Construction and application of a facultative anaerobic bacterial community with the functions of promoting the decomposition of straw directly returned to the field and promoting the growth of rice

Abstract

The present invention discloses the construction and application of a facultative anaerobic bacterial community that combines straw decomposition promotion for direct field return and rice growth promotion. The provided facultative anaerobic bacterial community with facultative anaerobic characteristics is suitable for promoting the decomposition of directly returned straw in double-cropping rice in the middle and lower reaches of the Yangtze River.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and specifically relates to the construction and application of a facultative anaerobic bacterial community with both straw promoting decay for direct field return and rice growth promotion. Technical Background

[0002] About 800 million tons of straw are produced in China every year, and rice straw accounts for about 25%. Due to problems such as low straw collection coefficient and high treatment cost, direct field return is currently the main utilization method. A large number of studies have shown that if straw cannot be rapidly degraded, it will affect the growth of the next crop, and applying a microbial agent for promoting decay is an effective way to accelerate straw degradation. CN103992958A discloses a mesophilic fungus Trichoderma pseudokoningii that can efficiently decompose agricultural waste rice straw and can efficiently decompose rice straw; CN 102409008 A discloses a microbial agent composed of Cellulomonas flavigena, Cytophaga fermentans, Bacillus pasteurii, Bacillus cereus, Butyrivibrio fibrisolvens, Fusarium sporotrichioides, etc., which can accelerate the degradation of rice straw, corn straw, etc.; CN 110846261A discloses a microbial agent composed of Trichoderma asperellum, Aspergillus niger, Aspergillus nidulans, Rhizopus oryzae, Saccharomyces cerevisiae, Bacillus subtilis, Bacillus amyloliquefaciens, etc., which can accelerate the degradation of wheat straw, corn straw, etc.

[0003] The main food crops in the middle and lower reaches of the Yangtze River are in a double-cropping rice mode, and it is required to sow late rice 7 - 10 days after the early rice harvest. Therefore, the degradation of early rice straw in double-cropping rice coincides with the planting of late rice. So, the degradation of early rice straw faces a relatively anaerobic situation with paddy field irrigation. However, most of the reported microbial agents for promoting straw decay are aerobic microorganisms, and there is no special facultative anaerobic microbial agent product for rice straw in the middle and lower reaches of the Yangtze River. Summary of the Invention

[0004] Aiming at the deficiencies of the above-mentioned existing technologies, the present application aims to provide the construction and application of a facultative anaerobic bacterial community with both straw promoting decay for direct field return and rice growth promotion.

[0005] The object of the present invention can be achieved by the following technical solutions:

[0006] The present invention provides a Bacillus siamensis 2-3C47, classified and named as Bacillus siamensis, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 10, 2024, with the deposit number CGMCC NO.29562.

[0007] The present invention provides a Bacillus siamensis 2-3C48, which is classified and named as Bacillus siamensis, and was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on January 10, 2024, with the deposit number CGMCC NO. 29563.

[0008] The present invention provides a Bacillus siamensis 2-4A51, which is classified and named as Bacillus siamensis, and was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on January 10, 2024, with the deposit number CGMCC NO. 29564.

[0009] The present invention provides a Bacillus australimaris 2-3C312, which is classified and named as Bacillus australimaris, and was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on January 10, 2024, with the deposit number CGMCC NO. 29561.

[0010] The facultative anaerobe described in the present invention is obtained by anaerobic enrichment culture with carbon dioxide filling and separation under aerobic conditions.

[0011] The present invention provides a facultative anaerobic bacterial community with both functions of directly returning straw to the field to promote straw decomposition and promoting rice growth. The bacterial community includes two or more of the Bacillus siamensis 2-3C47, Bacillus siamensis 2-3C48, Bacillus siamensis 2-4A51 or Bacillus australimaris 2-3C312 described in the present invention.

[0012] In some specific examples, the bacterial community described in the present invention is composed of the Bacillus siamensis 2-3C47, Bacillus siamensis 2-3C48, Bacillus siamensis 2-4A51 and Bacillus australimaris 2-3C312 described in the present invention.

[0013] In some more specific examples, the addition ratio of the cells of Bacillus siamensis 2-3C47, Bacillus siamensis 2-3C48, Bacillus siamensis 2-4A51 and Bacillus australimaris 2-3C312 in the bacterial community is 1:1:1:1.

[0014] The cultivation of the Bacillus siamensis 2-3C47, Bacillus siamensis 2-3C48, Bacillus siamensis 2-4A51 and Bacillus australimaris 2-3C312 described in the present invention can be obtained by culturing in LB liquid medium, and the specific cultivation method can follow the conventional methods in the art.

