A biocontrol strain of ginger wilt caused by enterobacter cloacae and a method for preventing and treating ginger wilt

Abstract

The present application relates to the field of microbial technology, and particularly relates to a ginger wilt disease biocontrol strain caused by Enterobacter cloacae and a ginger wilt disease prevention and treatment method. Bacillus cereus The ginger wilt disease biocontrol strain is named Bacillus cereus Y51, which is preserved in the China General Microbiological Culture Collection Center on May 9, 2024, and the preservation number is CGMCC NO.30536. Experiments prove that the Bacillus cereus Y51 exhibits good prevention effect on the Enterobacter cloacae on the ginger plant, and therefore the Bacillus cereus Y51 can be used for preventing and treating the ginger wilt disease, and has the advantages of environmental friendliness, no agricultural residues and green environmental protection. Bacillus cereus The biocontrol strain of the present application fills the blank of the existing ginger wilt disease biological control bacteria in the aspect of targeting specific pathogenic bacteria species.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and in particular to a biocontrol strain of ginger wilt caused by Enterobacter cloacae and a method for preventing and treating ginger wilt. BACKGROUND

[0002] Ginger wilt is widespread and serious, leading to ginger yield reduction, and even absolute yield reduction. At present, the main method for preventing and treating ginger soil-borne diseases is chemical prevention and treatment. However, a large amount of chemical pesticides will destroy the ecological environment, increase the drug resistance of diseases, and increase the amount of soil pesticide residues. Biological control by microorganisms and microbial metabolites has become the main development direction of plant soil-borne disease control. The main symptom of ginger wilt is plant yellowing and rotting of ginger tubers on a large scale, and finally only the ginger tuber skin is left. Once ginger wilt breaks out, there is almost no effective control measure, and even the surrounding plots and adjacent water bodies are always diseased.

[0003] Plant growth promoting rhizobacteria (PGPR) refers to beneficial bacteria that can colonize the rhizosphere of plants and directly or indirectly promote the growth and development of plants or regulate metabolism. The main mechanisms of PGPR are: more effective colonization in the root compared with other soil microorganisms, antagonistic effect on harmful microorganisms, advantage in nutrient competition especially iron competition in the rhizosphere, secretion of plant hormones to promote plant growth, induction of systemic resistance in plants, and secretion of enzymes to degrade pathogenic microorganisms.

[0004] Chinese patent application CN110760462B discloses an antagonistic bacterium YJ32 for preventing and treating ginger wilt and its application, which discloses a strain of Burkholderia ambifaria. The strain has strong inhibitory effect on the pathogenic bacteria Ralstonia solanacearum and Enterobacter mori causing ginger wilt and mulberry bacterial wilt, can target prevent ginger wilt, and whether it is effective for ginger wilt caused by Enterobacter cloacae is not recorded in the scheme. Since the strain is screened by taking Ralstonia solanacearum and Enterobacter mori JLIY2 as target pathogenic bacteria, it is inferred that the strain may not have good biocontrol efficacy for ginger wilt caused by Enterobacter cloacae. At present, most researchers focus on the primary pathogenic bacterium of ginger wilt, Ralstonia solanacearum, to develop corresponding biocontrol bacteria. Ginger wilt caused by Enterobacter cloacae can cause ginger root rot, plant growth retardation, and is not easy to be quickly detected, which is easily ignored by researchers. SUMMARY

[0005] (I) Technical problems to be solved

[0006] In view of the above-mentioned defects and deficiencies of the prior art, the present application provides a biological control strain of ginger wilt caused by Enterobacter cloacae and a method for preventing and treating ginger wilt, which fills the gap of the prior biological control bacteria of ginger wilt in targeting specific pathogenic bacteria.

