Bacillus subtilis, bacterial agent and application thereof for preventing and controlling konjac soft rot

Abstract

The invention provides a bacillus subtilis, a microbial agent and an application for preventing and treating konjac soft rot, and belongs to the technical field of biocontrol microbial agent development. The bacillus subtilis GY1 of the invention has the function of preventing and treating konjac soft rot, and the deposit number is CGMCC No.31159. The invention also develops a biocontrol microbial agent that can be directly applied based on the characteristics of the bacillus subtilis GY1, and performs microbial agent root irrigation treatment on konjac during the growth period of konjac, which can effectively prevent and treat konjac soft rot, increase konjac yield, improve soil quality in konjac planting areas, and alleviate konjac continuous cropping obstacles.

Description

Technical Field

[0001] The invention belongs to the technical field of biocontrol fungicide development, and in particular relates to a bacillus subtilis for preventing and treating konjac soft rot, a fungicide and an application thereof. Background Art

[0002] As a crop with great economic value, konjac has shown important uses in many fields such as food processing, medicine, and chemical industry. It is rich in various beneficial ingredients such as glucomannan and has broad market prospects and development potential.

[0003] Although konjac can bring high economic value, soft rot is very likely to occur during its cultivation, and konjac planted under the forest also shows continuous cropping obstacles like farmland planting. Related studies have shown that konjac generally has continuous cropping obstacles. With the expansion of artificial planting area and the increase of years, the disease incidence rate of konjac continuous cropping fields is 35%-50% higher than that of non-continuous cropping fields on average. Konjac soft rot is mainly caused by a combination of multiple pathogens, including but not limited to certain bacteria and fungi. Once konjac is attacked by soft rot, the plant will quickly show symptoms of rot, which may cause konjac to grow poorly and deteriorate in quality, or even cause a significant reduction in production or even a total crop failure, bringing unbearable economic losses to the majority of konjac growers. Among the current prevention and control methods, chemical agents are widely used. However, the long-term and large-scale use of chemical agents not only easily promotes pathogens to gradually develop drug resistance and reduce the prevention and control effect, but may also cause a series of environmental problems, such as soil pollution, water pollution, etc., threatening the balance and stability of the ecosystem.

[0004] In this context, the vigorous development of biotechnology has brought new hope for solving this problem, and biocontrol agents have emerged. Biocontrol agents can effectively inhibit the growth and reproduction of pathogens through their unique mechanism of action. Although biocontrol agents have achieved certain research results and application effects in the field of disease control of other crops, biocontrol agents specifically for konjac soft rot still have many shortcomings. Some existing biocontrol agents may not have ideal control effects and cannot effectively curb the spread of soft rot; or they may show unstable performance under different environmental conditions and are difficult to adapt to the complex and changeable konjac planting environment; or they may not be compatible with konjac plants and cannot fully exert their effectiveness.

[0005] Therefore, it is particularly important and urgent to develop a biocontrol agent specifically for konjac soft rot, which has high control ability, is safe, environmentally friendly and has stable performance. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a Bacillus subtilis, a bacterial agent and an application for preventing and controlling konjac soft rot, which can effectively prevent and control konjac soft rot, increase konjac yield, and improve soil quality.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] A Bacillus subtilis GY1 for preventing and treating konjac soft rot. The preservation number of the Bacillus subtilis GY1 is CGMCC No.31159.

[0009] The present invention also provides a Bacillus subtilis agent for preventing and treating konjac soft rot, wherein the active ingredient of the agent is the Bacillus subtilis GY1.

[0010] Preferably, the concentration of Bacillus subtilis GY1 in the bacterial agent is ≥ 900 million cfu / mL.

[0011] Preferably, the preparation method of the bacterial agent comprises the following steps: inoculating the Bacillus subtilis GY1 strain into a culture medium and culturing at room temperature, wherein the culture medium is a 1%-1.5% glucose solution, and 0.2%-0.3% cornmeal and 0.005%-0.01% sodium chloride are added.

[0012] More preferably, the inoculation amount of Bacillus subtilis GY1 is 0.5%-1%.

[0013] More preferably, the culturing process lasts for 10-14 days, with stirring 2-3 times a day.

[0014] The present invention also provides the use of the Bacillus subtilis and the bacterial agent in preventing and controlling konjac soft rot and alleviating the obstacle of konjac continuous cropping.

