Saline-alkaline tolerant bacillus velezensis capable of colonizing trichosanthes kirilowii maxim and application of bacillus velezensis

Abstract

The invention belongs to the technical field of crop control, and particularly relates to saline-alkaline tolerant bacillus velezensis capable of colonizing trichosanthes kirilowii maxim and application of the bacillus velezensis. The bacillus velezensis is bacillus velezensis MH24, the preservation number of the bacillus velezensis MH24 is CCTCC (China Center For Type Culture Collection) NO: M20221680, and the bacillus velezensis MH24 can be used for preventing and controlling diseases such as trichosanthes kirilowii maxim anthracnose, bacterial angular leaf spot, virus diseases, root rot and root-knot nematode. And the composition has no cross resistance with existing bactericides and insecticides. The strain has saline-alkaline tolerance and can be used for prevention and control and yield increase of soil-borne diseases of trichosanthes kirilowii maxim in salinized soil. Compared with the existing chemical synthetic pesticide for prevention and treatment, the compound is from the natural environment, has the advantages of high efficiency, low toxicity, no environmental pollution, low possibility of generating drug resistance and the like, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of crop prevention and control, and particularly relates to a salt-tolerant Velez subtilis strain and application thereof. Background Art

[0002] Trichosanthes kirilowii Maxim, also known as hanging or trichosanthes, is a perennial climbing herb of the Cucurbitaceae family, the genus Trichosanthes, which can grow up to 10 meters long. Its fruit, seed coat, seeds, and roots can all be used as medicine, with medicinal, edible, and health functions. Anthracnose, bacterial angular leaf spot, viral disease, root rot, and root-knot nematodes are the main soil-borne diseases that seriously restrict the production of Trichosanthes kirilowii, and the damage becomes more serious with the increase of the planting years of Trichosanthes kirilowii.

[0003] There are currently no registered fungicides for the above diseases in production, and chemical fungicides are usually used for prevention and control. The frequent use of chemical fungicides causes a series of problems such as pesticide residues, drug resistance and environmental pollution, and also destroys the structure of rhizosphere soil microbial communities. The prevention and control of Trichosanthes kirilowii diseases urgently needs safer and more effective prevention and control methods. As a new disease prevention and control measure, biological control not only has the effect of preventing diseases and promoting growth of plants, but also can effectively overcome the problems of pesticide residues and pathogen resistance in production. In addition, microbial agents have the function of regulating the structure of plant rhizosphere microbial communities.

[0004] Microorganisms need to colonize and reproduce effectively in the soil to function. In recent years, the application of chemical fertilizers has caused soil salinization. Excessive salt content in the soil is not conducive to the colonization and reproduction of microorganisms in the soil. In addition, microorganisms have certain host transformation properties, and their colonization ability in the rhizosphere of different hosts varies greatly.

[0005] In view of the above problems, the present invention isolates a new Bacillus velezensis MH24, which has good preventive effects on anthracnose, bacterial angular spot, viral disease, root rot and root-knot nematodes of Trichosanthes kirilowii, has good colonization ability in the root system of Trichosanthes kirilowii, and has certain salt and alkali resistance, and has certain application prospects. Summary of the invention

[0006] The invention aims to provide an isolated Bacillus Velez MH24, wherein the deposit number of the Bacillus Velez is CCTCC NO: M20221680.

[0007] Another object of the present invention is to provide the use of the above-mentioned Bacillus Velez MH24 in the preparation of a biocontrol agent for Trichosanthes kirilowii. The Bacillus Velez MH24 of the present invention can be used for the prevention and treatment of diseases such as anthracnose, bacterial angular spot, viral disease, root rot, and root-knot nematodes of Trichosanthes kirilowii.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A Bacillus isolated from the rhizosphere of Trichosanthes kirilowii was identified as Bacillus velezensis. The strain was sent to the China Center for Type Culture Collection for preservation on October 28, 2022, and the classification name is: Bacillus velezensis MH24; the preservation number is CCTCC NO: M20221680; location: Wuhan University, Wuhan, China.

[0010] The strain grew well on NA medium, with pale yellow single colonies ( Figure 1 ).

[0011] The protection scope of the present invention also protects:

[0012] The fermentation product of Bacillus Velez MH24 contains live bacteria of Bacillus Velez MH24.

