Biocontrol strain for ginger bacterial wilt caused by enterobacter cloacae and control method for ginger bacterial wilt

Abstract

The invention relates to the technical field of microorganisms, in particular to a ginger bacterial wilt biocontrol strain caused by enterobacter cloacae and a ginger bacterial wilt control method. The name of the ginger bacterial wilt biocontrol strain is bacillus cereus Y51, the strain is preserved in the China General Microbiological Culture Collection Center on May 9, 2024, and the preservation number of the strain is CGMCC NO.30536. The invention further discloses a preparation method of the ginger bacterial wilt biocontrol strain. Experiments prove that the bacillus cereus Y51 has a good prevention effect on the enterobacter cloacae on ginger plants, so that the bacillus cereus Y51 can be used for preventing and treating the ginger blast, and has the advantages of being environmentally friendly, free of pesticide residues and green and environmentally friendly. The biocontrol strain provided by the invention fills up the blank of the existing ginger bacterial wilt biological control strain in the aspect of targeting specific pathogenic bacteria species.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and specifically relates to a biocontrol strain for bacterial wilt of ginger caused by Enterobacter cloacae and a control method for bacterial wilt of ginger. Background Art

[0002] Bacterial wilt of ginger generally occurs severely, resulting in a reduction in ginger yield and even a complete crop failure. At present, the control methods for soil-borne diseases of ginger mainly rely on chemical control. However, a large amount of chemical pesticides will damage the ecological environment, increase the drug resistance of diseases, and increase the pesticide residue in the soil. Biological control with microorganisms and microbial metabolites has become the main development direction for the control of plant soil-borne diseases. The main symptoms of bacterial wilt of ginger are that the plants turn yellow and withered, the tubers rot and stink in large areas, and finally only the epidermis of the ginger pieces remains. Once bacterial wilt of ginger breaks out, there are almost no effective control measures, and even the surrounding plots and adjacent water bodies are often diseased throughout the year.

[0003] Plant growth promoting rhizobacteria (PGPR) refers to beneficial bacteria that can colonize the rhizosphere of plants and directly or indirectly promote plant growth and development or regulate metabolism. The action mechanisms of PGPR mainly include: being more effective in colonizing the roots compared with other soil microorganisms, having an antagonistic effect on harmful microorganisms, being in an advantageous position in the nutritional competition in the rhizosphere, especially for iron, being able to secrete plant hormones to promote plant growth, being able to induce plants to produce systemic resistance, and secreting enzymes to degrade pathogenic microorganisms, etc.

[0004] Chinese Patent Application CN110760462B discloses an antagonistic bacterium YJ32 for controlling bacterial wilt of ginger and its application, which discloses a strain of Burkholderia ambifaria. This strain has a strong inhibitory effect on the pathogenic bacteria Ralstonia solanacearum causing bacterial wilt of ginger and Enterobacter mori causing mulberry bacterial wilt, and can target the prevention of bacterial wilt of ginger. Whether it is effective against bacterial wilt of ginger caused by Enterobacter cloacae is not recorded in this scheme. Since this strain is screened using two pathogenic bacteria of bacterial wilt of ginger, Ralstonia solanacearum and Enterobacter mori JLIY2, as target pathogenic bacteria, it is inferred that this strain is likely to not have a good biocontrol effect on bacterial wilt of ginger caused by Enterobacter cloacae. At present, the vast majority of researchers focus on the primary pathogenic bacterium of bacterial wilt of ginger, Ralstonia solanacearum, to develop corresponding biocontrol bacteria. Bacterial wilt of ginger caused by Enterobacter cloacae will cause the root tips of ginger to rot, the plant growth to be slow, and it is not easy to be quickly detected, so it is easily overlooked by researchers. Summary of the Invention

[0005] (1) Technical problems to be solved In view of the above-mentioned disadvantages and deficiencies of the prior art, the present invention provides a biocontrol strain for bacterial wilt of ginger caused by Enterobacter cloacae and a method for preventing and treating bacterial wilt of ginger, which fills the blank in the aspect of targeting specific pathogenic bacteria species among the existing biological control bacteria for bacterial wilt of ginger.

