Facultative anaerobic bacterium 2-3C312 for promoting degradation of rice straws directly returned to field and application of facultative anaerobic bacterium 2-3C312

Abstract

The invention discloses facultative anaerobic bacteria 2-3C31 capable of promoting decomposition of straw directly returned to field and promoting growth of rice and application of the facultative anaerobic bacteria 2-3C31, four strains provided by the invention have good degradation effects on cellulose and lignin, also have the effects of promoting decomposition of straw directly returned to field and promoting growth of rice, have facultative anaerobic characteristics, and have good application prospects. The method is suitable for straw decay promotion of direct returning of double cropping rice in the middle and lower Yangtze River.

Description

[0001] This application is a divisional application of a Chinese invention patent with an application date of July 10, 2024, application number 202410926347.2, and the invention name is "A facultative anaerobic bacteria that promotes the degradation of rice straw directly returned to the field and its application." Technical Field

[0002] The invention belongs to the technical field of bioengineering and relates to facultative anaerobic bacteria for promoting the degradation of rice straw directly returned to the field and application thereof. Technical Background

[0003] my country produces about 800 million tons of straw every year, of which rice straw accounts for about 25%. Due to the low collection coefficient and high processing cost of straw, direct return to the field is currently the main way to use it. A large number of studies have shown that if straw cannot be quickly degraded, it will affect the growth of the next crop. Applying microbial agents that promote decomposition is an effective way to accelerate straw degradation. CN103992958A discloses a room-temperature fungus, Trichoderma pseudokoningii, which can efficiently decompose agricultural waste rice straw, and can efficiently decompose rice straw; CN 102409008 A discloses a bacterial agent composed of a compound of Xanthomonas erythrogenans, fermentative cellulophagus, Bacillus pasteurianus, Bacillus cereus, Butyrivibrio fibrinolyticus, Fusarium pseudosporioides, etc., which can accelerate the degradation of rice straw, corn straw, etc.; CN 110846261A discloses a bacterial agent composed of a compound of Trichoderma spinulosum, Aspergillus niger, Aspergillus nidulans, Rhizopus oryzae, Saccharomyces cerevisiae, Bacillus subtilis, Bacillus amyloliquefaciens, etc., which can accelerate the degradation of wheat straw, corn straw, etc.

[0004] The main food crop in the middle and lower reaches of the Yangtze River is a double-season rice model, which requires that late rice be sown 7 to 10 days after the early rice is harvested. Therefore, the degradation of early rice straw in the double-season rice coincides with the planting of late rice. Therefore, the degradation of early rice straw faces the relatively anaerobic situation of rice field irrigation. However, the straw-promoting microbial agents reported so far are mostly aerobic microorganisms, and there are no facultative anaerobic microbial agent products specifically for rice straw in the middle and lower reaches of the Yangtze River. Summary of the invention

[0005] In view of the deficiencies in the above-mentioned prior art, the present application aims to provide a facultative anaerobic bacteria and its application for promoting the degradation of rice straw when it is directly returned to the field.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] The present invention provides a siam Bacillus 2-3C47, which is classified and named as siam Bacillus (Bacillussiamensis), and is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 10, 2024, with a deposit number of CGMCC NO.29562.

[0008] The present invention provides a siam Bacillus 2-3C48, which is classified and named as siam Bacillus (Bacillussiamensis), and is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 10, 2024, with a deposit number of CGMCC NO.29563.

[0009] The present invention provides a siam Bacillus 2-4A51, which is classified and named as siam Bacillus (Bacillussiamensis), and is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 10, 2024, with a deposit number of CGMCC NO.29564.

[0010] The present invention provides a Bacillus australimaris 2-3C312, which is classified and named Bacillus australimaris (Bacillus australimaris), and is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on January 10, 2024, with a deposit number of CGMCC NO.29561.

[0011] The facultative anaerobic bacteria of the present invention are obtained by anaerobic enrichment culture with carbon dioxide and separation under aerobic conditions.

[0012] The present invention also provides the use of the siam Bacillus 2-3C47, siam Bacillus 2-3C48, siam Bacillus 2-4A51 or south China sea Bacillus 2-3C312 in the degradation of cellulose and / or lignin.

