Application of bacillus velezensis JB23 in promoting sugarcane bud germination under stress conditions

Abstract

The application provides an application of bacillus velezensis JB23 in promoting sugarcane bud germination under stress conditions, and belongs to the technical field of microorganisms.The bacillus velezensis JB23 provided by the application is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC M 20221134.The bacillus velezensis JB23 provided by the application can improve the germination rate of sugarcane, even under high-temperature and drought stress, can slow down the high-temperature stress on the sugarcane bud, and can guarantee the development of the stressed sugarcane bud to a certain extent.Meanwhile, under the field environment, the bacterial suspension of the JB23 strain can promote the growth of the plant height and stem diameter of the sugarcane stem, increase the aboveground and underground biomass and SPAD value, and improve the photosynthetic efficiency, and is a biological control strain which can also exhibit good growth promotion ability under the field condition, and provides a new strain resource for a sugarcane growth promotion microbial resource library.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to the application of Bacillus velezensis JB23 in promoting the germination of sugarcane buds under stress conditions. BACKGROUND

[0002] Sugarcane is a C4 crop with huge yield and biomass, and is the main sugar crop. It can also be used as a raw material in industry and light industry. However, various factors have restricted the further development of the sugarcane industry. After harvesting, sugarcane is often piled up in the field, and in winter in Guangxi, the temperature can reach 30℃, which will exacerbate the loss of water in sugarcane, drought, and other stress conditions, resulting in low emergence rate of new planting and ratoon sugarcane in the field, further leading to an inability to significantly improve yield per mu. Therefore, ensuring germination rate and emergence rate and cultivating strong seedlings are of great significance to improving sugarcane yield and sugar yield.

[0003] The use of chemical fertilizers and pesticides can effectively improve yield, but there are problems such as low utilization rate, leading to soil compaction and environmental pollution. The restriction of chemical pesticides is also a research focus in the development of Chinese agriculture. With the increasing importance of sustainable development, encouraging the reduction of chemical pesticide use and finding alternatives to chemical pesticide fungicides are gradually regarded as key methods to ensure food security and solve chemical pollution. More green and environmentally friendly microbial fertilizer has been researched and applied in sugarcane and has been proven to be an effective means of promoting growth.

[0004] Bacillus is a common raw material for microbial fertilizer, which has the functions of dissolving phosphorus and potassium, and can also produce a large amount of IAA, protease, cellulase and other promoting substances. The present application uses Bacillus velezensis JB23, which has been proven to have significant effects in the prevention and control of sugarcane diseases, to explore its role in promoting the germination of sugarcane and the growth of different varieties of sugarcane in the field, in order to provide scientific basis and technical reserves for the restriction of chemical pesticide use in sugarcane production and the green and sustainable development of the sugarcane industry. SUMMARY

[0005] Therefore, the present application aims to provide the application of Bacillus velezensis JB23 in promoting the germination of sugarcane buds under stress conditions. The Bacillus velezensis JB23 provided by the present application can improve the germination rate of sugarcane under stress conditions and promote the growth of sugarcane.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0007] The Bacillus velezensis JB23 is preserved in the China Center for Type Culture Collection, and the preservation time is July 18, 2022, the preservation address is Wuhan University China Center for Type Culture Collection in Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC M 20221134.

[0008] The Bacillus velezensis JB23 described in the application has been disclosed in a patent application with the application publication number “CN116254199A”.

[0009] In some embodiments, the stress conditions include but are not limited to high temperature stress, and / or drought stress. The Bacillus velezensis JB23 of the application can slow down the reduction of germination rate of sugarcane buds caused by high temperature stress, and to a certain extent, guarantee the development of sugarcane buds.

[0010] The application also provides the application of the Bacillus velezensis JB23 in promoting the germination of sugarcane buds, wherein the Bacillus velezensis JB23 is preserved in the China Center for Type Culture Collection, and the preservation time is July 18, 2022, the preservation address is Wuhan University China Center for Type Culture Collection in Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC M 20221134.

[0011] The application also provides the role of the Bacillus velezensis JB23 in promoting the growth of sugarcane, wherein the Bacillus velezensis JB23 is preserved in the China Center for Type Culture Collection, and the preservation time is July 18, 2022, the preservation address is Wuhan University China Center for Type Culture Collection in Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC M 20221134.

[0012] In some embodiments, the varieties of the sugarcane include but are not limited to Zhucen No. 9, Xintaitang No. 22, Liucheng 05136.