[0015] Preferably, during the growth period of konjac, the Bacillus subtilis agent is used for root irrigation once a month.

[0016] More preferably, the root irrigation dosage of the Bacillus subtilis agent is 200-300 mL / plant.

[0017] Preferably, the konjac is konjac konjac or konjac konjac.

[0018] Compared with the prior art, the present invention provides a Bacillus subtilis GY1 for preventing and treating konjac soft rot and a bacterial agent prepared therefrom, which has the following beneficial effects:

[0019] (1) Effectively improve the sustainable management of forest land: Through the "bacteria inhibiting bacteria" of Bacillus subtilis, the problem of increasing soil pathogens caused by continuous konjac cropping is effectively reduced, which helps to restore soil health, thereby improving forest productivity and land resource utilization, and is conducive to the sustainable management of forest land;

[0020] (2) Effectively improve the growth and yield of konjac: Apply Bacillus subtilis to the soil. Bacillus subtilis increases soil microbial diversity, maintains soil nutrient circulation, promotes konjac tuber absorption, and inhibits the growth of pathogenic bacteria, thereby increasing the growth and yield of konjac.

[0021] (3) Effectively reduce the incidence of konjac soft rot: Simply improving management measures and levels cannot effectively eliminate konjac soft rot. Based on green prevention and control, Bacillus subtilis is used to inhibit the konjac soft rot pathogen, effectively reducing the incidence of konjac soft rot.

[0022] (4) Improve the physical and chemical properties of the soil: This strain and microbial agent can solve the problem of increased soil pathogens and reduced microbial diversity caused by continuous konjac cropping, regulate the structure of soil microbial communities, improve soil fertility and physical and chemical properties, and significantly enhance soil fertility within a certain period of time, reduce soil pathogens, and help restore the functional diversity of microorganisms in the soil. It has a certain inhibitory effect on the increase in the number of pathogenic bacteria communities in the soil, prevents the degradation of the soil ecosystem, thereby improving the physical and chemical properties of the crop root microecological environment and reducing crop disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Bacillus subtilis of the present invention ( Bacillus subtilis ) Colony morphology of GY1;

[0024] Figure 2 Bacillus subtilis of the present invention ( Bacillus subtilis ) Microscopic morphology of GY1;

[0025] Figure 3 Bacillus subtilis of the present invention ( Bacillus subtilis ) Phylogenetic tree of 16s rDNA of GY1 strain.

[0026] The biological material of the present invention is Bacillus subtilis GY1, which belongs to Bacillus subtilis ( Bacillus subtilis ), the depository is the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC for short), the depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the deposit number is CGMCC No.31159, and the deposit date is July 3, 2024. DETAILED DESCRIPTION

[0027] The invention provides a Bacillus subtilis GY1 with a preservation number of CGMCC No.31159. The strain has an inhibitory effect on pathogenic bacteria of konjac soft rot, can effectively prevent and control the occurrence of konjac soft rot, and can also improve soil fertility, improve soil microbial levels, and alleviate konjac continuous cropping obstacles.

[0028] The present invention also provides a Bacillus subtilis agent for preventing and treating konjac soft rot, wherein the sterilizing active ingredient of the agent is the above-mentioned Bacillus subtilis GY1; preferably, the concentration of Bacillus subtilis GY1 in the agent is ≥ 900 million cfu / mL, further preferably 950 million to 1 billion cfu / mL, and more preferably 1 billion cfu / mL.

[0029] The present invention preferably comprises the following steps of preparing the bacterial agent: inoculating the Bacillus subtilis GY1 strain into the culture solution for normal temperature culture, and further preferably the strain inoculation amount is 0.5%-1% (m / v), and more preferably 0.75% (m / v). As an implementable method, 1-2g of Bacillus subtilis GY1 strain is inoculated per 200mL culture solution. Preferably, the culture solution is a 1%-1.5% glucose solution, with 0.2%-0.3% cornmeal and 0.005%-0.01% sodium chloride added; further preferably, it is a 1.25% glucose solution, with 0.25% cornmeal and 0.0075% sodium chloride added. As an implementable method, 200L of water is measured, 2500g of industrial glucose is weighed, 1.25% (m / v) industrial glucose solution is prepared, 500g of cornmeal and 15g of sodium chloride are weighed, and the culture solution is configured, and 1500g of Bacillus subtilis GY1 strain is inoculated, and stirred and cultured at room temperature. Preferably, the culture process lasts for 10-14 days with stirring 2-3 times a day.

