO:M 20221277.

[0005] The above-mentioned Bacillus Velez has been proven to have plant disease control effects, especially for gray mold, peanut white rot, tomato wilt, cucumber wilt and strawberry root rot and plant diseases caused by them, it has a significant control effect. To this end, the present invention provides the use of the above-mentioned Bacillus Velez in the control of gray mold, peanut white rot, tomato wilt, cucumber wilt or strawberry root rot and / or plant diseases caused by them; wherein, in this application, the control purpose can be diagnostic or non-therapeutic. Specifically, the present invention provides the use of the above-mentioned Bacillus Velez in the preparation of microbial preparations for the control of gray mold, peanut white rot, tomato wilt, cucumber wilt or strawberry root rot and / or plant diseases caused by them.

[0006] The above-mentioned Bacillus Velez has good salt tolerance and can significantly reduce the salt-alkali content in the soil. On this basis, the present invention provides the use of the above-mentioned Bacillus Velez in reducing the salt-alkali content in the soil.

[0007] The present invention further provides application of the above-mentioned Bacillus Velez in improving saline-alkali land.

[0008] The present invention further provides the use of the above-mentioned Bacillus Velez in promoting the growth of plants in saline-alkali land.

[0009] The above-mentioned Bacillus Velez subsp. has been shown to have good tolerance to chemical fungicides. Based on this, the present invention provides the use of the above-mentioned Bacillus Velez subsp. in an environment where chemical fungicides are applied; the application includes but is not limited to the following situations: (1) when applying chemical fungicides, the above-mentioned Bacillus Velez subsp. is applied to improve the soil microenvironment and prevent bacterial imbalance, which may cause soil compaction and other problems; (2) used together with chemical fungicides to prevent and control the above-mentioned gray mold, peanut white rot, tomato wilt, cucumber wilt or strawberry root rot.

[0010] The above-mentioned chemical fungicide is selected from one or more of cypermethrin, mefenamic acid and thiophanate-methyl.

[0011] The present invention provides a microbial preparation comprising the above-mentioned Bacillus Velezii, which can be used to prevent and control gray mold, peanut sclerotium, tomato wilt, cucumber wilt or strawberry root rot and / or plant diseases caused by them.

[0012] The above-mentioned microbial preparations include but are not limited to solid preparations or liquid preparations, such as powders or suspensions. If it is a suspension, the bacterial content of the Velez subtilis is ≥10 8 CFU / mL.

[0013] The present invention provides a fermentation culture of the above-mentioned Bacillus Velezii.

[0014] The method for preparing the above fermentation culture comprises the following steps:

[0015] The above-mentioned Bacillus Velez subtilis was fermented in an LB medium shaker for 2 to 3 days to obtain a fermentation culture; the LB medium was selected from the following components: 1% tryptone, 0.5% yeast extract, 1% NaCl, distilled water, and the pH was adjusted to 7.0.

[0016] In the fermentation culture, the bacterial content of the Bacillus Velez is ≥10 8 CFU / mL.

[0017] The beneficial effects of the present invention are:

[0018] The Velez Bacillus of the present invention has good tolerance to chemical fungicides and salts, can significantly reduce the salt-alkali content in the soil, and has a preventive effect on pathogens such as gray mold, peanut white rot, tomato wilt, cucumber wilt, and strawberry root rot. Therefore, the Velez Bacillus can be prepared into a microbial preparation and applied to the fields of pathogen control, saline-alkali land improvement, and plant growth promotion, with broad application prospects. In addition, the Velez Bacillus of the present invention can obtain fermentation products through short-term shaking culture; during the fermentation process, a large amount of fermentation products can be obtained without adjusting the pH value and supplementing the feed, which reduces the energy consumption during the fermentation process; the fermentation products do not need to be extracted and separated, and can be directly diluted for plant disease control or salinized soil improvement, with low production costs and no pollution to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the colony morphology of strain G-3-1;

[0020] Figure 2 Phylogenetic tree constructed based on 16S rDNA gene for strain G-3-1;

[0021] Figure 3 is the tolerance of strain G-3-1 to chemical fungicides and sea salt;

[0022] Figure 4 The antagonistic effect of strain G-3-1 on the target pathogen;

[0023] Figure 5 The control effect of the fermentation liquid of strain G-3-1 on peanut white rot;

[0024] Figure 6 The salt-alkali improvement effect and peanut growth promotion effect of strain G-3-1;