[0006] (2) Technical solutions In the first aspect, the present invention provides a biocontrol strain for bacterial wilt of ginger caused by Enterobacter cloacae, named Bacillus cereus ( Bacillus cereus ) Y51. This strain was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on May 9, 2024, with the deposit number CGMCC NO. 30536.

[0007] In the second aspect, the present invention provides a method for preventing and treating bacterial wilt of ginger caused by Enterobacter cloacae, which includes using the above-mentioned Bacillus cereus ( Bacillus cereus ) Y51 as a biocontrol bacterium to prevent and treat bacterial wilt of ginger.

[0008] According to a preferred embodiment of the present invention, the prevention and treatment method includes: using the fermentation broth of Bacillus cereus ( Bacillus cereus ) Y51 as a biocontrol bacterium solution, soaking the ginger tubers with this biocontrol bacterium solution for 1 - 4 hours, fishing out and air-drying indoors after soaking, and using the soaked ginger tubers as sowing tubers.

[0009] According to a preferred embodiment of the present invention, the prevention and treatment method includes: using the fermentation broth of Bacillus cereus ( Bacillus cereus ) Y51 as a biocontrol bacterium solution, and performing root irrigation and / or leaf spraying on ginger plants at the five-leaf stage with this biocontrol bacterium solution.

[0010] According to a preferred embodiment of the present invention, the prevention and treatment method includes: inoculating Bacillus cereus ( Bacillus cereus ) Y51 into a liquid medium, obtaining a fermentation broth after fermentation culture for 24 hours, diluting the fermentation broth by 10 times or 100 times to obtain a biocontrol bacterium solution; using the biocontrol bacterium solution to soak ginger tubers used as seeds, and performing root irrigation and / or leaf spraying on ginger plants at the five-leaf stage; wherein, when soaking ginger tubers, a 10-fold dilution solution is used; when performing root irrigation and / or leaf spraying on ginger plants at the five-leaf stage, a 100-fold dilution solution is used.

[0011] According to a preferred embodiment of the present invention, the prevention and treatment method includes: mixing Bacillus cereus ( Bacillus cereus ) Y51 with Burkholderia ambifaria with the deposit number CGMCC NO. 15922 (Burkholderia ambifaria ) After diluting the combined fermentation broth of YJ32, it is used as a biocontrol bacterial solution for bacterial wilt of ginger. Using the biocontrol bacteria against different pathogenic bacteria of bacterial wilt of ginger in combination can cover the bacterial wilt of ginger caused by the three currently prevalent pathogenic bacteria, covering almost all pathogenic bacteria species, and can greatly promote the emergence rate, growth and development speed, and yield of ginger tubers.

[0012] Preferably, the composition of the liquid medium is: corn steep liquor 50 g / L, sugarcane bagasse 20 g / L, soybean meal hydrolysate 15 mL / L, (NH 4 ) 2 SO 4 3 g / L and sterilized water. This liquid medium has a low cost (cost < 25 yuan / L), is a comprehensive utilization of agricultural by-products, is suitable for large-scale use, and can reduce costs. In addition, the liquid medium can also be one of LB medium, TSB medium, and kitchen waste hydrolysate medium.

[0013] (III) Beneficial effects The Bacillus cereus ( Bacillus cereus ) Y51 screened in the present invention has a good biocontrol effect on Enterobacter cloacae on ginger plants. The Bacillus cereus ( Bacillus cereus ) Y51 can be made into a microbial inoculant or the fermentation broth of Bacillus cereus ( Bacillus cereus ) Y51 is used to treat ginger tubers or plants for preventing and controlling bacterial wilt of ginger, which has the advantages of environmental friendliness, no agricultural residues, and green environmental protection. The biocontrol strain of the present invention fills the blank in targeting specific pathogenic bacteria species among the existing biological control bacteria for bacterial wilt of ginger. Description of the drawings

[0014] Figure 1 It is the in vitro leaf control effect results of different candidate biocontrol strains for bacterial wilt of ginger in Example 1.

[0015] Figure 2 It is the effect of different candidate biocontrol strains for bacterial wilt of ginger antagonizing pathogenic bacteria in Example 1.