[0007] (II) Technical solutions

[0008] In a first aspect, the present application provides a biological control strain of ginger wilt caused by Enterobacter cloacae, which is named Bacillus cereus (B. cereus) Y51, and the strain was deposited with the China General Microbiological Culture Collection Center on May 9, 2024, and the accession number is CGMCC NO.30536. Bacillus cereus

[0009] In a second aspect, the present application provides a method for preventing and treating ginger wilt caused by Enterobacter cloacae, which comprises using the above-mentioned B. cereus Y51 as a biological control bacteria to prevent and treat ginger wilt. Bacillus cereus

[0010] According to a preferred embodiment of the present application, the method for preventing and treating ginger wilt comprises using the fermentation broth of B. cereus Y51 as a biological control bacteria liquid, soaking ginger rhizome with the biological control bacteria liquid for 1-4 hours, taking out the ginger rhizome after soaking and drying it indoors, and using the soaked ginger rhizome as seed rhizome. Bacillus cereus According to a preferred embodiment of the present application, the method for preventing and treating ginger wilt comprises using the fermentation broth of B. cereus Y51 as a biological control bacteria liquid, and using the biological control bacteria liquid to irrigate the roots and / or spray the leaves of five-leaf-stage ginger plants.

[0011] According to a preferred embodiment of the present application, the method for preventing and treating ginger wilt comprises inoculating B. cereus Y51 into a liquid culture medium, obtaining a fermentation broth after 24 hours of fermentation culture, diluting the fermentation broth by 10 times or 100 times to obtain a biological control bacteria liquid, and using the biological control bacteria liquid to soak ginger rhizome as seed rhizome and to irrigate the roots and / or spray the leaves of five-leaf-stage ginger plants. Bacillus cereus According to a preferred embodiment of the present application, the method for preventing and treating ginger wilt comprises inoculating B. cereus Y51 into a liquid culture medium, obtaining a fermentation broth after 24 hours of fermentation culture, diluting the fermentation broth by 10 times or 100 times to obtain a biological control bacteria liquid, and using the biological control bacteria liquid to soak ginger rhizome as seed rhizome and to irrigate the roots and / or spray the leaves of five-leaf-stage ginger plants.

[0012] According to a preferred embodiment of the present application, the method for preventing and treating ginger wilt comprises inoculating B. cereus Y51 into a liquid culture medium, obtaining a fermentation broth after 24 hours of fermentation culture, diluting the fermentation broth by 10 times or 100 times to obtain a biological control bacteria liquid, and using the biological control bacteria liquid to soak ginger rhizome as seed rhizome and to irrigate the roots and / or spray the leaves of five-leaf-stage ginger plants. Bacillus cereus According to a preferred embodiment of the present application, the method for preventing and treating ginger wilt comprises inoculating B. cereus Y51 into a liquid culture medium, obtaining a fermentation broth after 24 hours of fermentation culture, diluting the fermentation broth by 10 times or 100 times to obtain a biological control bacteria liquid, and using the biological control bacteria liquid to soak ginger rhizome as seed rhizome and to irrigate the roots and / or spray the leaves of five-leaf-stage ginger plants.

[0013] According to a preferred embodiment of the present application, the method for preventing and treating ginger wilt comprises inoculating B. cereus Y51 into a liquid culture medium, obtaining a fermentation broth after 24 hours of fermentation culture, diluting the fermentation broth by 10 times or 100 times to obtain a biological control bacteria liquid, and using the biological control bacteria liquid to soak ginger rhizome as seed rhizome and to irrigate the roots and / or spray the leaves of five-leaf-stage ginger plants. Bacillus cereus ​​Y51 and Burkholderia bifidum with accession number CGMCC NO.15922 Burkholderia ambifaria The combined fermentation broth of YJ32 was diluted and used as a biocontrol solution for ginger wilt disease. The combined use of biocontrol bacteria targeting different pathogens of ginger wilt disease can cover the three currently prevalent pathogens causing ginger wilt, almost encompassing all pathogen types, and can greatly promote the emergence rate, growth rate, and yield of ginger tubers.

[0014] Preferably, the liquid culture medium comprises: 50 g / L corn steep liquor, 20 g / L sugarcane residue, 15 mL / L soybean meal hydrolysate, 3 g / L (NH4)2SO4, and sterile water. This liquid culture medium is low-cost (cost < 25 yuan / L), represents a comprehensive utilization of agricultural by-products, is suitable for large-scale use, and can reduce costs. Furthermore, the liquid culture medium can also be one of LB medium, TSB medium, or kitchen waste hydrolysate medium.