[0013] The culture of the siam Bacillus 2-3C47, siam Bacillus 2-3C48, siam Bacillus 2-4A51 and south China sea Bacillus 2-3C312 of the present invention can be obtained by culturing in LB liquid culture medium, and the specific culturing method can be obtained according to the conventional method in the art.

[0014] The present invention also provides the use of the Siamese Bacillus 2-3C47, Siamese Bacillus 2-3C48, Siamese Bacillus 2-4A51 or Nanhai Bacillus 2-3C312 in the degradation of returned straw, especially in promoting the decomposition of double-season rice straw in the middle and lower reaches of the Yangtze River.

[0015] In a specific example, the amount of strain added in the application is 1×10 6 ~5×10 6 CFU / g straw, in a specific example, the amount of strain added is 1×10 6 CFU / g straw.

[0016] The present invention also provides the use of the siam Bacillus 2-3C47, siam Bacillus 2-3C48, siam Bacillus 2-4A51 or south China sea Bacillus 2-3C312 in promoting rice growth.

[0017] Beneficial Effects

[0018] The present invention provides a facultative anaerobic Bacillus siamensis bacteria for promoting the degradation of rice straw directly returned to the field and its application. The selected strain has facultative anaerobic characteristics and is suitable for promoting the decomposition of double-season rice straw directly returned to the field in the middle and lower reaches of the Yangtze River. In addition, it also has the following specific advantages:

[0019] 1) Most of the current straw decomposing agents contain fungi, which need to be fermented in solid state and are very expensive. The strain screened in the present invention is a bacterium, which is more convenient in industrial production and can reduce costs.

[0020] 2) The bacterial community of the present invention can not only promote the rapid decomposition of the straw directly returned to the field, but also promote the growth of rice. It has the dual effects of promoting the decomposition of the straw directly returned to the field and promoting the growth of rice, and has good promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Determination of cellulase activity after different straw enrichment treatments.

[0022] Figure 2 The degradation rate of straw returned to the field for 40 days under different treatments.

[0023] Figure 3 Effects of different treatments on rice plant height at 50 days. DETAILED DESCRIPTION

[0024] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0025] The diameter of the plates (culture dishes) involved in the following examples is 90 mm.

[0026] The anaerobic enrichment medium formula involved in the following examples is (1L system): 20g rice straw powder (water content less than 2wt%), 10g peptone, 5g yeast extract powder, and 10g NaCl.

[0027] The TSB liquid culture medium formula involved in the following examples is (1L system): 17g of trypticase, 5g of sodium chloride, and 3g of soybean papain hydrolyzate.

[0028] The 1 / 10 TSB medium formula involved in the following examples is (1L system): 1.7 g of trypticase, 0.5 g of sodium chloride, 0.3 g of soybean papain hydrolysate, and 20 g of agar powder.

[0029] The CMC-Na culture medium formula involved in the following examples is (1L system): CMC-Na 20g, NH 4 NO 31g, KH 2 PO 4 1.5 g, Na 2 HPO 4 2.5g, yeast extract powder 0.5g, peptone 2.5g, agar powder 15g.

[0030] The PDA-aniline blue culture medium formula involved in the following examples is (1L system): 6g potato powder, 20g glucose, 0.2g aniline blue, and 18g agar powder.