[0013] A composition containing Bacillus Velez MH24 or a fermentation product thereof.

[0014] Use of Bacillus Velez MH24, a fermentation product of Bacillus Velez MH24 or a composition containing Bacillus Velez MH24 or a fermentation product thereof in the prevention and treatment of diseases of Trichosanthes kirilowii;

[0015] In the above application, preferably, the diseases include trichosanthes anthracnose, bacterial angular leaf spot, viral disease, root rot and / or root-knot nematode disease.

[0016] The above-mentioned application, preferably, the control method of the application comprises: using Bacillus Velez MH24, the fermentation product of Bacillus Velez MH24 or a composition containing Bacillus Velez MH24 or its fermentation product to irrigate the roots of Trichosanthes kirilowii or directly spray it.

[0017] Use of Bacillus Velez MH24, a fermentation product of Bacillus Velez MH24 or a composition containing Bacillus Velez MH24 or its fermentation product in the preparation of a biological control drug for Trichosanthes kirilowii;

[0018] In the above-mentioned application, preferably, the control target of the biocontrol drug in the application is biological diseases caused by Colletotrichum orbiculare, Fusarium oxysporum, Pseudomonas syringae, and / or root-knot nematodes.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] (1) The present invention provides a Velez subtilis strain, which is reported for the first time to be useful for simultaneously controlling anthracnose, bacterial angular spot, viral diseases, root rot and root-knot nematodes.

[0021] (2) The Bacillus Velezii MH24 of the present invention is isolated from the rhizosphere of Trichosanthes kirilowii and can colonize in Trichosanthes kirilowii, avoiding frequent root irrigation. Therefore, it can be used as a long-term biocontrol agent and has great promotion value.

[0022] (3) The Bacillus Velezii of the present invention has good tolerance in saline-alkali environments and has good colonization ability in salinized soils, and has no cross-resistance with existing fungicides and insecticides.

[0023] (4) Compared with the currently used chemical synthetic pesticides, since it comes from the natural environment, it has the advantages of high efficiency, low toxicity, no pollution to the environment, and is not easy to produce drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the colony morphology of Bacillus velez MH24.

[0025] Figure 2 Schematic diagram of the in vitro antibacterial activity of Bacillus velez MH24.

[0026] Figure 3 Schematic diagram of salt tolerance of Bacillus velez MH24.

[0027] Figure 4 Schematic diagram of the alkali resistance of Bacillus velez MH24.

[0028] Figure 5 Field effect of Bacillus Velez MH24 in controlling Trichosanthes root rot. DETAILED DESCRIPTION

[0029] In order to better explain the present invention, the main contents of the present invention are further illustrated below in conjunction with specific examples, but the contents of the present invention are not limited to the following examples. The technical solutions of the present invention, if not otherwise specified, are conventional techniques in the art, and the reagents or materials, if not otherwise specified, are all derived from commercial channels.

[0030] Embodiment 1:

[0031] Acquisition and Identification of Bacillus Velezii MH24

[0032] A strain of Bacillus isolated from the rhizosphere soil of Trichosanthes kirilowii was identified by molecular biology (whole genome sequencing) combined with physiological and biochemical identification and was identified as Bacillus velezensis ( Figure 1, Table 1, Table 2), the strain was sent to the China Center for Type Culture Collection for preservation on October 28, 2022, and the classification name is: Bacillus velezensis MH24; the preservation number is CCTCC NO: M20221680; location: Wuhan University, Wuhan, China.

[0033] In the present invention, Bacillus velezensis MH24 or simply referred to as MH24.

[0034] Table 1: Physiological and biochemical characteristics of strain MH24 - enzyme activity, carbon source oxidation

[0035]

[0036]

[0037] +: positive reaction; -: negative reaction;

[0038] Table 2: Physiological and biochemical characteristics of strain MH24 - Acid production using carbon sources

[0039]

[0040]

[0041] +: positive reaction; -: negative reaction; W: weak positive reaction

[0042] Embodiment 2:

[0043] In vitro antibacterial activity of Bacillus velezensis MH24 against pathogens of Trichosanthes kirilowii diseases

[0044] Activate the pathogens of the disease to be tested from the slant to the PDA plate and culture at 30℃ for 6 days. Transfer the MH24 single colony to the NB medium and culture it on a shaker at 35℃ and 180rpm for 12 hours. Drop 10μL of the fermentation liquid on both ends of the PDA plate. After 24 hours, use a 4mm hole puncher to access the above pathogenic bacteria plate. The culture medium bacteria plate is used as a blank control. The distance between the pathogen and the MH24 bacteria plate is 25mm. After the control plate is full of growth, the diameters of the colonies of each treatment and the control are counted, and the inhibition rate is calculated.