[0025] Figure 7 The growth-promoting effect of strain G-3-1 on Chinese cabbage, radish and strawberry seedlings;

[0026] Figure 8 The biofilm formed by strain G-3-1 in LB medium. DETAILED DESCRIPTION

[0027] Bacterial isolation and identification:

[0028] The strain is separated from the root tissue of Suaeda salsa collected from the wetland at the Yellow River estuary in Dongying City, and is obtained by applying a tissue separation method. The specific steps are as follows: the root tissue of Suaeda salsa is rinsed with tap water, dried, and cut into small pieces with a length of 5 cm; the root tissue is soaked in 75% ethanol for 2 minutes, rinsed with sterile water for 3 to 4 times, soaked in 4% sodium hypochlorite for 3 minutes, and rinsed with sterile water for 3 to 4 times; the root tissue is removed at both ends with sterile surgical scissors, cut into small pieces with a length of 0.5 cm, and implanted in an LB agar plate, 4 to 5 pieces are implanted in each dish, and inverted for culture at 28° C.; colonies of different forms are picked, and streaked on an LB agar plate containing 3% sea salt for separation, and the growth of the colonies is observed regularly; the plate dilution streaking method is used to further separate and purify the strain, and the strain is stored for standby use, and the strain is numbered G-3-1.

[0029] The strain G-3-1 was inoculated onto LB agar plates and cultured at 28°C for 2 days. Figure 1 As shown, the colony morphology of strain G-3-1 is: the colony surface is rough and opaque, the colony center has ridges and wrinkles, and is light yellow.

[0030] The results of 16S rDNA gene sequencing of the strain G-3-1 are as follows:

[0031]

[0032]

[0033] The sequence information was compared with the corresponding sequence information of known strains in the Genbank database, and a phylogenetic tree was constructed, such as Figure 2 As shown, the strain has the highest homology with the strain Bacillus velezensis FZB42 (Accession number: NR075005.2), which is 99.86%, and is thus identified as Bacillus velezensis. The strain was deposited in the China Center for Type Culture Collection on August 15, 2022, with the deposit number CCTCCNO: M 20221277.

[0034] Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0035] Example 1

[0036] Chemical fungicide and sea salt tolerance test:

[0037] Commercially available thiophanate-methyl, oxazolidinone and chlorpyrifos (main active ingredient: copper hydroxide) were selected as test chemical reagents. The specific test steps are as follows:

[0038] Thifluzamide, oxazolidinone, and chlorpyrifos were mixed with 45°C PDA medium, and 1, 10, and 20 times the dosage in the instructions were prepared into poisonous medium, wherein the concentrations of thifuramide were 0.16 g / L, 1.6 g / L, and 3.2 g / L, the concentrations of oxazolidinone were 0.32 g / L, 3.2 g / L, and 6.4 g / L, and the concentrations of chlorpyrifos were 0.3 g / L, 3 g / L, and 6 g / L, respectively; each culture medium was poured onto a 9 cm four-divided culture dish; strain G-3-1 was streaked on the plate and cultured at 28°C for 48 hours. The growth of the strain was observed (Chinese Journal of Biological Control, 2022, 38(03): 739-747).

[0039] LB agar medium with sea salt content of 1%, 5%, 10% and 15% was poured onto a 9 cm quadrant plate; strain G-3-1 was streaked on the plate and cultured at 28°C for 48 h. The growth of the strain was observed (Chinese Journal of Biological Control, 2022, 38(03):739-747).

[0040] The test results are as follows Figure 3 As shown:

[0041] Strain G-3-1 can grow at salinity of 1-15%; when the salt concentration reaches 5%, strain G-3-1 grows fastest, which shows that strain G-3-1 is a moderately salt-tolerant bacterium. Strain G-3-1 can grow in different concentrations of chemical fungicides, and it can still grow at a dose 20 times the field dose, indicating that strain G-3-1 has good tolerance to chemical fungicides thiophanate-methyl, oxadiazine and chlorpyrifos. Therefore, strain G-3-1 is suitable for the comprehensive prevention and control of diseases in salinized soil or protected land.

[0042] Example 2

[0043] Pathogen antagonism test:

[0044] The plate confrontation method was used to determine the in vitro antibacterial activity. The test strains were as follows: Botrytis cinerea, Fussarium oxysporumf.sp.Lycopersici Snyder et Hansen, and Strawberry root rot

[0045] (Fusarium oxysporium Schlecht), cucumber wilt (Fusarinm oxysporun), peanut white rot (Sclerotium rolfsii Sacc.), the above plant pathogens were isolated and preserved by the Green Pesticide Creation and Evaluation Laboratory of Qingdao Agricultural University.

