Application of bacillus velezensis JB-23 in promotion of sugarcane bud germination under stress condition

Abstract

The invention provides application of bacillus velezensis JB-23 in promotion of sugarcane bud germination under stress conditions, and belongs to the technical field of microorganisms. The bacillus velezensis JB-23 provided by the invention is preserved in the China Center for Type Culture Collection, and the preservation number of the bacillus velezensis JB-23 is CCTCC M 20221134. The bacillus velezensis JB-23 provided by the invention can improve the germination rate of sugarcane, can slow down high-temperature stress suffered by sugarcane buds even under high-temperature and drought stress, and ensures the development of the stressed sugarcane buds to a certain extent. Meanwhile, in a field environment, a bacterial suspension of the JB-23 strain can promote the plant height and stem diameter growth of sugarcane stems, increase the biomass and SPAD value of the overground part and the underground part, improve the photosynthetic efficiency and the like, and is a biological control strain which can also show good growth promoting ability under the field condition; and a new strain resource is provided for a sugarcane growth-promoting microbial resource library.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to application of Bacillus velez JB-23 in promoting germination of sugarcane buds under stress conditions. Background Art

[0002] As a C4 crop, sugarcane has huge yield and biomass. It is the main sugar crop and can also be used as raw materials in industry and light industry. However, the influence of various factors has restricted the further development of the sugarcane industry. After harvesting, sugarcane will not be planted again immediately, but will often be piled up in the fields. In winter, Guangxi will also have a temperature of 30℃, which will aggravate the loss of water in sugarcane. The influence of drought, high temperature and other stresses will make the emergence rate of newly planted and perennial sugarcane in the field low, further resulting in the inability to significantly increase the yield per mu. Therefore, ensuring the germination rate and emergence rate and cultivating strong seedlings are of great significance to increasing sugarcane yield and sucrose yield.

[0003] The use of chemical fertilizers and pesticides can effectively increase production, but there are also problems such as low utilization rate, soil compaction, and environmental pollution. The restricted use of chemical pesticides is also a research focus in China's agricultural development process. As the importance of sustainable development is gradually being recognized, people are encouraged to reduce the use of chemical pesticides and find alternatives to chemical pesticides and fungicides. It is gradually regarded as a key method to ensure food security and solve chemical pollution. Greener and more environmentally friendly microbial fertilizers have been studied and applied on sugarcane and have also been proven to be an effective means of promoting growth.

[0004] Bacillus is a common raw material for microbial fertilizer. It has the functions of dissolving phosphorus and potassium, and can also produce a large amount of growth-promoting substances such as IAA, protease, cellulase, etc. The present invention will use Bacillus Velez JB-23, which has been proven to have significant effects in the prevention and control of sugarcane diseases, to explore its role in sugarcane germination and the growth promotion of different varieties of sugarcane in the field, in order to limit the use of chemical pesticides in sugarcane production and provide a scientific basis and technical reserves for the green and sustainable development of the sugarcane industry. Summary of the invention

[0005] In view of this, the object of the present invention is to provide an application of Bacillus Velez JB-23 in promoting the germination of sugarcane sprouts under stress conditions. The Bacillus Velez JB-23 provided by the present invention can increase the germination rate of sugarcane under stress conditions and promote the growth of sugarcane.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The application of Bacillus Velez JB-23 in promoting sugarcane bud germination under stress conditions. The Bacillus Velez JB-23 is deposited in the China Type Culture Collection on July 18, 2022. The deposit address is China Type Culture Collection of Wuhan University, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC M 20221134.

[0008] The Bacillus velezensis JB-23 described in the present invention has been disclosed in a patent application with application publication number “CN116254199A”.

[0009] In certain embodiments, the stress conditions include, but are not limited to, high temperature stress and / or drought stress. The Bacillus Velez JB-23 of the present invention can slow down the reduction in germination rate of sugarcane buds caused by high temperature stress and ensure the development of sugarcane buds to a certain extent.

[0010] The present invention also provides an application of Bacillus Velez JB-23 in promoting the germination of sugarcane sprouts. The Bacillus Velez JB-23 is deposited in the China Type Culture Collection on July 18, 2022. The deposit address is China Type Culture Collection of Wuhan University, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC M 20221134.

[0011] The present invention also provides the role of Bacillus Velez JB-23 in promoting sugarcane growth. The Bacillus Velez JB-23 is deposited in the China Type Culture Collection on July 18, 2022. The deposit address is China Type Culture Collection of Wuhan University, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC M 20221134.

[0012] In certain embodiments, the sugarcane varieties include but are not limited to Zhongzhe No. 9, Xintaitang No. 22, and Liucheng 05136.