[0013] In some embodiments, the promotion of the growth of sugarcane includes but is not limited to: promoting the growth of the plant height of sugarcane; promoting the growth of the stem diameter; promoting the biomass of the aboveground and underground parts of sugarcane; and improving the relative chlorophyll content SPAD value of sugarcane. The Bacillus velezensis JB23 of the application can secrete IAA, protease, cellulase, and amylase, and can improve the biomass of sugarcane in the field, which is conducive to the yield increase of sugarcane.

[0014] The application also provides a bacterial agent containing Bacillus velezensis JB23, which is preserved in the China Center for Type Culture Collection on July 18, 2022, and has a preservation number of CCTCC M 20221134.

[0015] The bacterial agent can be applied to any one or more of the following:

[0016] (1) promoting sugarcane bud germination;

[0017] (2) promoting sugarcane bud germination under stress conditions;

[0018] (3) promoting sugarcane growth.

[0019] In some embodiments, the bacterial agent is prepared by inoculating 50% (v / v) glycerol mixed bacterial solution of Bacillus velezensis JB23 strain preserved in a-80 DEG C refrigerator into LB broth medium at an amount of 0.1% (v / v) for activation, culturing in a 37 DEG C shaker for 24 hours to obtain a seed solution, and further expanding the seed solution to obtain the bacterial agent through centrifugal filtration.

[0020] The application also provides a method for promoting sugarcane bud germination under stress conditions, which comprises soaking sugarcane seeds in a suspension of Bacillus velezensis JB23 or the bacterial agent described in the above technical solution for 1 hour, and then planting the seeds after drying.

[0021] In some embodiments, when the suspension is a suspension of Bacillus velezensis JB23, the OD 600 value of the suspension is 1.

[0022] Beneficial technical effects: the application provides the application of Bacillus velezensis JB23 in promoting sugarcane bud germination under stress conditions, which is preserved in the China Center for Type Culture Collection on July 18, 2022, and has a preservation number of CCTCC M 20221134. The Bacillus velezensis JB23 provided by the application can improve the germination rate of sugarcane, slow down the high-temperature stress on sugarcane buds even under high-temperature and drought stress, and to a certain extent, guarantee the development of stressed sugarcane buds. Meanwhile, under field conditions, the bacterial suspension of the JB23 strain can promote the growth of sugarcane stem height and stem diameter, increase the aboveground and underground biomass and relative chlorophyll content SPAD value, and improve photosynthetic efficiency, etc. It is a biological control strain that can also exhibit good growth-promoting ability under field conditions, and provides a new strain resource for a sugarcane growth-promoting microbial resource library. Attached Figure Description

[0023] Figure 1 Phenotype of Bacillus belyss JB23 on protease medium;

[0024] Figure 2 Phenotypic characteristics of Bacillus belyssus JB23 on cellulase medium;

[0025] Figure 3 Phenotype of Bacillus belyss JB23 on starch hydrolase medium;

[0026] Figure 4 The effect of Bacillus berberis JB23 bacterial suspension on sugarcane seedling height;

[0027] Figure 5 The effects of Bacillus belyss JB23 bacterial suspension treatment on sugarcane budding and growth under high temperature and drought stress are shown in Figure a. The left side of Figure a shows the phenotype of sugarcane after JB23 treatment, and the right side shows the control treatment. Figure b shows the statistical results of plant height.

[0028] Figure 6 The effects of JB23 bacterial suspension treatment on the physiological indicators of Zhongzhe 9, ROC22, and Liucheng 05136 sugarcane in the field were investigated. Among them, a represents the statistics on seedling emergence, b represents the statistics on plant height, c represents the statistics on stem diameter, d represents the statistics on aboveground biomass, e represents the statistics on underground biomass, and f represents the effect on the SPAD value of the first leaf of sugarcane. Detailed Implementation

[0029] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments. Unless otherwise specified, the materials, reagents, etc., used in the embodiments and experimental examples of the present invention can be obtained commercially; unless otherwise specified, the methods used in the embodiments and experimental examples of the present invention are conventional methods.

[0030] Example 1: Determination of the growth-promoting properties of JB23

[0031] (1) IAA production capacity determination

[0032] This invention tested the ability of strain JB23 to produce indole-3-acetic acid (IAA). Specifically, strain JB23 was cultured in LB medium containing 0.2 g / L tryptophan at 37°C with shaking for 48 h. After centrifugation at 12000 rpm for 5 min, the supernatant was collected and mixed with an equal volume of Salkowski colorimetric solution. After incubation in the dark for half an hour, the absorbance at 530 nm was measured. A standard curve was prepared using IAA solutions of different concentrations. Finally, the OD value was... 530Substituting the values ​​into the standard curve, the amount of IAA produced by strain JB23 was calculated.

