Bacillus gibsonii HMF12 and its application

Abstract

The present invention relates to a Bacillus mojavensis ( Oceanobacillus jeddahense ), namely HMF12, with a preservation number of CGMCC No. 30478. This strain can promote the seed germination of plants and the growth of plantlets under salt stress environments, and can also effectively prevent and control crop diseases, especially Botrytis cinerea of strawberries.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microorganisms, and specifically relates to a Bacillus jidensis HMF12 and its application. Background Art

[0002] The salinization of cultivated land and frequent plant diseases have long restricted the development of sustainable agriculture. Excessive soil salinity affects almost all life processes of plants, such as photosynthesis, protein synthesis, energy metabolism, and lipid metabolism of plants. At the same time, it will inhibit plant growth and affect the germination and emergence of plant seeds.

[0003] In cucumber, tomato, eggplant, sweet pepper, and strawberry under protected cultivation, gray mold often occurs, which is a disease caused by Botrytis cinerea ( Botrytis cinerea ). Botrytis cinerea is a world-wide distributed fungus that overwinters in the soil with sclerotia, conidia, and mycelia along with diseased and residual tissues. Most of its hosts are dicotyledonous plants. It mainly invades the host through wounds, natural orifices, and young tissues. After the disease occurs, a large number of conidia can be produced, which are spread by wind and rain for multiple secondary infections, causing gray mold. Flowers, fruits, leaves, and stems can all be diseased, resulting in crop wilting and death, affecting yield and quality. It is one of the most difficult important diseases to control in production.

[0004] Therefore, finding a microorganism that can not only effectively control crop diseases but also improve the seed germination rate of crops under salt stress or promote plant growth is the main direction of biological control. Summary of the Invention

[0005] The purpose of the present invention is to provide a Bacillus jidensis HMF12 and its application, which can promote the seed germination of plants under salt stress environment and the growth of plants, and can also effectively prevent and control crop diseases, especially strawberry gray mold.

[0006] The present invention adopts the following technical solutions:

[0007] A Bacillus jidensis ( Oceanobacillus jeddahense ) HMF12, with the preservation number of CGMCC No. 30478, is preserved in the China General Microbiological Culture Collection Center, with the address in Beijing, China, and the preservation date is April 30, 2024.

[0008] Further, the Bacillus jidensis HMF12 can antagonize Botrytis cinerea.

[0009] Further, the Bacillus jidensis HMF12 can tolerate salinity and colonize in saline-alkali soil.

[0010] A microbial inoculum, which comprises the above-mentioned Bacillus jidensis HMF12.

[0011] Furthermore, each gram of the microbial inoculant contains 4×10 9 cfu~7×10 9 cfu of Bacillus mojavensis HMF12 spores.

[0012] Application of Bacillus mojavensis HMF12 in promoting the germination of plant seeds under salt stress environment.

[0013] Application of Bacillus mojavensis HMF12 in promoting the growth of plants under salt stress environment.

[0014] Application of Bacillus mojavensis HMF12 in controlling gray mold of crops.

[0015] The beneficial effects of the present invention are as follows: Bacillus mojavensis HMF12 of the present invention has the characteristics of strong salt and alkali tolerance and high colonization rate in saline-alkali soil. The microbial inoculant prepared by using this strain can improve the emergence rate of crops under saline-alkali soil conditions, promote the growth of crops, and can also prevent and control gray mold of crops caused by Botrytis cinerea, and the control effect on strawberry gray mold reaches 86.45%. Description of the Drawings

[0016] Figure 1 It is the confrontation culture result of strain HMF12 and Botrytis cinerea.

[0017] Figure 2 It is the colony morphology of Bacillus mojavensis HMF12.

[0018] Figure 3 It is the cell morphology of Bacillus mojavensis HMF12.