[0015] (III) Beneficial Effects

[0016] The Bacillus cereus strain screened in this invention ( Bacillus cereus Y51 exhibits good biocontrol activity against Enterobacter cloacae on ginger plants and can inhibit Bacillus cereus (…). Bacillus cereus Y51 can be made into a microbial inoculant or used with Bacillus cereus ( Bacillus cereus The fermentation broth of Y51 is used to treat ginger tubers or plants for the prevention and control of ginger wilt disease. This method is environmentally friendly, pesticide-free, and green. The biocontrol strain of this invention fills the gap in existing biocontrol bacteria for ginger wilt disease that target specific pathogens. Attached Figure Description

[0017] Figure 1 The results show the in vitro leaf control efficacy of different candidate biocontrol strains for ginger wilt in Example 1.

[0018] Figure 2 The effect of different candidate biocontrol strains for ginger wilt disease on the antagonistic pathogen in Example 1 is shown.

[0019] Figure 3 for Bacillus cereus Morphological image of Y51 on LB solid medium. Detailed Implementation

[0020] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] Example 1

[0022] This embodiment relates to Bacillus cereus ( Bacillus cereusThe screening and identification process of Y51 is as follows:

[0023] 1. Experimental materials

[0024] (1) Collection of soil samples

[0025] The soil samples used for the isolation and screening of biocontrol bacteria for ginger wilt were collected by random sampling from the rhizosphere of different plants in test fields in various leagues and cities of Inner Mongolia, including rhizosphere tissues and root soil. These samples have been preserved in the strain library of the Plant Pathology Laboratory of Inner Mongolia Agricultural University. Enterobacter cloacae was detected in the soil of Inner Mongolia, and relevant studies have shown that Enterobacter cloacae is one of the pathogenic bacteria of ginger wilt (ginger rot).

[0026] (2) Preparation of culture medium and reagents

[0027] LB medium: 10 g / L proteose peptone, 5 g / L yeast, 10 g / L NaCl, pH 7.0-7.5, 121°C steam sterilization for 20 min. Solid medium requires the addition of 20 g of agar powder.

[0028] 2. Experimental methods

[0029] (1) Preliminary screening of ginger wilt biocontrol strains

[0030] The strains to be tested preserved in the strain library of the Plant Pathology Laboratory of Inner Mongolia Agricultural University were evenly spread on LB medium for activation and cultured at 28°C for 48h. The activated strains (strains with biocontrol potential) were inoculated with a 5mm bacterial cake using a sterile gun head into 30ml of LB liquid medium, incubated at 28°C with constant temperature shaking at 180rpm for 24h, then diluted 10 times to obtain the bacterial solution. Healthy leaves of ginger with consistent leaf age and growth were washed with sterile water and soaked in the bacterial solution for 10min. After the leaves were dried, Enterobacter cloacae bacterial cakes with a diameter of 5mm were inoculated on the central part of the leaves. Each leaf was inoculated with one bacterial cake, a total of 5 leaves, with 3 replicates. Leaves soaked in sterile water for 10min served as negative controls, and leaves soaked in sterile water and inoculated with Enterobacter cloacae served as positive controls. The leaves were placed on sterile water-soaked filter paper in a light incubator at 28°C with 8 hours of light and 8 hours of darkness, and the expansion of the lesions was observed. The results of the in vitro leaf control effect are shown in Table 1. Figure 1

[0031] The calculation method of in vitro leaf control effect is shown in the following formula:

[0032]

[0033] ​The test results show that the strain named Y51 has the best control effect, and the leaves remain green and healthy without disease 7 days after inoculation of pathogenic bacteria. Other strains such as 14-5, 3-11-2 and X37 have the second best control effect, and the leaves remain green and healthy without disease 5 days after inoculation of pathogenic bacteria, but the leaves start to turn yellow on the 6th day. The leaves soaked in the rest of the strains successively get sick and die within 3 days after inoculation of pathogenic bacteria (Table 1 is the control effect of the in vitro leaf method).

[0034] Table 1: Control effect of in vitro leaf method

[0035]

[0036] Note: CK is the negative control of water soaking; CK1 is the positive control of water soaking inoculated with pathogenic bacteria.