[0015] The present invention also provides the application of the bacterial community in the degradation of straw returned to the field, especially in the promotion of straw decomposition of double-season rice straw returned to the field in the middle and lower reaches of the Yangtze River.

[0016] In a specific example, the total amount of the strains added in the application is 1×10 6 ~5×10 6 CFU / g of straw. In a specific example, the total amount of the strains added is 1×10 6 CFU / g of straw.

[0017] The present invention also provides the application of the bacterial community in promoting the growth of rice.

[0018] Beneficial effects

[0019] The present invention provides a construction and application of a facultative anaerobic bacterial community that combines the rapid decomposition of directly returned straw and the promotion of rice growth. The constructed bacterial community has facultative anaerobic characteristics and is suitable for the rapid decomposition of directly returned straw in double-cropping rice in the middle and lower reaches of the Yangtze River. In addition, it has the following specific advantages:

[0020] 1) Currently, most straw decomposing agents contain fungi, and fungi need solid fermentation, which has a high cost; all the bacterial communities of the present invention are bacteria, which are more convenient in industrial production and can reduce costs.

[0021] 2) The bacterial community of the present invention can not only promote the rapid decomposition of directly returned straw, but also promote the growth of rice, combining the dual functions of promoting the decomposition of directly returned straw and promoting the growth of rice, and has good popularization value. Description of the drawings

[0022] Figure 1 Determination of cellulase activity treated with different straw enrichment solutions.

[0023] Figure 2 Degradation rate of straw returned to the field for 40 days under different treatments.

[0024] Figure 3 Effect of different treatments on the plant height of rice at 50 days. Specific implementation manners

[0025] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0026] The diameter of the plates (culture dishes) involved in the following examples is 90 mm.

[0027] The formula of the anaerobic enrichment medium involved in the following examples is (1 L system): 20 g of rice straw powder (water content less than 2 wt%), 10 g of peptone, 5 g of yeast extract powder, and 10 g of NaCl.

[0028] The TSB liquid medium formula involved in the following examples is (1L system): 17g of tryptone, 5g of sodium chloride, and 3g of soy peptone.

[0029] The 1 / 10 TSB medium formula involved in the following examples is (1L system): 1.7g of tryptone, 0.5g of sodium chloride, 0.3g of soy peptone, and 20g of agar powder.

[0030] The Rose Bengal medium formula involved in the following examples is (1L system): 1g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate (MgSO 4 ·7H 2 O), 100 mL of 1 / 3000 Rose Bengal solution, 0.1g of chloramphenicol, and 20g of agar powder.

[0031] The PDA medium formula involved in the following examples is (1L system): 6g of potato powder, 20g of glucose, and 18g of agar powder.

[0032] The PDA liquid medium formula involved in the following examples is (1L system): 6g of potato powder and 20g of glucose.

[0033] The CMC-Na medium formula involved in the following examples is (1L system): 20g of CMC-Na, 1g of NH 4 NO 3, 1.5g of KH 2 PO 4 1.5g of Na 2 HPO 4 2.5g, 0.5g of yeast extract, 2.5g of peptone, and 15g of agar powder.

[0034] The PDA-aniline blue medium formula involved in the following examples is (1L system): 6g of potato powder, 20g of glucose, 0.2g of aniline blue, and 18g of agar powder.

[0035] Enrichment and isolation of the strain in Example 1

[0036] In Qidu Village, Jinjiang Town, Yujiang District, Yingtan City, Jiangxi Province, rice straws (about 20 cm long) with stubbles (open-air placed for about 1 month, high water content (30wt%-40wt%), numbered 1) and cut by a harvester (low water content (less than 15wt%), numbered 2) were buried in the 0-20 cm soil (the soil after rice harvesting). The No. 1 samples were buried in the soil numbered 1-1, 1-2, 1-3, and those not buried in the soil were numbered 1-4; the No. 2 samples were buried in the soil numbered 2-1, 2-2, 2-3, and those not buried in the soil were numbered 2-4. After 3 months, the rice straw samples were taken out. Weighed 10 g of rice straw samples and added them to 90 ml of sterile water, shook at 170 rpm at room temperature for 2 h, and left to stand for 10 min to obtain a bacterial suspension. Absorbed 0.5 ml of the supernatant of the bacterial suspension and added it to a culture flask containing 50 ml of anaerobic enrichment medium. Each sample was repeated 3 times, numbered A, B, and C respectively, such as 1-1A, 1-1B, 1-1C. Created an anaerobic environment by filling carbon dioxide into the culture flask through a special equipment carbon dioxide cylinder, and statically cultured at 37°C for 14 d to obtain a straw enrichment solution. Absorbed the supernatant of the straw enrichment solution and measured the cellulase activity in the supernatant of the straw enrichment solution (the cellulase activity was measured using a cellulase activity kit from Suzhou Keming Biotechnology Co., Ltd.). The cellulase activities in the supernatants of different straw enrichment solutions are as Figure 1 shown. For each treatment, the supernatant of the straw enrichment solution with high cellulase activity (1-1C, 1-2A, 1-3C, 1-4A, 2-1C, 2-2B, 2-3C, 2-4A) was selected for the isolation of microorganisms. The supernatant of the straw enrichment solution was diluted to 10 -1 , 10 -2 , 10 -3 at three gradients respectively, and then 0.1 ml was absorbed and spread on Rose Bengal medium to screen for fungi. After culturing at 28°C for 2 d, the different colonies were selected and purified on PDA medium at 28°C for standby. The supernatant of the straw enrichment solution was diluted to 10 -4 , 10 -5 , 10 -6 at three gradients respectively, and then 0.1 ml was absorbed and spread on 1 / 10 TSB medium to screen for bacteria. After culturing at 28°C for 2 d, the different colonies were selected and purified on 1 / 10 TSB medium at 28°C for standby.