[0033] The standard curve was constructed as follows: Standard solutions with concentrations of 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL were prepared using IAA standards. The absorbance of these standard solutions at 530 nm was measured. A standard curve was then plotted based on the measured absorbance values ​​and the corresponding IAA standard concentrations: y = 0.0327x + 0.0181, R² = 0.9983, where y represents the OD value. 530 The absorbance value is given, where x represents the corresponding IAA concentration (μg / mL). The final IAA concentration of JB23 was calculated to be 0.97 μg / mL based on the standard curve.

[0034] (2) Protease detection

[0035] The protease was detected using sterilized skim milk powder culture medium (12g skim milk powder, 20g agar, ddH2O to a final volume of 1000mL). The OD... 600 Inoculate 10 μL of JB23 bacterial suspension with a value of 1 into the center of the plate, incubate at 37°C for 5 days, and observe whether a clear zone appears around the colony.

[0036] The results are as follows Figure 1 As shown, a clear zone appeared around strain JB23, indicating that strain JB23 can produce protease.

[0037] (3) Cellulase detection

[0038] The detection of cellulase involves measuring the OD... 600 10 μL of JB23 bacterial culture with a pH of 1 was inoculated into cellulose medium (10 g sodium carboxymethyl cellulose, 10 g peptone, 20 g agar, 5 g yeast extract, 1 g KH2PO4, 5 g NaCl, ddH2O to a final volume of 1000 mL, pH 7.0) and cultured at 37°C for 5 days. After inoculation, the culture was stained with 1% Congo red for 15 min, and then rinsed twice with 1 M NaCl solution. The presence of a yellow halo around the colony was observed.

[0039] The results are as follows Figure 2 As shown, a yellow halo appeared around strain JB23, indicating that it has the ability to produce cellulase.

[0040] (4) Amylase detection

[0041] Prepare starch hydrolysis medium (yeast extract 5 g / L, peptone 10 g / L, NaCl 5 g / L, soluble starch 30 g / L, agar 15 g / L, pH 7.6), sterilize at 115℃ for 20 min, and then pour the medium onto a plate for later use. Take 10 μL of OD200 medium. 600Fresh JB23 bacterial liquid with OD600=1 was inoculated in the center of starch hydrolysis medium plate, and after 24h incubation at 37℃, Lugol's iodine solution was used to stain and observe. If transparent hydrolysis ring appeared around the colony, it proved that the strain could hydrolyze starch.

[0042] Figure 3 As shown in the figure, transparent hydrolysis ring appeared around JB23, indicating that it had the ability to secrete amylase.

[0043] Example 2 Promoting effect of JB23 strain on sugarcane sprouting

[0044] (1) Preparation of Bacillus velezensis JB23 bacterial liquid:

[0045] The 50% (v / v) glycerol mixed bacterial liquid of Bacillus velezensis JB23 strain stored at -80℃ was inoculated in LB broth medium at an amount of 0.1% (v / v) for activation; after 24h incubation at 37℃ on a shaker at 220r / min, fresh seed liquid was obtained; in a fermentation tank, LB broth medium powder was added at a concentration of 20g / L, and after high-temperature sterilization at 121℃ for 30min, the seed liquid was added to the fermentation tank at 1% (v / v) and incubated at 37℃ at 180r / min for 72h to obtain fresh fermentation liquid; the fresh JB23 bacterial liquid was centrifuged at 5000rpm for 30min, the waste liquid was discarded, and the precipitated bacterial cells were resuspended in sterilized water; after centrifugation at 4000rpm for 20min, the waste liquid was discarded, and the precipitated bacterial cells were washed twice with sterilized deionized water; then, the OD600 value of JB23 strain was adjusted to OD600=1 with purified water for standby use.

[0046] (2) Sugarcane setts treatment:

[0047] Guangxi University self-bred variety Zhacane 9 was used, and single bud segments were cut, then the sugarcane single bud segments were completely immersed in the JB23 strain suspension with OD600=1 prepared in advance for 1h, and immersion in sterile water for 1h was used as a control. After the immersion was completed, the samples were naturally air-dried, then placed in a 30℃ incubator for 2 weeks, and water was poured every 2 days to keep the planting pots in a humid state. The germination and growth were observed. Each treatment was repeated three times, and each repetition had 8 sugarcane buds.

[0048] As shown in Table 1 and Figure 4 the strain treated with JB23 could improve the germination rate of sugarcane buds and increase the plant height of sugarcane seedlings.