[0016] Figure 3 It is for Bacillus cereus The morphological diagram of Y51 on LB solid medium. Specific implementation manners

[0017] In order to better explain the present invention for easy understanding, the present invention will be described in detail below with reference to the drawings through specific implementation manners.

[0018] Example 1 This example relates to the screening and identification process of Bacillus cereus ( Bacillus cereus ) Y51, which is as follows: 1. Experimental materials (1)Collection of soil samples Soil samples for the isolation and screening of biocontrol bacteria against bacterial wilt of ginger were collected from the rhizospheres of different plants in experimental fields in various leagues and cities of Inner Mongolia Autonomous Region by the random sampling method, including rhizosphere tissues and rhizosphere soils. These samples have been stored in the strain bank of the Plant Pathology Laboratory of Inner Mongolia Agricultural University. Enterobacter cloacae has been detected in Inner Mongolia soils, and relevant studies have proven that Enterobacter cloacae is one of the pathogenic bacteria of bacterial wilt of ginger (ginger rot).

[0019] (2)Preparation of culture media and reagents LB medium: Peptone 10 g / L, yeast 5 g / L, NaCl 10 g / L, pH 7.0 - 7.5, sterilized by steam at 121 °C for 20 min. 20 g of agar powder needs to be added to the solid medium.

[0020] 2. Experimental methods (1)Primary screening of biocontrol strains against bacterial wilt of ginger The test strains stored in the strain bank of the Plant Pathology Laboratory of Inner Mongolia Agricultural University were evenly spread on LB medium for activation and cultured at 28 °C for 48 h. The activated strains (strains with biocontrol potential) were inoculated with a 5 - mm - diameter bacterial cake into 30 ml of LB liquid medium using a sterile pipette tip, cultured at 28 °C with constant shaking at 180 rpm for 24 h, and then diluted 10 - fold to obtain a bacterial solution. Healthy ginger leaves with the same leaf age and growth vigor were selected, rinsed with sterile water, soaked in the bacterial solution for 10 min, and after the leaves were air - dried, a bacterial cake of Enterobacter cloacae was punched out and a 5 - mm - diameter bacterial cake was inoculated in the center of the leaf. One bacterial cake was inoculated on each leaf, and a total of 5 leaves were inoculated. The experiment was set up with 3 replicates. Leaves soaked in sterile water for 10 min were used as negative controls, and leaves soaked in sterile water and inoculated with Enterobacter cloacae were used as positive controls. They were placed on filter paper moistened with sterile water and placed in a light incubator at 28 °C with 8 - hour light and 8 - hour darkness to observe the expansion of disease spots. As Figure 1 shown, it is the control effect result of detached leaves.

[0021] The calculation method of the control effect of detached leaves is shown in the following formula:

[0022] The test results showed that the strain named Y51 had the best control effect, and the leaves remained green, healthy, and disease - free 7 days after inoculating the pathogenic bacterial cake. The control effects of other strains such as 14 - 5, 3 - 11 - 2, and X37 were the second. The leaves were green, healthy, and disease - free 5 days after inoculating the pathogenic bacteria, but started to turn yellow on the 6th day. The leaves of the remaining strains died of disease successively within 3 days after inoculating the pathogenic bacteria (Table 1 shows the control effect by the detached leaf method).

[0023] Table 1: Control effect by the detached leaf method

[0024] Note: CK is the negative control of water immersion; CK1 is the positive control of water immersion inoculated with pathogens.