[0019] Figure 4 The shown is the phylogenetic tree constructed by Bacillus mojavensis HMF12 based on 16S rDNA. Detailed Embodiments

[0020] The present invention will be further described below in conjunction with the embodiments and the drawings. The protection scope of the present invention is not limited to the embodiments, and any changes made by those skilled in the art within the scope defined by the claims also belong to the protection scope of the present invention.

[0021] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The reagents used in the following embodiments are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0022] Example 1 Isolation, purification and screening of the strain

[0023] Soil samples were collected from saline-alkali soil in Huanghua City, Hebei Province, a total of 6 samples, with 100 g taken from each. The 6 collected soils were evenly mixed. 10 g of the mixed soil sample was added to 100 mL of distilled water and stirred evenly. After standing for 1 min, 1 mL of the supernatant was taken and added to a test tube containing 9 mL of sterile water to obtain a 1×10 -1 dilution, and gradient dilution was carried out, successively diluted to 1×10 -4 , 1×10 -5 , 1×10 -6 concentrations. From the dilutions of 1×10 -4 , 1×10 -5 , 1×10 -6 concentrations, 0.1 μL of soil solutions with different dilution degrees was taken and placed on a high-saline-alkali selective medium plate, spread evenly, and then cultured at 37 °C for 24 - 48 h. According to the differences in colony and cell morphology, different strains were respectively transferred to the slant of the preservation medium test tube and cultured at 37 °C for 1 - 2 d. After the bacterial lawn grew, it was stored in a 4 °C refrigerator for standby. A total of 24 salt-tolerant strains were screened out, numbered 01 - 24.

[0024] The formula of the high-saline-alkali selective medium is: peptone 10 g, yeast extract 5 g, compound inorganic salts (NaCl∶KCl∶MgCl2 = 10∶1∶1) 30 g, agar 20 g, distilled water 1000 mL, pH 9.0, sterilized at 121 °C for 20 min.

[0025] The formula of the preservation medium is: peptone 10 g, yeast powder 5 g, NaCl 10 g, agar 20 g, distilled water 1000 mL, pH 7.0, sterilized at 121 °C for 20 min.

[0026] Confrontation test: The 24 strains that had been numbered and activated on the LB solid medium were respectively inoculated on both sides of the PDA medium plate, and then a 5-mm diameter Botrytis cinerea mycelial disc that had been activated was inoculated in the middle of the medium. The PDA plate inoculated only with the pathogen was used as a control. It was placed in an incubator at 28 °C for 96 h. When the control grew over the entire plate, the antibacterial effects of the 24 strains against Botrytis cinerea were observed, and the strains with inhibition zones were selected for re-screening. 3 bacteria strains with antagonistic effects against Botrytis cinerea were initially screened from the 24 strains, numbered 04, 12, and 17 respectively.

[0027] Continuing with Botrytis cinerea as the indicator bacterium, the strains numbered 04, 12, and 17 that were screened out were separately spot-inoculated at the four corners 3 cm away from the center of the petri dish. An activated Botrytis cinerea bacterial block with a diameter of 5 mm was inoculated at the center of the petri dish at the same time. Each strain was repeated three times. It was cultured at a constant temperature of 28 °C for 5 days. The diameter of the antibacterial circle was measured and recorded, and the average inhibition rate was calculated. The strain with the strongest antagonistic effect against Botrytis cinerea was selected as strain 04. The results of the confrontation culture are as Figure 1 shown. The Botrytis cinerea used in the experiment was provided by the Institute of Biology, Hebei Academy of Sciences.

[0028] Taking into account the growth rate and the antagonistic ability against the pathogenic bacterium of gray mold shown by the 24 salt-tolerant strains screened above when cultured on a high-salt medium, one of the strains, numbered 04, which has a strong antagonistic effect against Botrytis cinerea and a high growth rate on a high-salt and alkali selective medium petri dish, was named HMF12.