[0037] (2) Determination of biocontrol function of strains Y51, 14-5, 3-11-2 and X37

[0038] According to the above method, the biocontrol strains whose leaves remain healthy and green 5 days after inoculation of pathogenic bacteria are selected for plate confrontation experiment. Enterobacter cloacae QK is inoculated into 50 mL of LB liquid medium, and incubated at 28°C, 180 rpm for 48 h. After being diluted 100 times with sterile distilled water, 30 μL is spotted on a filter paper disc with a diameter of 5 mm and dried in a sterile operation table. Strains Y51, 14-5, 3-11-2 and X37 are inoculated into 50 mL of LB liquid medium, and incubated at 28°C, 180 rpm for 48 h. 50 μL of the above culture is evenly spread on the surface of LB solid medium. After the surface culture liquid is dried, the filter paper disc inoculated with Enterobacter cloacae is taken with sterile forceps and placed in the center of the culture medium. The experiment is repeated 3 times, and the size of the inhibition zone is observed after 3 days of incubation at 28°C in an incubator (the effect of biocontrol strain antagonizing Enterobacter cloacae). Figure 2 The test results show that strains Y51, Y57, 14-5, 3-11-2 and X37 do not produce inhibition zones when antagonizing Enterobacter cloacae QK, indicating that strain Y51 does not directly antagonize Enterobacter cloacae QK, and its biocontrol effect may be achieved by inducing antagonism.

[0039] (3) Results of pot experiment to verify the biocontrol ability of biocontrol strains

[0040] Ginger tubers with sprouts are placed in pots and planted at 28°C for 50 days. After streak culture on LB medium, the biocontrol strains are cultured in liquid medium for 48 h. Then 3 mL of biocontrol bacterial solution is added to the pot after being diluted 100 times. After 3 days of adding the biocontrol bacterial solution, pathogenic bacteria fermentation broth is added, and the potting disease is observed. The test results show that the strain named Y51 has the best control effect.

[0041] (4) Identification results of strain Y51

[0042] 1. Morphological characteristics identification results

[0043] Strain Y51 is round and smooth on LB solid medium, with slight wrinkles and white color. As shown in the figure, it is the morphology of strain Y51 on LB solid medium. Figure 3

[0044] 2. Physiological and biochemical characteristics identification results

[0045] Strain Y51 is gram-positive.

[0046] 3. 16s rDNA sequence sequencing results, and construction of phylogenetic tree. According to NCBI database comparison and analysis, strain Y51 is Bacillus cereus, Chinese name is Bacillus cereus Y51. The strain has been preserved by the applicant in China General Microbiological Culture Collection Center on May 9, 2024, with the preservation number of CGMCC NO.30536.

[0047] Example 2

[0048] This example is the field application of Bacillus cereus Y51. The application method is as follows:

[0049] (1) Seed soaking with biocontrol bacteria

[0050] Strain Bacillus cereus Y51 is inoculated in liquid medium at an inoculation ratio of 5% for fermentation culture. The liquid medium can be LB medium or agricultural by-product medium, the purpose is to obtain a large amount of bacteria and metabolites. The composition of the agricultural by-product medium is: corn syrup 50g / L, sugar residue 20g / L, soybean meal hydrolysate 15mL / L, (NH4)2SO4 3g / L and sterilized water. In this example, the agricultural by-product medium is used for fermentation for 24h, and the fermented liquid is diluted 10 times to soak ginger rhizome for 2h. After soaking, the seed is taken out and dried indoors.

[0051] (2) Root irrigation and leaf spraying with biocontrol bacteria

[0052] The soaked ginger rhizome is planted in the field and managed according to the conventional planting technology.

[0053] Part of the test field of ginger is managed as follows after germination: at the five-leaf stage of ginger, the diluted liquid of the fermentation liquid of Bacillus cereus Y51 is used for root irrigation and leaf spraying. Specifically, the fermentation liquid of step (1) is diluted 100 times after fermentation for 24h, and 100 times diluted liquid is used for root irrigation and leaf spraying. Each column is sprayed with 10L, and the root irrigation is 20L.

[0054] ​The ginger wilt disease incidence was compared between the case of only biocontrol bacteria seed soaking treatment and the case of combined biocontrol bacteria seed soaking and 2-8 times of later root irrigation and leaf spraying.

[0055] (3) Ginger above-ground growth data analysis

[0056] Table 2: Ginger above-ground growth data analysis

[0057]

[0058] Note: CK represents the blank control group inoculated with Enterobacter cloacae; B represents the treatment group inoculated with Enterobacter cloacae + biocontrol bacteria.

[0059] According to the data in Table 2, Bacillus cereus Y51 has a significant promoting effect on the growth of ginger above-ground. In this test, 25 samples were taken from each column, and the average stem diameter and height of the 25 samples were calculated, as well as the total number of branches. The results showed that the stem diameter and height of the treatment group were higher than those of the CK group, and the total number of branches was significantly higher than that of the CK group. The use of biocontrol bacteria seed soaking can promote the growth of ginger above-ground.