[0046] The specific experimental steps are as follows:

[0047] The above-mentioned plant pathogenic fungi were activated and inoculated onto PDA plates and cultured at 25-28°C for 5 days; a 5-mm-diameter cake was punched along the edge of the colony, and the pathogenic fungus was placed in the center of a 9-cm-diameter PDA plate; the strain G-3-1 was inoculated on both sides of the center at an equal distance and cultured at 25-28°C; the pathogenic fungus was inoculated alone as a control (CK); when the control mycelium grew to almost cover the plate, the colony diameter and inhibition zone were measured, and the inhibition rate was calculated (BIOLOGICAL CONTROL, 2021, 10.1016), inhibition rate (%) = (R1-R2) / R1×100%; wherein R1 is the colony radius of the blank control; R2 is the colony radius on the opposite side of the treated group strain.

[0048] The test results are shown in Table 1 and Figure 4 As shown:

[0049] Table 1

[0050]

[0051] The results of plate confrontation showed that strain G-3-1 had a significant antagonistic effect on the mycelial growth of the five tested pathogenic fungi, with inhibition rates ranging from 54.3% to 65.7% and inhibition zone widths ranging from 3 to 9 mm.

[0052] Example 3

[0053] Antibacterial application test:

[0054] The specific experimental steps are as follows: strain G-3-1 was inoculated into LB medium, cultured in a shaking incubator at 28°C and 180 rpm for 48 h, and the fermentation broth was diluted to a bacterial concentration of 10 8CFU / mL, the bacterial solution is reserved. Take the above bacterial solution and add it to the peanut potted plants that have been cultured for 14 days, 200mL / pot, use carbendazim and Bacillus subtilis as positive controls, LB medium as negative control, 5 pots for each treatment group, humidity is 90%, and culture in a greenhouse at 30℃; after 24h, inoculate toothpicks with peanut white rot fungus (5 pieces / pot) at the base of the peanut stem, and continue to culture in the greenhouse; count the incidence of the disease at 7d, 14d, and 21d after inoculation of the pathogen, and calculate the prevention effect (Henan Agricultural Science, 2022, 51(02):97-103).

[0055] Peanut disease index: Level 0: no symptoms; Level 1: less than 1 / 4 of the leaves of the plant show wilting symptoms, the base of the stem is asymptomatic, and the plant grows normally; Level 2: 1 / 4-1 / 2 of the leaves of the plant show wilting symptoms, browning occurs below 1 / 2 of the stem base, and the plant is dwarfed; Level 3: more than 1 / 2 of the leaves of the plant show wilting symptoms, browning occurs above 1 / 2 of the stem base, and the plant is obviously dwarfed; Level 4: The whole plant wilts and dies.

[0056] Prevention effect (%) = ∑ (disease index × number of diseased plants) / (highest disease index × total number of plants) × 100%;

[0057] The test results are shown in Table 2 and Figure 5 As shown:

[0058] Table 2

[0059]

[0060] The fermented dilution of strain G-3-1 has a significant protective effect against peanut white rot, and the protective effect reaches 100% 21 days after inoculation, which is significantly higher than Bacillus subtilis and carbendazim, indicating that the fermented liquid of strain G-3-1 has a growth-promoting effect on peanut plants.

[0061] Example 4

[0062] Salt-alkali improvement and plant growth promotion experiment:

[0063] The strain G-3-1 was inoculated into LB medium and cultured in a shaking incubator at 28°C and 180 rpm for 48 h. The fermentation broth was diluted to a bacterial concentration of 10 8 CFU / mL, the bacterial solution was reserved; peanuts were planted in saline soil (salinity of 4.4g / kg, pH=8.06) for 14 days, with 2 peanuts in each pot; when the potted peanuts were fully grown, the above 200mL bacterial solution was taken for root irrigation; Bacillus subtilis inoculant was used as the positive control, LB medium was used as the negative control, and every five pots were a treatment group, the humidity was 90%, and the culture was carried out in a greenhouse at 30℃; the soil salinity was measured at 7d, 14d, and 21d respectively; and the dry weight and wet weight of the peanut plants were counted after 21d (Henan Agricultural Science, 2022, 51(02):97-103).