[0030] The present invention also provides the use of the Bacillus subtilis agent in preventing and controlling konjac soft rot and alleviating konjac continuous cropping obstacles; the prevention and control of konjac soft rot refers to reducing the incidence of konjac soft rot in the field, and the alleviating konjac continuous cropping obstacles refers to improving the soil fertility of the konjac continuous cropping field, improving the soil microbial level, and promoting the growth and production of konjac.

[0031] In the present invention, preferably, the Bacillus subtilis agent is used for root irrigation during the growth period of konjac, once a month. More preferably, the root irrigation dosage of the Bacillus subtilis agent is 200-300 mL / plant, more preferably 300 mL / plant.

[0032] The present invention preferably uses konjac as flower konjac or konjac with bulbils. The present invention has no special restrictions on the specific varieties of konjac or konjac with bulbils.

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1

[0035] Isolation and Storage of Bacillus subtilis GY1

[0036] Bacillus subtilis GY1 strain was isolated and cultured from semi-decomposed eucalyptus bark in a natural pile state after peeling in a wood processing factory in Jinggu County, Pu'er City, Southwest Forest Production Area, Yunnan Province. A single colony of GY1 strain was obtained from it by dilution plate method. Degenerate primers (primer information F: AGRGTTTGATYNTGGCTCAG, as shown in SEQ ID NO.1; R: TASGGHTACCTTGTTASGACTT, as shown in SEQ ID NO.2) were used to amplify the 16S rDNA 27F-1492R (V1 to V8) region of the colony by PCR, and then DNA sequencing and sequence alignment were performed to identify it as Bacillus subtilis ( Bacillus subtilis The original strain was stored in 30% glycerol solution at -80°C.

[0037] The DNA sequence of Bacillus subtilis GY1 strain is shown in SEQ ID NO. 3, and has a length of 1523 bp.

[0038] Example 2

[0039] Identification of Bacillus subtilis GY1

[0040] The morphological characteristics of Bacillus subtilis GY1 strain are as follows Figure 1 The microscopic morphological features are shown in Figure 2 The 16s rDNA phylogenetic tree of the strains is shown in Figure 3 As shown, clustering was performed at a similarity level of 0.97, which was homologous to the known Bacillus subtilis DNA sequence (KJ469796.1) included in the NCBI database.

[0041] Example 3

[0042] Cultivation and Activation of Bacillus subtilis GY1

[0043] Culture: Take out the strain in 30% glycerol solution from -80℃ and restore it to room temperature. Use a pipette to gently aspirate and evenly spread it, then take 100μl and spread it on a plate containing TSA solid culture medium. Place the plate open in a biosafety cabinet, cover the culture dish after the bacterial solution is completely absorbed, seal it with sealing film, and invert it to culture at 37℃ for 24h.

[0044] Activation: Dip a single colony with a sterile toothpick under sterile conditions, then place it in 100 ml TSA liquid culture medium to clean the toothpick stained with plaque, and then place the TSA liquid culture medium in a constant temperature and uniform speed culture at 37°C for 24 hours (speed 200r / min).

[0045] Example 4

[0046] A preparation method of a Bacillus subtilis GY1 biocontrol agent comprises the following steps: using 1.25% (m / v) industrial glucose solution as a carrier, adding 0.25% (m / v) cornmeal and 0.0075% (m / v) sodium chloride to prepare a culture solution; inoculating the Bacillus subtilis GY1 strain at 0.75% (m / v), culturing with stirring at room temperature for 10-14 days, stirring 2-3 times a day, and preparing the Bacillus subtilis GY1 biocontrol agent, wherein the effective content of the strain is ≥900 million cfu / mL.

[0047] Example 5

[0048] A preparation method of a Bacillus subtilis GY1 biocontrol agent comprises the following steps: using 1% (m / v) industrial glucose solution as a carrier, adding 0.2% (m / v) cornmeal and 0.005% (m / v) sodium chloride to prepare a culture solution; inoculating the Bacillus subtilis GY1 strain at 0.5% (m / v), stirring and culturing at room temperature for 10-14 days, stirring 2-3 times a day, and preparing the Bacillus subtilis GY1 biocontrol agent, wherein the effective content of the strain is ≥900 million cfu / mL.