[0029] Example 2 Identification of Strain HMF12

[0030] 1. Morphological identification

[0031] Observation of the colony morphology found that the colony morphology of HMF12 was as Figure 2 , presenting a circular shape, with a neat edge and milky white on the Gibson medium. Through microscopic observation, HMF12 was a Gram-positive bacterium, as Figure 3 shown. It was preliminarily judged that strain HMF12 was a bacillus.

[0032] 2. Physiological and biochemical identification

[0033] Referring to "Bergey's Manual of Determinative Bacteriology" and "Manual of Systematic Identification of Common Bacteria" edited by Dong Xiuzhu and Cai Miaoying, the physiological and biochemical tests of the screened strain HMF12 were carried out. The results are shown in Table 1. Combining with the morphological observation, HMF12 was preliminarily identified as a strain of the genus Bacillus.

[0034] Table 1 Physiological and biochemical characteristics of strain HMF12

[0035] .

[0036] 3. Molecular biology identification

[0037] The DNA of strain HMF12 was extracted. After determining the fragment by PCR reaction, 16S rRNA gene sequencing was carried out. The sequence was compared by Blast on the NCBI website and a phylogenetic tree was constructed (see Figure 4 ). From the results of the phylogenetic tree, combined with the morphological observation and physiological and biochemical experiments, the antagonistic bacterium HMF12 was determined to be Bacillus oceanisediminis Jeddah ( Oceanobacillus jeddahense ).

[0038] The strain HMF12 was deposited with the China General Microbiological Culture Collection Center at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101, with the deposit number CGMCC No. 30478, and the deposit date was April 30, 2024.

[0039] Example 3 Colonization ability of Bacillus jiddaensis HMF12

[0040] Three bacterial strains HM-1, HM-3, and HM-6 that the applicant has used in actual production and showed good performance were selected for comparison, and the colonization ability of Bacillus jiddaensis HMF12 was evaluated through pot experiments.

[0041] (1) The soil for the pot experiment was taken from the applicant's experimental base, which was cinnamon soil. After air-drying, it was sterilized at high temperature to make sterile soil 1. The saline-alkali soil for the pot experiment was taken from the saline-alkali soil in Huanghua City, Hebei Province. After testing, the soil salt content was 0.31%. After air-drying, it was sterilized at high temperature to make sterile soil 2.

[0042] (2) The strains HM-1, HM-3, HM-6, and HMF12 were respectively inoculated into LB liquid medium, cultured at 30 °C and a rotation speed of 160 r / min for 48 h to obtain fermentation broth, and then the fermentation broth was formulated into a bacterial suspension with a viable bacteria content of 1.0×10 9 CFU / mL.

[0043] (3) The sterile soil 1 and sterile soil 2 were respectively and evenly mixed with the solid fermentation medium in a ratio of 4:1, and filled into flowerpots with a diameter of 10 cm and a height of 11 cm.

[0044] (4) 200 mL of the prepared bacterial suspension was watered, the flowerpots were placed in a tray filled with water, and the water was sucked up through the bottom holes to moisten the surface soil. The water in the tray was removed, and the flowerpots were placed in the greenhouse at a temperature of 20-28 °C. After 15 days, 1 g of soil was sampled from 6 cm deep to detect the number of effective viable bacteria contained. Each strain was repeated 5 times. During this period, water was poured once every 4 days.

[0045] Solid fermentation medium: wheat bran: rice husk: 3% glucose water = 7:3:3, urea 2.16%, potassium dihydrogen phosphate 2.77%. After mixing evenly, it was bagged and sterilized at 121 °C under high-pressure moist heat for 60 min, and intermittently sterilized 2 times.

[0046] The colonization number of each strain in each gram of soil was detected, and the colonization rate was statistically analyzed. As shown in Table 2.

[0047] Colonization rate (%) = colonization number ÷ inoculation number × 100.

[0048] Table 2 Detection results of the colonization rate of strains

[0049] .