[0064] The experimental steps for determining soil salinity are as follows:

[0065] Prepare 0.01mol / L, 0.05mol / L, 0.10mol / L, 0.15mol / L and 0.20mol / L NaCl standard solutions; use a conductivity meter to measure the conductivity of the NaCl standard solutions and draw a standard salinity-conductivity standard curve; measure the conductivity value of the soil leachate, calculate the NaCl concentration of the solution according to the standard curve, and convert the soil salinity.

[0066] The test results are as follows Figure 6 and Figure 7 As shown:

[0067] After treatment with the fermentation liquid of strain G-3-1, the soil salt content decreased from 4.4 g / kg to 2 g / kg and the pH decreased from pH = 8.06 to pH = 7.26 after 21 days. Figure 6 As shown in C, this indicates that strain G-3-1 can significantly reduce soil salt content and soil pH value. It can reduce soil salt content by more than 50%, and the effect is very significant.

[0068] The dry weight and wet weight of peanut plants treated with the fermentation liquid of strain G-3-1 were significantly higher than those of the negative control (CK) and the positive control Bacillus subtilis inoculant ( Figure 6 B, the number of peanut fibrous roots in the treatment group was significantly higher than that in the control group ( Figure 6 A), which shows that strain G-3-1 can not only improve saline-alkali land, but also promote the growth of peanut plants in saline-alkali environment. Moreover, under the same experimental conditions, strain G-3-1 can also significantly promote the growth of Chinese cabbage, radish and strawberry seedlings in saline soil. Figure 7 This indicates that the strain is helpful to increase the yield and income of cash crops in saline-alkali land.

[0069] Example 5

[0070] Biofilm formation test:

[0071] The strain G-3-1 was inoculated into LB medium and cultured at 28°C for 24 h; 9 mL of bacterial solution was aspirated and placed in a 10 mL centrifuge tube and cultured at 28°C for 24 h; the culture solution was discarded, the centrifuge tube was washed with deionized water, and stained with 1% crystal violet for 2 min, the crystal violet solution was discarded, and the tube was washed with water to observe the biofilm formation.

[0072] The grading standards for biofilm formation are as follows (China Plant Protection Guide, 2022, 42(07):10-15): Grade 0: No biofilm is formed, and no purple ring is found after staining; Grade 1: A biofilm is initially formed, and a purple ring is vaguely visible after staining; Grade 2: A more obvious biofilm is formed, and a purple ring can be seen after staining; Grade 3: A more obvious biofilm is formed, and a clearer purple ring is seen after staining; Grade 4: An obvious biofilm is formed, and a very clear purple ring can be seen after crystal violet staining.

[0073] The test results are as follows Figure 8 As shown:

[0074] Strain G-3-1 can form a very clear purple ring, reaching the 4th level standard of biofilm; the production of biofilm is beneficial to improving the tolerance of strain G-3-1, and the formation of biofilm is beneficial to the colonization of strain G-3-1 in plant roots or salinized soil.

[0075] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A Bacillus velezensis, characterized in that: The strain was deposited in the China Center for Type Culture Collection on August 15, 2022, with the deposit number being CCTCC NO:M 20221277.

2. Use of the Bacillus Velezii described in claim 1 in preventing and controlling Botrytis cinerea, Sclerotium rolfsii, Fusarium wilt of tomato, Fusarium wilt of cucumber or strawberry root rot and / or plant diseases caused by them.

3. Use of the Bacillus Velez subtilis according to claim 1 in reducing the salinity and alkali content in soil.

4. Use of the Bacillus Velez subtilis described in claim 1 in improving saline-alkali land.

5. Use of the Bacillus Velez subtilis according to claim 1 in promoting the growth of plants in saline-alkali land.

6. Use of the Bacillus Velezii according to claim 1 in an environment where chemical fungicides are applied; the chemical fungicides are selected from one or more of cypermethrin, mefenamic acid and thiophanate-methyl.

7. A microbial preparation, characterized in that: Contains the Bacillus Velezii according to claim 1.

8. The fermentation culture of Bacillus Velezii according to claim 1.

9. The method for preparing the fermentation culture according to claim 8, characterized in that: The steps are as follows: fermenting the Bacillus Velez subtilis described in claim 1 in an LB medium shaker for 2 to 3 days to obtain a fermentation culture; the LB medium is composed of the following components: 1% tryptone, 0.5% yeast extract, 1% NaCl, distilled water, and the pH is adjusted to 7.0.

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