[0060] (4) Ginger below-ground test result statistics

[0061] Table 3: Ginger below-ground test result statistics

[0062]

[0063] Note: CK represents the blank control group inoculated with Enterobacter cloacae; B represents the treatment group inoculated with Enterobacter cloacae + biocontrol bacteria.

[0064] According to the data in Table 3, Bacillus cereus Y51 has a significant promoting effect on the growth of ginger below-ground. The total number of fleshy roots and fine roots of 25 samples in each treatment was calculated, and the results showed that the use of biocontrol bacteria seed soaking and later spraying and irrigation can significantly increase the number of fleshy roots and fine roots of ginger tubers, and improve the yield. In the case of pathogenic bacteria soaking ginger tubers, the use of biocontrol bacteria seed soaking can increase the number of fleshy roots by 26.44%, the number of fine roots by 16.56%, and the yield by 31.15%. In the case of soil spraying pathogenic bacteria, the use of biocontrol bacteria seed soaking can increase the number of fleshy roots by 32.17%, the number of fine roots by 8.64%, and the yield by 4.71%.

[0065] (5) Biocontrol bacteria treatment incidence

[0066] From the data of Table 4, Bacillus cereus Y51 has good control effect on ginger rot caused by Enterobacter cloacae. In the case of soaking and later root irrigation and foliar spraying of biocontrol bacteria, the incidence of new ginger tubers can be reduced. After inoculation of Enterobacter cloacae, the incidence of new ginger tubers was as high as 42%. If combined with biocontrol bacteria soaking and later eight times of biocontrol bacteria root irrigation and foliar spraying, the incidence can be reduced to 7.7%.

[0067] Table 4: Incidence of biocontrol bacteria treatment

[0068]

[0069] Finally, it should be noted that: the above examples are used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements, or in the case of technical features in the above examples do not conflict with each other, can be combined in the manner recorded in the examples, and these modifications, replacements or combinations do not make the essence of the corresponding technical solution deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. Application of Bacillus cereus in preventing and treating ginger wilt caused by Enterobacter cloacae; the name of the biocontrol strain of the ginger wilt is Bacillus cereus Y51, which was preserved in the China General Microbiological Culture Collection Center on May 9, 2024, with the preservation number of CGMCC NO. 30536; the ginger wilt is ginger wilt caused by Enterobacter cloacae; the application method is as follows: The fermentation broth of Bacillus cereus Y51 is used as a biocontrol bacterial liquid, the biocontrol bacterial liquid is used to soak ginger tubers for 1-4 h, and the soaked ginger tubers are taken out and dried indoors after soaking; the soaked ginger tubers are used as seed tubers; Or, the fermentation broth of Bacillus cereus Y51 is used as a biocontrol bacterial liquid, the biocontrol bacterial liquid is used for root irrigation and leaf spraying of ginger plants, and the root irrigation and leaf spraying are performed for more than 2 times.

2. Use according to claim 1, characterized in that, The prevention method comprises: inoculating Bacillus cereus Y51 into a liquid culture medium, and after 24 h of fermentation culture, a fermentation broth is obtained, the fermentation broth is diluted by 10 times or 100 times to obtain a biocontrol bacterial liquid; the biocontrol bacterial liquid is used for soaking ginger tubers as seeds, and for root irrigation and leaf spraying of five-leaf-stage ginger plants; wherein, the 10-fold diluted liquid is used for soaking ginger tubers; the 100-fold diluted liquid is used for root irrigation and leaf spraying of five-leaf-stage ginger plants.

3. Use according to claim 1, characterized in that, The prevention method comprises: diluting the combined fermentation broth of Bacillus cereus Y51 and Burkholderia ambifaria YJ32 with the preservation number of CGMCC NO. 15922 to obtain a biocontrol bacterial liquid for ginger wilt.

4. Use according to any one of claims 1 to 3, characterized in that, The composition of the liquid culture medium for culturing Bacillus cereus Y51 is: corn syrup 50 g / L, sugar residue 20 g / L, soybean meal hydrolysate 15 mL / L, (NH4)2SO4 3 g / L, and sterilized water.

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