[0050] As can be seen from Table 2, Bacillus mojavensis HMF12 has better colonization ability than other control strains. In fluvo-aquic soil, the colonization number of HMF12 is 1.64×10 9 CFU / g soil, while in saline-alkali soil, the colonization number of HMF12 is 1.54×10 9 CFU / g soil, with little difference between the two, indicating that saline-alkali soil has very little impact on the colonization of Bacillus mojavensis HMF12, and this strain can colonize and grow well in saline-alkali soil.

[0051] Example 4 Promoting growth test of Bacillus mojavensis HMF12 on wheat germination

[0052] Inoculate strain HMF12 into LB liquid medium, culture at 30 °C and 160 r / min for 48 h to obtain fermentation broth, and then prepare the fermentation broth into a bacterial suspension with a bacterial content of 1.0×10 9 CFU / mL.

[0053] Prepare the above-prepared bacterial suspension and sterile water into 7 concentrations according to the volume ratios of 1∶0, 1∶10, 1∶20, 1∶30, 1∶40, 1∶50, 0∶1, and record them as T0, T1, T2, T3, T4, T5, CK respectively, with a total of 7 treatments. Disinfect wheat seeds (Hanji wheat variety Jiemai 19) with 75% ethanol for 3 min and 1% sodium hypochlorite for 10 - 15 min. Soak the wheat seeds in the bacterial suspension with different concentrations for 24 h. Place 2 layers of filter paper on the petri dish, add 15 mL of sterile water to moisten, put it in an incubator at 28 °C, and measure the seed germination rate after 24 h, and also measure the seed germination rate at 3 d, 5 d, and 7 d.

[0054] Table 3 Effect of fermentation dilution of Bacillus mojavensis HMF12 on wheat seed germination rate

[0055] .

[0056] As shown in Table 3, the fermentation broth of Bacillus mojavensis HMF12 has a certain promoting effect on wheat seed germination. Without dilution, the germination rate of wheat seeds soaked in the fermentation broth reached 81.0% on the 7th day, which was 4% higher than the control. As the dilution ratio of the fermentation broth increased, the germination rate of wheat seeds showed different changes. When the fermentation broth was diluted 20 times and 30 times, the germination rates of wheat seeds reached 96% and 97% respectively, which were 19% and 20% higher than the control of 77%.

[0057] Example 5 Promoting growth test of Bacillus mojavensis HMF12 on wheat germination under salt stress conditions

[0058] 1) Preparation of bacterial suspension: Pick a loopful of inoculum from the preservation medium and inoculate it onto an LB solid plate medium. Activate it at 30 °C for 48 h. After single colonies grow, use forceps to hold a 100-µL yellow pipette tip to pick a single colony and put it into a 100-mL Erlenmeyer flask containing 30 mL of LB liquid medium. Culture it at 30 °C and 180 r / min for 72 h to obtain the fermentation broth of Bacillus jiddaensis HMF12. Dilute it with sterile distilled water, and use a spectrophotometer to adjust the absorbance value (OD) of the bacterial suspension to 0.5 at a wavelength of 600 nm. Set it aside for use.

[0059] 2) Test seeds and treatments: The test material is the seeds of wheat variety Nongda 212, purchased from the local seed company of the applicant. Select wheat seeds of the same size, surface sterilize them with 0.1% mercuric chloride for 3 min, and rinse them 3 times with sterile distilled water. Soak them in sterile water (CK) and the prepared bacterial suspension (HMF12) at room temperature for 2 h. After soaking, rinse the seeds 2 to 3 times with distilled water, arrange the seeds neatly in a petri dish (put 2 layers of moist sterile filter paper at the bottom of the dish), with 20 seeds in each petri dish, and 5 replicates in each group. Water them with 0%, 0.4% (low salt concentration), and 0.8% (high salt concentration) NaCl solutions respectively, and place them in an artificial climate chamber for cultivation. The light / dark cycle is 12 h / 12 h, the day / night temperature is 28 °C / 23 °C, and the relative humidity is 70%.