[0049] Example 6

[0050] A preparation method of a Bacillus subtilis GY1 biocontrol agent comprises the following steps: using 1.5% (m / v) industrial glucose solution as a carrier, adding 0.3% (m / v) cornmeal and 0.01% (m / v) sodium chloride to prepare a culture solution; inoculating the Bacillus subtilis GY1 strain at 1% (m / v), stirring and culturing at room temperature for 10-14 days, stirring 2-3 times a day, and preparing the Bacillus subtilis GY1 biocontrol agent, wherein the effective content of the strain is ≥900 million cfu / mL.

[0051] Example 7

[0052] Control effect of Bacillus subtilis GY1 biocontrol agent on soft rot of konjac

[0053] The preparation method of Bacillus subtilis GY1 biocontrol agent has the following steps: using 1.25% (m / v) industrial glucose solution as a carrier, adding 0.25% (m / v) cornmeal and 0.0075% (m / v) sodium chloride to prepare a culture solution, inoculating Bacillus subtilis GY1 strain at 0.75% (m / v), stirring and culturing at room temperature, stirring 3 times a day, using the plate count method to count, and culturing to obtain biocontrol agents with effective strain contents of 9, 9.5, and 1 billion cfu / mL as test agents, and setting the following treatments for field biocontrol tests:

[0054] Treatment 1: clean water (CK), application rate was 250 mL / plant;

[0055] Treatment 2: 0.1% thiophanate-methyl, application rate 250 mL / plant;

[0056] Treatment 3: 900 million cfu / mL biocontrol agent, application rate 200 mL / plant;

[0057] Treatment 4: 950 million cfu / mL biocontrol agent, application rate 250mL / plant;

[0058] Treatment 5: 1 billion cfu / mL biocontrol agent, application rate is 300 mL / plant.

[0059] The implementation period lasts for 5 consecutive months, including 1 month before the rainy season in the konjac cultivation cycle, and 4 months from the rainy season to the harvest; in the early morning of each month, the test sites are irrigated with pesticides and fungal agents and samples are taken.

[0060] The method and standard for determining the incidence rate: Statistics are collected once a month for 5 consecutive months, including the 1 month before the rainy season in the konjac cultivation cycle, and the 4 months from the rainy season to harvest. The number of statistical samples in each group is 600 plants, and the incidence rate is the cumulative incidence rate for 5 consecutive months.

[0061] Among them, the incidence rate = (total number of cases in 5 months / number of samples) × 100%.

[0062] The symptoms of disease are that the above-ground part turns yellow, black, becomes soft and falls over, or the underground part becomes soft and rots.

[0063] The test treatments and results are shown in Table 1.

[0064] Table 1 Different control methods, costs and control effects on konjac soft rot

[0065]

[0066] Soil probiotics have an antagonistic effect on harmful bacteria. As shown in Table 1, in the clean water (CK) control, the probiotics in the original soil inhibited the growth and reproduction of harmful bacteria to a certain extent. As a broad-spectrum fungicide, thiophanate-methyl can not only kill harmful bacteria in the soil, but also kill probiotics in the soil, so that the soil lacks the protection of probiotics, causing the incidence of soft rot to increase instead of decrease. Compared with the traditional chemical agent thiophanate-methyl, the use of Bacillus subtilis GY1 biocontrol agent to control konjac soft rot is cheaper and more effective. And with the increase of the effective content and dosage of the strains in the agent, the incidence of konjac soft rot was significantly reduced, and the control effect reached 41.1%-74.2%.

[0067] Example 8

[0068] Effects of Bacillus subtilis GY1 biocontrol agent on konjac yield

[0069] After the field biocontrol test in Example 7 was completed, the underground tubers of each group were harvested, the underground root length and the aboveground crown width were measured, and the fresh weight of the underground tubers was measured. The results are shown in Table 2.

[0070] Table 2 Effects of different control methods on the growth and yield of konjac

[0071]

[0072] As shown in Table 2, compared with the control and traditional chemical agent thiophanate-methyl, root irrigation with Bacillus subtilis GY1 biocontrol agent is more conducive to the growth and yield of konjac. Compared with the control treatment, the aboveground crown growth of the biocontrol agent treatment increased by 34.24%-63.08%, the underground root length growth increased by 18.66%-36.68%, and the underground tuber fresh weight increased by 41.75%-61.66%.