[0045] Inhibition rate = [(control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter)] × 100%

[0046] Pseudomonas syringae was streaked from the glycerol tube to obtain a single colony, and then transferred to NB liquid culture medium and cultured at 37℃ at 180r / min for 48h before use. NA culture medium was melted and poured into the plate at about 45℃, and then the Ralstonia culture solution was added, with the ratio of NA to the culture solution being 50:1, and mixed thoroughly. Then 10μL of MH24 fermentation solution was dripped into the plate, with 3 spots on each plate, and cultured at 37℃ for 48h to observe whether an inhibition zone was produced. The results are as follows: Figure 2 As shown, there is an obvious inhibition zone.

[0047] The in vitro antibacterial test showed that MH24 had good antibacterial activity against pathogens of Trichosanthes kirilowii, such as Colletotrichum cucurbitae, Fusarium oxysporum, and Pseudomonas syringae. Figure 2 ). The antibacterial rates against cucurbit anthracnose and Fusarium oxysporum reached 79.35% and 78.62% respectively.

[0048] Example 3: Control of Trichosanthes anthracnose by Bacillus Velezii MH24

[0049] After activation at 30°C, the MH24 slant strain was inoculated into a loop of LB liquid medium and cultured for 16 hours at 30°C and 180 r / min as seeds for shake flask fermentation. The seeds were inoculated into the shake flask fermentation medium at a 1% (v / v) inoculation rate and cultured at 30°C and 180 r / min until 20% of the spores fell off, and the fermentation liquid spore count was 10 billion cfu / mL, which was used in the following examples.

[0050] The composition of the fermentation medium is as follows: sweet potato powder 11 g / L, fish meal 12 g / L, yeast powder 14 g / L, soybean meal powder 6 g / L, magnesium chloride 0.5 g / L, pH 7.

[0051] The experiment was conducted in a greenhouse for the cultivation of Trichosanthes kirilowii in Xinzhou, Wuhan. Before the onset of anthrax on May 10, 2022, MH24 was diluted 200 times and sprayed on the leaves. The leaves were sprayed evenly on both sides until the leaves were saturated, and the second spraying was performed after an interval of 10 days. The control was irrigated with clean water, and each treatment was repeated 3 times, with 10 plants in each repeat. On June 20, after the control was fully infected, 4 points were randomly selected in each plot for investigation, 2 plants were checked at each point, and all leaves of each plant were investigated. The number of diseased leaves at each level was recorded, and the disease index and control effect were calculated.

[0052] The grading standard for Trichosanthes anthracnose is as follows: Grade 0, no lesions; Grade 1, lesion area accounts for less than 5% of the total leaf area; Grade 3, lesion area accounts for 6% to 10% of the total leaf area; Grade 5, lesion area accounts for 11% to 25% of the total leaf area; Grade 7, lesion area accounts for 26% to 50% of the total leaf area; Grade 9, lesion area accounts for more than 50% of the total leaf area.

[0053] Disease index = ∑ (number of plants at each level × level) / (total number of plants surveyed × highest representative level) × 100.

[0054] Control effect = (control disease index - treatment disease index) / control disease index × 100%

[0055] Small-scale field trials showed that MH24 had a good control effect on anthrax, with a control efficiency of 63.42% (Table 3).