[0060] Start observing and recording the germination number every day from the first day of salt stress, and calculate the germination rate. Consider it germinated when the radicle length reaches half of the seed length. Record the germination number at 7 d as the number of finally germinated seeds. Calculate the germination rate according to the following formula. Germination rate (%) = (number of finally germinated seeds / number of test seeds) × 100. The test results are shown in Table 4.

[0061] Table 4 Germination rate of wheat seeds soaked with the fermentation broth of Bacillus jiddaensis HMF12 under different salt stresses

[0062] .

[0063] As can be seen from Table 4, under salt stress, the germination rates of wheat seeds soaked with HMF12 are 15% (salt concentration 0%), 29% (salt concentration 0.4%), and 22% (salt concentration 0.8%) higher than those soaked with sterile water respectively, indicating that soaking seeds with HMF12 can alleviate salt stress damage to a certain extent, and the effect under low salt (salt concentration 0.4%) stress is better than that under high salt (salt concentration 0.8%) stress.

[0064] Example 6 Bacillus jiddaensis HMF12 promotes the growth of maize plants

[0065] A pot experiment with maize was conducted to evaluate the growth-promoting effect of Bacillus mojavensis HMF12 on maize plants. The potting soil was a 1:1 mixture of loam soil from Changli area and coastal saline-alkali soil from Changli, and its salt content was detected to be 0.33%. The pot experiment was set up with a CK group (without HMF12 fermentation broth) and a fermentation broth group (with HMF12 fermentation broth). There were 15 maize seedlings in each treatment, 3 plants per pot, and a total of 5 pots. Each group used flowerpots with an inner diameter of 12.5 cm and a height of 11.5 cm, and each pot was filled with 0.9 kg of soil. When the maize seedlings grew to 3 leaves, those with consistent growth vigor were selected for transplanting. In the fermentation broth group, HMF12 fermentation broth (1.0×10 9 CFU / mL) was added to the roots of the maize at the time of transplanting. After adding the HMF12 bacterial suspension, the final inoculation amount per gram of soil in each pot was 5.2×10 6 CFU / g soil. The CK group was applied with an equal amount of sterile physiological saline as a control. The second application of bacteria was carried out after 7 days, and the same amount of bacterial solution and sterile physiological saline were applied to the maize potting soil. When the maize grew into the jointing stage, the plant indexes were measured. This was the stage with the fastest growth vigor, and the maize with the application of strain HMF12 showed obvious growth differences compared with the CK group.

[0066] The plant height, stem diameter, and root dry weight of all plants in each treatment were measured as shown in Table 5. The results showed that the plant height, root dry weight, and leaf dry weight of the maize plants with the application of HMF12 increased by 35.59%, 83.44%, and 80.50% respectively compared with the CK group. It indicated that Bacillus mojavensis HMF12 could significantly promote the growth of maize plants in the saline-alkali soil environment.

[0067] Table 5 Measurement results of plant height and biomass of maize plants in different treatments at the jointing stage

[0068] .

[0069] Example 7 Application of Bacillus mojavensis HMF12 bacterial agent in strawberry field planting

[0070] The Bacillus mojavensis HMF12 strain was inoculated into a 30 L fermenter containing 15 L of PB liquid medium and cultured at a constant temperature of 28 °C and 180 r / min for 36 h. The spores of Bacillus mojavensis were obtained by centrifugation, and then spray-dried. The spore dry powder of the bacterium was mixed with soluble starch to form a spore preparation with a final concentration of 4×10 9 cfu / g to 7×10 9 cfu / g.