[0013] In certain embodiments, the promoting sugarcane growth includes but is not limited to: promoting the growth of sugarcane plant height; promoting the growth of stem diameter; promoting the biomass of the aboveground and underground parts of sugarcane; and increasing the relative chlorophyll content SPAD value of sugarcane. The Velez sp. JB-23 of the present invention can secrete IAA, protease, cellulase, and amylase, and can increase the biomass of sugarcane in the field, which is beneficial to increasing the yield of sugarcane.

[0014] The present invention also provides a bacterial agent, wherein the bacterial agent contains Bacillus Velez JB-23, wherein the Bacillus Velez JB-23 is deposited in the China Center for Type Culture Collection on July 18, 2022, at the China Center for Type Culture Collection of Wuhan University, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC M 20221134;

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

[0016] (1) Promote the germination of sugarcane buds;

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

[0018] (3) Promote the growth of sugarcane.

[0019] In certain embodiments, the preparation method of the bacterial agent is to inoculate 0.1% (v / v) of a 50% (v / v) glycerol mixed bacterial solution of Bacillus Velez JB-23 strain stored in a -80°C refrigerator into an LB broth medium for activation, culture the mixture in a shaker at 37°C for 24 hours to obtain a seed solution, further expand the seed solution, and centrifuge and filter to obtain the bacterial agent.

[0020] The present invention also provides a method for promoting the germination of sugarcane buds under stress conditions, wherein the method comprises soaking the sugarcane seeds in a suspension of the Bacillus Velez JB-23 or the bacterial agent described in the above technical solution for 1 hour, taking out and drying them, and then germinating and planting them.

[0021] In certain embodiments, when the suspension is a suspension of Bacillus velez JB-23, the OD of the suspension is 600 The value is 1.

[0022] Beneficial technical effects: The present invention provides an application of Bacillus Velez JB-23 in promoting the germination of sugarcane buds under stress conditions. The Bacillus Velez JB-23 is deposited in the China Type Culture Collection Center on July 18, 2022, and the deposit address is the China Type Culture Collection Center of Wuhan University, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC M 20221134. The Bacillus Velez JB-23 provided by the present invention can improve the germination rate of sugarcane, and even under high temperature and drought stress, it can also slow down the high temperature stress of sugarcane buds, and to a certain extent, ensure the development of stressed sugarcane buds. At the same time, in the field environment, the bacterial suspension of the JB-23 strain can promote the growth of plant height and stem diameter of sugarcane stems, increase the biomass of the aboveground and underground parts and the relative chlorophyll content SPAD value, improve photosynthetic efficiency, etc. It is a biological control strain that can also show good growth-promoting ability under field conditions, and provides a new strain resource for the sugarcane growth-promoting microbial resource library. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the phenotype of Bacillus velez JB-23 on protease medium;

[0024] Figure 2 The phenotype of Bacillus velez JB-23 in cellulase medium;

[0025] Figure 3 is the phenotype of Bacillus velez JB-23 on starch hydrolyzing enzyme medium;

[0026] Figure 4 The effect of Bacillus Velez JB-23 bacterial suspension on sugarcane seedling height;

[0027] Figure 5 The effect of Bacillus Velez JB-23 bacterial suspension on sugarcane germination and growth under high temperature and drought stress. The left side of a is the phenotype of sugarcane after JB-23 treatment, and the right side is the control treatment. b is the statistics of plant height.

[0028] Figure 6 The results show the effects of JB-23 bacterial suspension treatment on the physiological parameters of Zhongzhe No. 9, ROC22 and Liucheng 05136 sugarcanes in the field; a is the statistics of seedling emergence, b is the statistics of plant height, c is the statistics of stem diameter, d is the statistics of aboveground biomass, e is the statistics of underground biomass, and f is the effect on the SPAD value of relative chlorophyll content in one leaf of sugarcane. DETAILED DESCRIPTION

[0029] In order to better understand the present invention, the content of the present invention is further explained in conjunction with the following examples, but the content of the present invention is not limited to the following examples. The materials, reagents, etc. used in the examples and test examples of the present invention, unless otherwise specified, can be obtained from commercial channels; the methods used in the examples and test examples of the present invention, unless otherwise specified, are conventional methods.

[0030] Example 1 Determination of JB-23 Growth-Promoting Properties

[0031] (1) Determination of IAA production capacity

[0032] The present invention detects the ability of JB-23 to produce indole-3-acetic acid (IAA), specifically: JB-23 strain is cultured in LB medium containing 0.2g / L tryptophan at 37°C for 48h, centrifuged at 12000rpm for 5min, the upper liquid is collected, mixed with an equal volume of Salkowski colorimetric solution, treated in the dark for half an hour, and the absorbance at 530nm is detected. At the same time, different concentrations of IAA solution are used to make a standard curve, and finally OD 530 The values ​​were substituted into the standard curve to calculate the amount of IAA produced by the JB-23 strain.