[0056] Table 3: Field efficacy of MH24 fungicide against anthracnose of Trichosanthes kirilowii

[0057] deal with Disease index Prevention effect (%) MH24 12.14 63.42 Comparison 33.19 -

[0058] Embodiment 4:

[0059] Control of Trichosanthes kirilowii root rot with Bacillus Velezii MH24

[0060] The experiment was conducted in a greenhouse for the cultivation of Trichosanthes kirilowii in Xinzhou, Wuhan. Before the onset of Trichosanthes kirilowii wilt on June 10, 2022, the MH24 bacterial agent prepared in Example 3 was diluted 200 times and irrigated the roots, 400 mL / tree, and the second root irrigation was performed at an interval of 10 days. The control was irrigated with clear water, and each treatment was repeated 3 times, with 12 plants in each repetition. On July 30, after the control was fully infected, the disease index of the treatment and control was statistically calculated, and the control effect was calculated.

[0061] The severity grading standard of cucumber wilt disease is as follows: Level 0, no symptoms; Level 1, mild symptoms on stems and leaves; Level 2, mild wilt of plants, necrotic spots on stems, and yellowing of leaves; Level 3, moderate wilt of plants, drooping and yellowing of leaves; Level 4, severe wilt of plants, lodging and death.

[0062] Disease index = ∑ (number of plants at each level × level) / (total number of plants surveyed × highest representative level) × 100.

[0063] Control effect = (control disease index - treatment disease index) / control disease index × 100%

[0064] Field efficacy tests showed that MH24 had a good control effect on Trichosanthes anthracnose, with a control rate of 87.57% (Table 4, Figure 5 ).

[0065] Table 4: Field efficacy of MH24 fungicide against Trichosanthes root rot

[0066] deal with Disease index Prevention effect (%) MH24 9.23 87.57 Comparison 74.28 -

[0067] Example 5: Control of Trichosanthes kirilowii bacterial angular leaf spot by Bacillus Velezii MH24

[0068] The test was conducted in a greenhouse for the cultivation of Trichosanthes kirilowii in Xinzhou, Wuhan. Before the onset of bacterial angular leaf spot on June 10, 2022, the MH24 bacterial agent prepared in Example 3 was diluted 200 times and sprayed on the leaves, and the front and back of the leaves were evenly sprayed until the leaves were saturated. The second spraying was performed at an interval of 10 days. The control was irrigated with clear water, and each treatment was repeated 3 times, with 10 plants in each repetition. After the control was fully infected on June 20, 5 random sampling points were taken in each plot, and all leaves of 3 plants were investigated at each point. The number of diseased leaves at each level was recorded, and the disease index and control effect were calculated.

[0069] The grading standard is: Level 0: no lesions; Level 1: lesion area accounts for 5% or less of the total leaf area; Level 3: lesion area accounts for 6% to 10% of the total leaf area; Level 5: lesion area accounts for 11% to 20% of the total leaf area; Level 7: lesion area accounts for 21% to 50% of the total leaf area; Level 9: lesion area accounts for more than 51% of the total leaf area.

[0070] Disease index = ∑ (number of plants at each level × level) / (total number of plants surveyed × highest representative level) × 100.

[0071] Control effect = (control disease index - treatment disease index) / control disease index × 100%

[0072] Field efficacy tests showed that MH24 had a good control effect on bacterial angular leaf spot of Trichosanthes kirilowii, with a control efficiency of 62.89% (Table 5).

[0073] Table 5: Field efficacy of MH24 fungicide against bacterial angular leaf spot of Trichosanthes kirilowii

[0074] deal with Disease index Prevention effect (%) MH24 10.09 62.89 Comparison 27.19 -

[0075] Example 6: Control of Trichosanthes root-knot nematode disease by Bacillus Velezii MH24

[0076] The experiment was conducted in a greenhouse for the cultivation of Trichosanthes kirilowii in Xinzhou, Wuhan. Before the onset of Trichosanthes kirilowii wilt on June 10, 2022, the MH24 bacterial agent prepared in Example 3 was diluted 200 times and irrigated the roots, 400 mL / tree, and the second root irrigation was performed at an interval of 10 days. The control was irrigated with clear water, and each treatment was repeated 3 times, with 12 plants in each repetition. On July 30, after the control was fully infected, the disease index of the treatment and control was statistically calculated, and the control effect was calculated.

[0077] There are 5 levels in total: Level 0, no root knots; Level 1, 0-10% of the root systems have root knots; Level 2, 11%-30% of the root systems have root knots; Level 3, 31%-50% of the root systems have root knots; Level 4, 51%-75% of the root systems have root knots; Level 5, more than 75% of the root systems have root knots.