[0025] (2) Determination of biocontrol functions of strains Y51, 14-5, 3-11-2, and X37 According to the above method, the biocontrol strains whose leaves remain healthy and green 5 days after inoculation with pathogens were screened for plate confrontation experiments. Enterobacter cloacae QK was inoculated into 50mL LB liquid culture medium, cultured at a constant temperature of 28℃ and 180rpm for 48h, diluted 100 times with sterile distilled water, and 30μL was applied on a filter paper with a diameter of 5 mm, and air-dried in a sterile operating table. Strains Y51, 14-5, 3-11-2, and X37 were inoculated into 50mL LB liquid culture medium, cultured at a constant temperature of 28℃ and 180rpm for 48h, and 50μL of the above culture solution was evenly applied on the surface of LB solid culture medium. After the surface culture solution was dried, the filter paper inoculated with Enterobacter cloacae was picked up with sterilized tweezers and placed in the center of the culture medium. The experiment was repeated 3 times, and the size of the inhibition zone was observed in a constant temperature incubator at 28℃ for 3d. Figure 2 The results showed that strains Y51, Y57, 14-5, 3-11-2, and X37 did not produce inhibition zones when antagonizing Enterobacter cloacae QK, indicating that strain Y51 did not directly antagonize Enterobacter cloacae QK, and its biocontrol effect may be achieved through induced antagonism.

[0026] (3) Potted plant experiment to verify the biocontrol ability of biocontrol bacteria Ginger tubers with buds were placed in pots and planted at 28°C for 50 days. After the strain was streaked on LB medium, it was cultured in a liquid medium shaker for 48 hours, and then 3 mL of biocontrol bacteria solution was diluted 100 times and added to the pots. 3 days after adding the biocontrol bacteria solution, the pathogen fermentation solution was added and the disease of the pots was observed. The test results showed that the strain named Y51 had the best prevention effect.

[0027] (4) Identification results of strain Y51 1. Morphological characteristics identification results The colonies of strain Y51 on LB solid medium are round, smooth, slightly wrinkled, and white. Figure 3 Shown is the morphology of strain Y51 on LB solid medium.

[0028] 2. Physiological and biochemical characteristics identification results Strain Y51 was Gram-positive.

[0029] 3. 16S rDNA sequence sequencing results and construction of phylogenetic tree. According to the alignment and analysis in the NCBI database, it was found that strain Y51 is Bacillus cereus, with the Chinese name being Bacillus cereus Y51. This strain has been preserved by the applicant at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on May 9, 2024, with the preservation number CGMCC NO. 30536.

[0030] Example 2

[0031] This example is about the field application of Bacillus cereus Y51. The application method is as follows: (1)Biocontrol bacteria seed soaking The Bacillus cereus Y51 strain was inoculated into a liquid medium for fermentation culture at an inoculation ratio of 5%. The liquid medium can be LB medium or an agricultural by - product medium, with the aim of obtaining a large number of bacteria and metabolites. The composition of the agricultural by - product medium is: corn steep liquor 50 g / L, sugarcane bagasse 20 g / L, soybean meal hydrolyzate 15 mL / L, (NH 4 ) 2 SO 4 3 g / L and sterilized water. In this example, the agricultural by - product medium was used for fermentation for 24 h, and the fermentation broth was diluted 10 times to soak ginger tubers for 2 h. After the seed soaking, the tubers were fished out and air - dried indoors.

[0032] (2)Biocontrol bacteria root irrigation and leaf spraying The soaked ginger tubers were sown in the field and managed according to conventional planting techniques.

[0033] For the ginger in some test fields, the following management was carried out after emergence: Starting from the five - leaf stage of ginger, a diluted solution of the fermentation broth of Bacillus cereus Y51 was used for root irrigation and leaf spraying. Specifically, after diluting the fermentation broth fermented for 24 h in step (1) by 100 times, the 100 - fold diluted solution was used for root irrigation and leaf spraying. 10 L was sprayed per row, and 20 L was used for root irrigation.

[0034] Compare the incidence of ginger blast when only biocontrol bacteria seed soaking treatment is carried out and when combined with biocontrol bacteria seed soaking and subsequent 2 - 8 times of root irrigation and leaf spraying under the condition of using pathogenic bacteria treatment.

[0035] (3)Data analysis of the above - ground growth of ginger Table 2: Statistics of the test results of the above - ground growth of ginger

[0036] Note: CK represents the blank control group inoculated with Enterobacter cloacae; B represents the treatment group inoculated with Enterobacter cloacae + biocontrol bacteria.