[0071] A field experiment on Bacillus agents against Botrytis cinerea was carried out in strawberry continuous cropping greenhouses in Changli County and Funing District, Hebei Province. Before the flowering stage and at the early fruit stage, the Bacillus preparation was suspended in water at a weight ratio of 1:300 and sprayed on the plant leaves and / or the early fruits of strawberries in two times. The situation of Botrytis cinerea was investigated seven days after the second spraying. The dosage was 100 grams of Bacillus preparation per mu (the final concentration of the sprayed bacterium agent was 1.62×10 7 cfu / ml); carbendazim is an effective pesticide for controlling Botrytis cinerea of strawberries. The conventional control was to spray 300-fold liquid of 25% carbendazim wettable powder in two times before the flowering stage and at the early fruit stage; the blank control did not apply control drugs but applied the same weight of water as the experimental group. Each treatment had 3 replicates, and the incidence of Botrytis cinerea of strawberries was investigated.

[0072] Investigation method: At the mature stage, the diagonal five-point sampling method was used. 20 plants were fixed at each point, and a total of 100 plants were sampled to investigate the incidence and disease index of the plants, and the total number of investigated plants and the total number of diseased plants were recorded.

[0073] Grading standard for the disease index of Botrytis cinerea of strawberries:

[0074] Grade 0: No symptoms;

[0075] Grade 1: The diseased area accounts for less than 5% of the whole fruit surface;

[0076] Grade 3: The diseased area accounts for 6% - 10% of the whole fruit surface;

[0077] Grade 5: The diseased area accounts for 11% - 25% of the whole fruit surface;

[0078] Grade 7: The diseased area accounts for 26% - 50% of the whole fruit surface;

[0079] Grade 9: The diseased area accounts for more than 50% of the whole fruit surface.

[0080] Calculation method of control effect:

[0081] Incidence rate (%) = total number of diseased plants / total number of investigated plants × 100;

[0082] Disease index (%) = ∑(number of diseased plants × the grade value of the diseased plant) / (total number of investigated plants × the highest grade value) × 100;

[0083] Control effect (%) = (disease index of the control area - disease index of the treatment area) / disease index of the control area × 100.

[0084] The control effect is shown in Table 6. Using this Bacillus agent can reduce the incidence rate and disease index of Botrytis cinerea of strawberries, and the disease prevention effect can reach more than 94.71% in 7 days.

[0085] Table 6 Control effect of Bacillus jiddaensis HMF12 agent on Botrytis cinerea of strawberry

[0086] 。

[0087] It can be seen from the above results that the strain HMF12 of the present invention has a good antagonistic effect on Botrytis cinerea of strawberry, and its spore agent can effectively prevent and control the occurrence of Botrytis cinerea in strawberry greenhouses, and is superior to the control effect of using carbendazim.

[0088] The above is only the preferred embodiment of the present invention, rather than a limitation on its protection scope. Any improvement made by those skilled in the art to the present invention without creative work shall be considered within the protection scope of the present invention.

Claims

1. A Jeddah Bacillus ( Oceanobacillus jeddahense ) HMF12, characterized in that The deposit number is CGMCC No.30478.

2. The Bacillus jeddahense HMF12 according to claim 1, characterized in that It can antagonize Botrytis cinerea.

3. The Bacillus jeddahense HMF12 according to claim 1, characterized in that It is resistant to salt and alkali.

4. A microbial agent, characterized in that: It includes the Bacillus jeddah HMF12 as claimed in claim 1.

5. The microbial agent according to claim 4, characterized in that: It contains 4×10 9 cfu / g~7×10 9 cfu / g of Bacillus jeddahcensis HMF12 spores.

6. A use of the Bacillus jeddahense HMF12 as claimed in claim 1 in promoting wheat seed germination under salt stress environment.

7. A use of the Bacillus jeddahense HMF12 as claimed in claim 1 in promoting the growth of wheat or corn under salt stress environment.

8. Use of the Bacillus jeddahii HMF12 as claimed in claim 1 in preventing and controlling gray mold of crops caused by Botrytis cinerea.

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