[0033] The method for drawing the standard curve is: use the IAA standard to prepare standard solutions with concentrations of 10μg / mL, 20μg / mL, 30μg / mL, 40μg / mL, and 50μg / mL, and measure the absorbance of these standard solutions at 530nm. Based on the measured absorbance and the corresponding concentration of the IAA standard, a standard curve y=0.0327x+0.0181 is drawn, R2=0.9983, y represents OD 530 The absorbance value of the sample was 0.97 μg / mL, and x was the corresponding IAA concentration (μg / mL).

[0034] (2) Protease detection

[0035] The skim milk powder culture medium (skim milk powder 12g, agar 20g, ddH2O to 1000mL) was sterilized and the OD 600 Inoculate 10 μL of the JB23 bacterial solution with a value of 1 to the center of the plate, culture at 37°C for 5 days, and observe whether a transparent zone appears around the colonies.

[0036] The results are as follows Figure 1 As shown, a transparent zone appeared around the JB-23 strain, indicating that the JB-23 strain was able to produce protease.

[0037] (3) Cellulase detection

[0038] The detection of cellulase is to measure the OD 60010 μL of JB23 bacterial liquid with a pH value of 1 was inoculated into cellulose culture medium (10 g sodium carboxymethyl cellulose, 10 g peptone, 20 g agar, 5 g yeast powder, 1 g KH2PO4, 5 g NaCl, ddH2O to 1000 mL, pH 7.0) and cultured at 37°C for 5 days. The culture was then stained with 1% Congo red for 15 min and rinsed twice with 1 M NaCl solution to observe whether a yellow halo appeared around the colonies.

[0039] The results are as follows Figure 2 As shown, a yellow halo appeared around the JB-23 strain, indicating that it has the ability to produce cellulase.

[0040] (4) Amylase detection

[0041] Prepare starch hydrolysis medium (yeast extract 5g / L, peptone 10g / L, NaCl 5g / L, soluble starch 30g / L, agar 15g / L, PH=7.6), sterilize at 115℃ for 20min, then pour into plates for later use. Take 10μL of the concentration as OD 600 = 1 fresh JB-23 bacterial liquid was inoculated in the center of the starch hydrolysis medium plate, incubated at 37°C for 24 hours, and then stained with Lugol's iodine solution for observation. If a transparent hydrolysis zone appears around the colony, it proves that the strain can hydrolyze starch.

[0042] Figure 3 As shown, a transparent hydrolysis zone appeared around JB-23, indicating that it has the ability to secrete amylase.

[0043] Example 2 Effect of JB-23 strain on promoting budding of sugarcane

[0044] (1) Preparation of Bacillus Velez JB-23 bacterial solution:

[0045] A 50% (v / v) glycerol mixed bacterial solution of the Velez Bacillus JB-23 strain stored at -80°C was inoculated into an LB broth medium at a rate of 0.1% (v / v) for activation; the mixture was cultured at 37°C in a shaker at 220 r / min for 24 hours to obtain a fresh seed solution; purified water was added to a fermenter, LB broth medium powder was added at a concentration of 20 g / L, and the mixture was sterilized at 121°C for 30 minutes, and then the seed solution was added to a fermenter at a rate of 1% (v / v) and cultured at 37°C and 180 r / min for 72 hours to obtain a fresh fermentation solution; the fresh JB-23 bacterial solution was centrifuged at 5000 rpm for 30 minutes, the waste liquid was discarded, the precipitated bacterial body was resuspended with sterile water, the mixture was centrifuged again at 4000 rpm for 20 minutes, the waste liquid was discarded, the precipitate was rinsed twice with sterile deionized water, and the OD600 value of the JB-23 strain was adjusted to OD600=1 with purified water for standby use.

[0046] (2) Sugarcane stem treatment:

[0047] Use the self-bred variety Zhongzhe No. 9 of Guangxi University, cut into single bud segments, and completely immerse the sugarcane single bud segments in the JB-23 strain suspension prepared in advance with OD600=1 for 1 hour, and soak in sterile water for 1 hour as a control. After the soaking, take it out and dry it naturally. Then put it in a 30℃ incubator for 2 weeks, water it every 2 days to keep the planting pot moist. Observe the germination and growth. Each treatment was repeated three times, with 8 sugarcane buds in each repeat.

[0048] The results are shown in Table 1 and Figure 4 As shown, the strain treated with JB-23 can improve the germination rate of sugarcane buds and increase the plant height of sugarcane seedlings.

[0049] Table 1 Effect of JB-23 strain on germination rate of fresh sugarcane buds

[0050]

[0051] Example 3 Protective effect of strain JB-23 on sugarcane sprouts

[0052] The preparation method of JB-23 bacterial solution is the same as the preparation method of bacterial solution in step (1) of Example 2.