[0037] It can be seen from the data in Table 2 that Bacillus cereus Y51 has an obvious promoting effect on the growth of the above-ground part of ginger. In this experiment, 25 plants were sampled from each column, and the average stem diameter, plant height, and total number of branches of these 25 plants were counted. The results showed that the stem diameter and plant height of the treatment group were higher than those of the CK group, and the total number of branches was significantly increased compared with the CK group. Soaking the seeds with the biocontrol bacteria can promote the growth of the above-ground part of ginger.

[0038] (4)Statistics of the test results of the underground part of ginger Table 3: Statistics of the test results of the underground part of ginger

[0039] Note: CK represents the blank control group inoculated with Enterobacter cloacae; B represents the treatment group inoculated with Enterobacter cloacae + biocontrol bacteria.

[0040] It can be seen from the data in Table 3 that Bacillus cereus Y51 has an obvious promoting effect on the growth and yield of the underground part of ginger. The total number of fleshy roots and fine roots of 25 plants in each treatment was calculated. The results showed that soaking the seeds with the biocontrol bacteria, and later spraying and irrigation with the biocontrol bacteria could significantly increase the number of fleshy roots and fine roots of ginger tubers and improve the yield. When the ginger tubers were soaked with the pathogenic bacteria, soaking the seeds with the biocontrol bacteria could increase the number of fleshy roots of the ginger plants by 26.44%, the number of fine roots by 16.56%, and the yield by 31.15%. When the pathogenic bacteria were sprayed on the soil, soaking the seeds with the biocontrol bacteria could increase the number of fleshy roots by 32.17%, the number of fine roots by 8.64%, and the yield by 4.71%.

[0041] (5)Incidence rate of biocontrol bacteria treatment It can be seen from the data in Table 4 that Bacillus cereus Y51 has a good control effect on ginger rot caused by Enterobacter cloacae. Soaking with the biocontrol bacteria, and later irrigating the roots and spraying the biocontrol bacteria on the leaves could reduce the incidence rate of new ginger tubers. After inoculating with Enterobacter cloacae, the incidence rate of new ginger tubers was as high as 42%. If soaking the seeds with the biocontrol bacteria and later irrigating the roots and spraying the biocontrol bacteria eight times in total, the incidence rate could be reduced to 7.7%.

[0042] Table 4: Incidence rate of biocontrol bacteria treatment

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements, or in the case where the technical features in the above embodiments do not conflict with each other, can be combined in the manner described in the embodiments, and these modifications, replacements or combinations do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Bacillus cereus in preventing and controlling ginger blight caused by Enterobacter cloacae; the name of the ginger blight biocontrol strain is Bacillus cereus ( Bacillus cereus ) Y51, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 9, 2024, with the deposit number CGMCC NO.30536; the ginger blight is caused by Enterobacter cloacae; Bacillus cereus ( Bacillus cereus ) The fermentation liquid of Y51 is used as the biocontrol liquid, and the ginger tubers are soaked in the biocontrol liquid for 1-4 hours. After the soaking, the ginger tubers are taken out and air-dried indoors, and the soaked ginger tubers are used as sowing tubers; Bacillus cereus ( Bacillus cereus ) The fermentation liquid of Y51 was used as the biocontrol liquid, and the ginger plants were irrigated with the liquid and sprayed on the leaves. The root irrigation and leaf spraying were performed more than 2 times.

2. The use according to claim 1, characterized in that: The control method comprises: introducing Bacillus cereus ( Bacillus cereus ) Y51 is inoculated into a liquid culture medium, and fermented for 24 hours to obtain a fermentation liquid, which is diluted 10 times or 100 times to obtain a biocontrol solution; the biocontrol solution is used to soak ginger tubers as seeds, and the ginger plants at the five-leaf age are irrigated with roots and sprayed with leaves; wherein, when soaking ginger tubers, a 10-fold dilution is used; when irrigating the roots and spraying the leaves of the ginger plants at the five-leaf age, a 100-fold dilution is used.

3. The use according to claim 1, characterized in that: The control method comprises: introducing Bacillus cereus ( Bacillus cereus )Y51 and the bidirectional Burkholderia with the deposit number of CGMCC NO.15922 ( Burkholderia ambifaria ) The combined fermentation liquid of YJ32 was diluted and used as the biocontrol liquid for ginger blight.

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

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