[0073] Example 9

[0074] Effects of Bacillus subtilis GY1 biocontrol agent on heavy metal contents in konjac field soil

[0075] After the field biocontrol test of Example 7 was completed, soil from each treated plot was collected to determine the copper content in the soil. Detection method: diethylenetriaminepentaacetic acid (DTPA) extraction method (NY / T 890-2004).

[0076] Soil copper content results: Group 1 13-24 mg / kg, Group 2 29-36 mg / kg, Groups 3-5 17-25 mg / kg.

[0077] The above results show that compared with the thiophanate-copper fungicide, the biocontrol agents in groups 3-5 reduced the input of heavy metals in the soil of konjac fields.

[0078] Example 10

[0079] Effects of Bacillus subtilis GY1 biocontrol agent on microbial communities in konjac fields

[0080] After the field biocontrol test in Example 7 was completed, soil from each treatment plot was collected, and the number of soil microbial species in each treatment was: 202 species in Group 1, 148 species in Group 2, 631 species in Group 3, 638 species in Group 4, and 659 species in Group 5. According to the soil ecological function corresponding to the abundance analysis of soil microbial gene sequences, Groups 1 and 2 were positively correlated with plant pathogenicity, and Groups 3, 4, and 5 were negatively correlated with plant pathogenicity; Groups 1 and 2 were negatively correlated with soil environmental stress tolerance, and Groups 3, 4, and 5 were positively correlated with soil environmental stress tolerance.

[0081] The above results show that the chemical fungicides in group 2 reduced the diversity of soil microorganisms, which is not conducive to the balance of soil microecology; while the biocontrol agents in groups 3-5 increased the diversity of soil microorganisms, making the soil ecosystem healthier.

[0082] Embodiment 11

[0083] Effects of Bacillus subtilis GY1 biocontrol agent on soil fertility in konjac fields

[0084] After the field biocontrol test in Example 7 was completed, soil from each treatment plot was collected, and soil pH (NY / T1377-2007), organic matter (NY / T1121.6-2006), total nitrogen (LY / T 1228-2015), total phosphorus (LY / T 1232-2015), total potassium (LY / T 1234-2015), hydrolyzable nitrogen (LY / T1228-2015), available phosphorus (LY / T 1232-2015), available potassium (LY / T 1234-2015), exchangeable calcium and magnesium (LY / T 1245-1999), mercury (GB / T22105.1 2008), arsenic (GB / T22105.2-2008), lead and cadmium (GB / T 17141-1997) contents were determined according to relevant standards. Among them, the following indicators showed significant differences among the treatment groups, and the results are shown in Table 3.

[0085] Table 3 Effects of different control methods on soil fertility in konjac fields

[0086]

[0087] The above results show that the biocontrol agents in groups 3-5 can also supplement soil organic matter, macro-element nitrogen, medium-element magnesium and sulfur, and the increase is significant compared with the chemical fungicides in group 2.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Bacillus subtilis for preventing and treating konjac soft rot ( Bacillus subtilis ) GY1, the preservation number of the Bacillus subtilis GY1 is CGMCC No.31159.

2. A Bacillus subtilis agent for preventing and treating konjac soft rot, characterized in that: The active ingredient of the bacterial agent is the Bacillus subtilis GY1 described in claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial concentration of Bacillus subtilis GY1 in the bacterial agent is ≥ 900 million cfu / mL.

4. The bacterial agent according to claim 2 or 3, characterized in that: The preparation method of the bacterial agent comprises the following steps: inoculating the Bacillus subtilis GY1 strain into a culture solution and culturing at room temperature; the culture solution is a 1%-1.5% glucose solution, and 0.2%-0.3% cornmeal and 0.005%-0.01% sodium chloride are added.

5. The bacterial agent according to claim 4, characterized in that The inoculation amount of the Bacillus subtilis GY1 is 0.5%-1%.

6. The bacterial agent according to claim 4, characterized in that The culture process lasts for 10-14 days, with stirring 2-3 times a day.

7. Use of the Bacillus subtilis GY1 according to claim 1 or the Bacillus subtilis agent according to any one of claims 2 to 6 in preventing and treating konjac soft rot and alleviating the obstacle of konjac continuous cropping.

8. The use according to claim 7, characterized in that: During the growth period of konjac, the Bacillus subtilis agent is used for root irrigation once a month.

9. The use according to claim 8, characterized in that: The root irrigation dosage of the Bacillus subtilis agent is 200-300 mL / plant each time.

10. The use according to claim 7, characterized in that: The konjac is flower konjac or bulbil konjac.

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