[0078] Disease index = ∑ (number of plants at each level × level) / (total number of plants surveyed × highest representative level) × 100.

[0079] Control effect = (control disease index - treatment disease index) / control disease index × 100%

[0080] Field efficacy tests showed that MH24 had a good control effect on Trichosanthes anthracnose, with a control efficiency of 60.46% (Table 6).

[0081] Table 6: Field efficacy of MH24 microbial agent against root-knot nematode disease in Trichosanthes kirilowii

[0082] deal with Disease index Prevention effect (%) MH24 17.62 60.46 Comparison 44.56 -

[0083] Example 7: Salt and alkali tolerance of Bacillus Velez MH24

[0084] The MH24 fermentation broth prepared in Example 3 was streaked on 5% and 10% NaCl NA plates, with water as a control, to observe the growth of MH24. The MH24 fermentation broth was streaked on pH 12 NA plates, with water as a control, to observe the growth of MH24.

[0085] The experiment showed that the MH24 strain grew well on 10% NaCl and pH 12 NA plates ( Figure 3 ,4), has a certain salt-alkali tolerance, which helps it to colonize and reproduce in salinized soils and saline-alkali lands.

[0086] Example 8: Colonization ability of Bacillus Velezii MH24 in the roots of Trichosanthes kirilowii

[0087] The resistant mutants of MH24 were screened on LB plates containing rifampicin at different gradient concentrations to obtain resistant mutant strains. The mutant strains had the same physiological and biochemical properties as those of Bacillus Velezii MH24 except for rifampicin resistance.

[0088] Bacillus Velez MH24 and rifampicin-resistant mutant of Bacillus Velez MH24 were fermented according to Example 3. The fermentation liquid prepared in Example 3 was diluted 300 times and used to irrigate the roots of Trichosanthes kirilowii and watermelon at 10 mL / tree. After 14 days, the roots were taken out and repeatedly rinsed with sterile water for 3 times. The roots were cut and 1 g was weighed. After grinding and diluting, the roots were applied to a 300 μg mL -1 The colonization ability of MH24 in the roots of Trichosanthes kirilowii and watermelon was determined by LB plates containing rifampicin and incubating at 37°C for 24 h.

[0089] The results showed that the colonization rate of MH24 in the roots of Trichosanthes kirilowii was 7.18×10 6 cfu / g, while in watermelon roots it was only 2.7×10 2 cfu / g. The colonization ability of MH24 is helpful for the prevention and control of soil-borne diseases of Trichosanthes kirilowii.

Claims

1. An isolated strain of Bacillus velez Bacillus velezensis ) MH24, the deposit number of the strain is CCTCC NO: M20221680.

2. The fermentation product of Bacillus Velez subtilis MH24 according to claim 1, wherein the fermentation product contains live bacteria of Bacillus Velez subtilis MH24.

3. A composition comprising the Bacillus Velezii MH24 or a fermentation product thereof according to claim 1.

4. Use of the Bacillus Velez MH24 according to claim 1, the fermentation product of the Bacillus Velez MH24 according to claim 2, or the composition according to claim 3 in the prevention and treatment of diseases of Trichosanthes kirilowii.

5. The use according to claim 4, wherein the diseases in the use include trichosanthes anthracnose, bacterial angular leaf spot, viral disease, root rot and / or root knot nematode disease.

6. The use according to claim 4, wherein the control method of the use comprises: The Bacillus Velez MH24 of claim 1, the fermentation product of Bacillus Velez MH24 of claim 2 or the composition of claim 3 is used to irrigate the roots of Trichosanthes kirilowii or directly spray them.

7. Use of the Bacillus Velez MH24 of claim 1, the fermentation product of the Bacillus Velez MH24 of claim 2, or the composition of claim 3 in the preparation of a biocontrol agent for Trichosanthes kirilowii.

8. The use according to claim 7, wherein the biocontrol agent in the use is used to control anthracnose fungi ( Colletotrichum orbiculare ), Fusarium oxysporum ( Fusariumoxysporum ), Pseudomonas syringae ( Pseudomonas syringae ), and / or biological diseases caused by root-knot nematodes.

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