[0049] Table 1 Effect of JB23 strain on the germination rate of fresh sugarcane buds

[0050]

[0051] Example 3 Protective effect of JB23 strain on sugarcane buds

[0052] The preparation method of JB23 bacterial solution is the same as the preparation method of bacterial solution in step (1) of Example 2.

[0053] Sugarcane seed stalk treatment: Using the Guangxi University-bred variety Zhongzhe No. 9, cut into single bud segments, and completely immersing the sugarcane single bud segments in pre-prepared OD... 600 The sugarcane stalks were immersed in a bacterial suspension of strain JB23 (1 = 1) for 1 hour, with a control of soaking in sterile water for 1 hour. After drying, the sugarcane stalks were placed in a 40℃ oven for 72 hours of high-temperature stress, and then planted in a high-temperature sterilized culture medium (nutrient humus and vermiculite 2:1). The soil was watered once with sterile water to keep it moist, and then cultured in a 30℃ incubator for 20 days. Watering was repeated on the fifth day, and no further watering was performed. Germination and growth were observed. Each treatment was replicated three times, with six buds per replicate.

[0054] The results are as follows Figure 5 As shown, after being treated with JB23 bacterial solution, 12 sugarcane stalks still sprouted after high-temperature stress, with a sprouting rate of 66.7%. In contrast, only 3 sugarcane stalks treated with sterile water sprouted after high-temperature stress, with a sprouting rate of 16.7%. This result indicates that JB23 bacterial solution can protect sugarcane buds to a certain extent and alleviate the high-temperature and drought stress on sugarcane stalks.

[0055] Example 4: Growth-promoting effect of strain JB23 on sugarcane in the field.

[0056] The preparation method of JB23 bacterial solution is the same as the preparation method of bacterial solution in step (1) of Example 2.

[0057] Sugarcane stalk treatment: Three sugarcane varieties were used: Zhongzhe 9 (ZZ9, a variety bred by Guangxi University), Xintai Sugar 22 (ROC22, laboratory-preserved germplasm), and Liucheng 05136 (LC-05136, laboratory-preserved germplasm). Sugarcane stalks of uniform growth were selected and cut into double-bud segments. These double-bud segments were then completely immersed in pre-prepared OD (dimethylformaldehyde) solution. 600 The sugarcane was immersed in a JB23 bacterial suspension (1 oz) for 1 hour, with a control of immersion in sterile water for 1 hour. The dried double-bud segments were then planted in prepared furrows and watered regularly. Sugarcane growth indicators were measured after 7 months. Each treatment was performed in triplicate, with 15 double-bud segments per replicate.

[0058] The results are as follows Figure 6 As shown, the three sugarcane varieties treated with JB23 exhibited significantly higher plant number, plant height, stem diameter, aboveground biomass, and belowground biomass than the control (CK). Furthermore, the SPAD value of the first leaf in the JB23-treated sugarcane was also higher than that in the control (CK). These results indicate that JB23 has a strong growth-promoting effect on different sugarcane varieties in the field and provides practical benefits for field application.

[0059] The above merely preferred embodiments of the present application, it should be noted that for those of ordinary skill in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered within the scope of the present application.

Claims

1. Use of Bacillus velezensis JB23 in promoting sugarcane bud germination under stress conditions, characterized in that, The Bacillus velezensis JB23 is preserved in the China Center for Type Culture Collection, the preservation time is July 18, 2022, the preservation address is Wuhan University China Center for Type Culture Collection, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC M 20221134; the stress condition is high temperature stress.

2. Use of Bacillus velezensis JB23 in promoting the growth of sugarcane, characterized in that, The Bacillus velezensis JB23 is preserved in the China Center for Type Culture Collection, the preservation time is July 18, 2022, the preservation address is Wuhan University China Center for Type Culture Collection, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC M 20221134.

3. Use according to claim 2, characterized in that, The sugarcane varieties include but are not limited to Zhongzhe No. 9, Xintaitang No. 22 and Liucheng 05136.

4. Use according to claim 2 or 3, characterized in that, The promotion of the growth of the sugarcane includes but is not limited to: promoting the growth of the plant height of the sugarcane; promoting the growth of the stem diameter; promoting the aboveground and underground biomass of the sugarcane; and improving the relative chlorophyll content SPAD value of the sugarcane.

5. A method of promoting sugarcane bud germination under stress conditions, characterized by, The method is to soak the sugarcane seeds in the suspension of the Bacillus velezensis JB23 in claim 1 for 1-3 hours, take out and dry, and then germinate and plant; and the stress condition is high temperature stress.

6. The method of claim 5, wherein, OD of the suspension 600 value of 1.

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