[0031] Example 1 Enrichment and separation of bacterial strains

[0032] In Qidu Village, Jinjiang Town, Yujiang District, Yingtan City, Jiangxi Province, rice straw (about 20 cm long) left in the open air (placed in the open air for about 1 month, high water content (30wt%-40wt%), numbered 1) and cut by harvester (low water content (less than 15wt%), numbered 2) were buried in 0-20 cm soil (soil after rice harvest). Sample No. 1 was buried in the soil and numbered 1-1, 1-2, 1-3, and not buried in the soil was numbered 1-4; Sample No. 2 was buried in the soil and numbered 2-1, 2-2, 2-3, and not buried in the soil was numbered 2-4. The rice straw samples were taken out after 3 months. 10g of rice straw sample was weighed and added to 90ml of sterile water, shaken at room temperature at 170rpm for 2h, and left to stand for 10min to obtain a bacterial suspension. Pipette 0.5 ml of the supernatant of the bacterial suspension and add it to a culture bottle containing 50 ml of anaerobic enrichment medium. Repeat 3 times for each sample and number them A, B, and C, such as 1-1A, 1-1B, and 1-1C. Use a special equipment carbon dioxide cylinder to fill the culture bottle with carbon dioxide to create an anaerobic environment. Incubate at 37°C for 14 days to obtain a straw enrichment solution. Pipette the supernatant of the straw enrichment solution and determine the cellulase activity in the supernatant of the straw enrichment solution (the cellulase activity is determined using the cellulase activity kit of Suzhou Keming Biotechnology Co., Ltd.). The cellulase activity in the supernatant of different straw enrichment solutions is as follows: Figure 1 As shown. For each treatment, the supernatant of the straw enriched liquid with high cellulase activity (1-1C, 1-2A, 1-3C, 1-4A, 2-1C, 2-2B, 2-3C, 2-4A) was selected for microbial separation. The supernatant of the straw enriched liquid was diluted to 10 -4 , 10 -5 , 10 -6After three gradients, 0.1 ml was respectively applied on 1 / 10 TSB medium to screen bacteria. After culturing at 28°C for 2 days, differential colonies were selected and purified on 1 / 10 TSB medium at 28°C for later use.

[0033] Example 2 Determination of cellulose and lignin degradation ability of bacterial species

[0034] The purified bacterial strains obtained in Example 1 were cultured in CMC-Na medium and PDA-aniline blue medium at 28°C for 4 days, and their cellulose degradation ability and lignin degradation ability were determined. The cellulose degradation ability was characterized by the ratio of cellulose hydrolysis circle to colony diameter, and the lignin degradation ability was characterized by the ratio of lignin hydrolysis circle to colony diameter. As shown in Table 1, 71 strains of bacteria showed a certain degradation ability for cellulose or lignin. Two strains (strain numbers 2-3C47, 2-3C48, 2-3C312 and 2-4A51) with a ratio of cellulose hydrolysis circle to colony diameter greater than 2 and a ratio of lignin hydrolysis circle to colony diameter greater than 1.5 were selected for preservation:

[0035] The strain Bacillus siamensis 2-3C47 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 10, 2024, with the deposit number CGMCC NO.29562;

[0036] The strain Bacillus siamensis 2-3C48 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 10, 2024, with the deposit number CGMCC NO.29563;

[0037] The strain Bacillus australimaris 2-3C312 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 10, 2024, with the deposit number CGMCC NO.29561;

[0038] The strain Bacillus siamensis 2-4A51 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 10, 2024, with the deposit number CGMCC NO.29564.

[0039] Table 1 Ratio of cellulose and lignin hydrolysis circles to colony diameters of screened strains

[0040]

[0041]

[0042]

[0043]

[0044] Example 3 Degradation rate of returned straw after 40 days of treatment with different strains

[0045] The strains Bacillus siamensis 2-3C47, Bacillus siamensis 2-3C48, Bacillus australimaris 2-3C312 and Bacillus siamensis 2-4A51 were numbered A, B, C and D, respectively. The control was made up of no added strains, and there were 5 treatments in total.

[0046] The soil used in the potted plant experiment was sterile soil (pH 4.92): it was taken from Yingtan City, Jiangxi Province, dried in the sun, passed through a 10-mesh net, and sterilized by gamma rays.

[0047] Rice straw: taken from Yingtan City, Jiangxi Province, with a water content of about 15wt%, cut into 4-5cm length.

[0048] Preparation of bacterial agent: The activated bacterial strains (strain 2-3C47, strain 2-3C48, strain 2-3C312 and strain 2-4A51) were inoculated into TSB liquid medium and cultured at 25°C and 170 rpm for 2 days to obtain fermentation liquid, and then the cell concentration of the fermentation liquid was adjusted to 1×10 9 CFU / mL (number of bacteria counted after dilution and coating) was used to obtain the bacterial agent.

[0049] The rice variety is Huanghuazhan. The bottom of the sterilized culture dish is covered with filter paper, and sterilized rice seeds are placed in it. Sterilized water is used to soak and submerge the rice seeds to half their height, and the seeds are cultured at 28°C for 2 days, and germinated for use.