[0053] Sugarcane stem treatment: Use the Guangxi University self-bred variety Zhongzhe No. 9, cut into single bud segments, and completely immerse the sugarcane single bud segments in the prepared OD 600 =1 JB-23 strain bacterial suspension for 1 hour, and soaked in sterile water for 1 hour as a control. After drying, the cane stems were placed in a 40℃ oven. After high temperature stress for 72 hours, they were planted in a high temperature sterilized culture matrix (nutrient humus and vermiculite 2:1). Sterile water was poured once during planting to keep the soil moist. After that, they were cultured in a 30℃ incubator for 20 days. After watering again on the fifth day, no more watering was done to observe the germination and growth. Each treatment was repeated three times, with 6 buds in each repeat.

[0054] The results are as follows Figure 5 As shown in the figure, after the sugarcane stems treated with JB-23 bacterial solution were subjected to high temperature stress, 12 sugarcane stems still germinated, with a germination rate of 66.7%, while after the sugarcane stems treated with sterile water were subjected to high temperature stress, only 3 sugarcane stems germinated, with a germination rate of 16.7%. This result shows that the JB-23 bacterial solution can protect sugarcane buds to a certain extent and alleviate the high temperature stress and drought stress of sugarcane stems.

[0055] Example 4: Effect of JB-23 strain on sugarcane growth in the field

[0056] The preparation method of JB-23 bacterial solution is the same as the preparation method of bacterial solution in step (1) of Example 2.

[0057] Sugarcane stem treatment: Three sugarcane varieties, namely, Zhongzhe No. 9 (ZZ9, a variety bred by Guangxi University), ROC22 (ROC22, a germplasm preserved in the laboratory), and Liucheng 05136 (LC-05136, a germplasm preserved in our laboratory), were used. Sugarcane stems with uniform growth were selected and cut into double-bud segments. The double-bud segments were completely immersed in the prepared OD 600 = 1 JB-23 bacterial suspension for 1 hour, and soaked in sterile water for 1 hour as a control. The dried double-bud segments were planted in the dug furrows and watered regularly. The growth index of sugarcane was counted after 7 months. Each treatment was repeated three times, with 15 double-bud segments in each repeat.

[0058] The results are as follows Figure 6 As shown in the figure, the three varieties of sugarcane treated with JB-23 had significantly higher plant numbers, plant height, stem diameter, aboveground biomass and underground biomass than the control CK sugarcane, and the relative chlorophyll content SPAD value of the first leaf of the sugarcane treated with JB-23 was also higher than that of the control CK. The above results show that JB-23 also has a strong growth-promoting effect on different varieties of sugarcane in the field, and it also has practical benefits for field application.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of Bacillus Velez JB-23 in promoting germination of sugarcane buds under stress conditions, characterized in that: The Bacillus Velezii JB-23 was deposited in the China Type Culture Collection on July 18, 2022. The deposit address is China Type Culture Collection, Wuhan University, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC M20221134.

2. The use according to claim 1, characterized in that: The stress conditions include, but are not limited to, high temperature stress, and / or drought stress.

3. The application of Bacillus Velez JB-23 in promoting the germination of sugarcane buds, characterized in that: The Bacillus Velezii JB-23 was deposited in the China Type Culture Collection on July 18, 2022. The deposit address is China Type Culture Collection, Wuhan University, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC M 20221134.

4. The effect of Bacillus Velez JB-23 in promoting sugarcane growth is characterized in that: The Bacillus Velezii JB-23 was deposited in the China Type Culture Collection on July 18, 2022. The deposit address is China Type Culture Collection, Wuhan University, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC M 20221134.

5. The use according to claim 4, characterized in that: The sugarcane varieties include but are not limited to Zhongzhe No. 9, Xintaitang No. 22, and Liucheng 05136.

6. The use according to claim 4 or 5, characterized in that: The method of promoting the growth of sugarcane includes but is not limited to: promoting the growth of plant height of sugarcane; promoting the growth of stem diameter; promoting the biomass of the aboveground and underground parts of sugarcane; and increasing the SPAD value of the relative chlorophyll content of sugarcane.

7. A bacterial agent, characterized in that: The bacterial agent contains Bacillus Velez JB-23, which is deposited in China Center for Type Culture Collection on July 18, 2022, at China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC M 20221134; The microbial agent can be applied to any one or more of the following: (1) Promote the germination of sugarcane buds; (2) Promote sugarcane bud germination under stress conditions; (3) Promote the growth of sugarcane.

8. A method for promoting sugarcane bud germination under stress conditions, characterized in that: The method comprises soaking the sugarcane seeds in the suspension of the Bacillus Velez JB-23 described in claims 1 to 2 or the bacterial agent described in claim 7 for 1 to 3 hours, taking them out, drying them, and then germinating and planting them.

9. The method according to claim 8, characterized in that When the suspension is a suspension of Bacillus Velez JB-23, the OD 600 The value is 1.

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