[0050] Each pot was first filled with soil to a depth of about 2 cm, and then 3 bags of rice straw were added (the rice straw bags were equipped with traction ropes), each bag of rice straw was 4 g, and sterile water / microbial agent was poured on the surface of the rice straw according to the treatment, and the inoculation amount was 1 ml / g rice straw, that is, 12 ml of sterile water / microbial agent was poured on each pot, and then soil was continued to be added, the total depth of the soil was about 12 cm, and a total of about 2000 g of soil was filled in each pot. Each pot was placed in a greenhouse, and after 2 days, an equal amount of sterile water was added to each pot until it was completely soaked, and rice seeds with consistent bud length were selected and sown in the pot, with a sowing depth of 1-2 cm, and 5 seeds were sown in each pot. During the planting period, the lighting time was 8:00-20:00, the temperature was controlled at 20℃-25℃ in the first week, and then the temperature was controlled at 25℃-30℃, and the same amount of water was added as needed. Sterile water was poured every day in the first week to keep the soil surface moist; after that, sterile water was poured every 1 day, and the soil was kept flooded by 2-3 cm each time. 40 days after sowing, use the rice straw bag traction rope to carefully remove the rice straw bag from the outside of the soil and measure the rice straw degradation rate. The rice straw degradation rate determination method is as follows: After the rice straw is taken out, carefully rinse off the soil on the surface of the rice straw with clean water, dry it at 105°C to constant weight, and weigh the remaining weight. The difference between the initial dry weight and the remaining weight divided by the initial dry weight is the rice straw degradation rate. Figure 2 As shown in the figure, the straw degradation rate of CK without adding strains was the lowest, only 2.83%. The straw degradation rates of treatments with different strains were about 40%, which were significantly better than the control treatment. Among them, the degradation rate of adding facultative anaerobic bacteria Bacillussiamensis 2-3C48 was the highest, reaching 41.42%.

[0051] Example 4 Effects of different bacterial strain treatments on rice plant height on the 50th day of growth

[0052] Following Example 3, 50 days after sowing, the rice plant height was measured. The specific measurement method is as follows: Use a ruler to measure the height from the root of the rice to the highest leaf tip. Figure 3 As shown in the figure, the rice plant height of the CK treatment without adding bacteria was the lowest, only 51.2 cm. The straws treated with different bacterial communities were significantly better than the control treatment, with an average plant height of about 59.6 cm. Among them, the plant height of the straw treated with facultative anaerobic bacteria Bacillus siamensis 2-3C48 was the highest, reaching 59.87 cm.

[0053] In summary, the facultative anaerobic bacteria Bacillus siamensis2-3C48 obtained by anaerobic enrichment-aerobic separation has the best effect on rice straw degradation and rice growth promotion. The present invention obtains a facultative anaerobic Bacillus siamensis bacterium that has the functions of directly returning straw to the field to promote decomposition and promoting rice growth.

Claims

1. A Bacillus australimaris 2-3C312, classified and named Bacillus australimaris, with a deposit number of CGMCC NO.29561.

2. Use of the Bacillus nanhaiense 2-3C312 according to claim 1 in the degradation of cellulose and / or lignin.

3. Use of the Bacillus nanhaiense 2-3C312 according to claim 1 in the degradation of returned straw.

4. The use according to claim 3, characterized in that: The amount of strain added in the application is 1×10 6 ~5×10 6 CFU / g straw.

5. The use according to claim 4, characterized in that: The total amount of strain added in the application is 1×10 6 CFU / g straw.

6. Use of the Nanhai Bacillus 2-3C312 described in claim 1 in promoting the decay of double-season rice straw in the middle and lower reaches of the Yangtze River.

7. The use according to claim 6, characterized in that: The amount of strain added in the application is 1×10 6 ~5×10 6 CFU / g straw.

8. The use according to claim 7, characterized in that: The total amount of strain added in the application is 1×10 6 CFU / g straw.

9. Use of the Bacillus nanhaiense 2-3C312 according to claim 1 in promoting rice growth.

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