[0037] Example 2 Determination of the cellulose and lignin degradation abilities of strains

[0038] The purified strains (fungi, bacteria) obtained in Example 1 were cultured in CMC-Na medium and PDA-aniline blue medium at 28°C for 4 days, and then their cellulose degradation ability and lignin degradation ability were measured. The cellulose degradation ability was characterized by the ratio of the cellulose hydrolysis zone to the colony diameter, and the lignin degradation ability was characterized by the ratio of the lignin hydrolysis zone to the colony diameter. As shown in Table 1 and Table 2, 71 bacterial strains and 24 fungal strains showed certain degradation ability to cellulose or / and lignin. For bacteria, four bacterial strains with a ratio of cellulose hydrolysis zone to colony diameter greater than 2 and a ratio of lignin hydrolysis zone to colony diameter greater than 1.5 (Strain 2-3C47, Strain 2-3C48, Strain 2-3C312, and Strain 2-4A51) were selected for the next experiment. For fungi, four fungal strains with a ratio of cellulose hydrolysis zone to colony diameter greater than 1.0 and a ratio of lignin hydrolysis zone to colony diameter greater than 1.5 (Strain 1-1322, Strain 1-3221, Strain 2-4316, and Strain 2-4311) were selected for the next experiment.

[0039] Table 1 Ratio of cellulose hydrolysis zone to colony diameter and ratio of lignin hydrolysis zone to colony diameter of the selected bacterial strains

[0040]

[0041]

[0042] Table 2 Ratio of cellulose hydrolysis zone to colony diameter and ratio of lignin hydrolysis zone to colony diameter of the selected fungal strains

[0043]

[0044]

[0045] Example 3 Preservation and combination of strains

[0046] The strains 2-3C47, 2-3C48, 2-3C312, and 2-4A51 in Table 1 were preserved respectively, as follows:

[0047] Strain Bacillus siamensis 2-3C47 was preserved in the China General Microbiological Culture Collection Center on January 10, 2024, with the preservation number CGMCC NO.29562;

[0048] Strain Bacillus siamensis 2-3C48 was preserved in the China General Microbiological Culture Collection Center on January 10, 2024, with the preservation number CGMCC NO.29563;

[0049] The strain Bacillus australimaris 2-3C312 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 10, 2024, with the deposit number CGMCC NO.29561;

[0050] The strain Bacillus siamensis 2-4A51 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 10, 2024, with the deposit number CGMCC NO.29564;

[0051] 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, and the fungal strains 1-1322, 1-3221, 2-4316 and 2-4311 were numbered 1, 2, 3, and 4 respectively. According to the short rod theory, as shown in Table 3, a total of 38 treatments were formed, including 0 strains (control), 1 strain, 2 strains, 4 strains and 8 strains.

[0052] Table 3 Grouping of microbial communities

[0053]

[0054] Example 4 Degradation rate of returned straw after 40 days under different microbial community treatments

[0055] Based on the 37 microbial communities combined in Example 3 and 1 control treatment, a pot experiment was carried out with 3 replicates for each treatment.

[0056] The soil used in the pot experiment was sterile soil (pH 4.92): collected from Yingtan City, Jiangxi Province, dried and passed through a 10-mesh sieve, and sterilized by gamma rays.

[0057] Rice straw: collected from Yingtan City, Jiangxi Province, with a water content of about 15 wt%, and cut into 4-5 cm lengths.

[0058] Preparation of microbial agents:

[0059] Single bacterial agent: The activated bacterial strains (strains 2-3C47, 2-3C48, 2-3C312 and 2-4A51) were inoculated into TSB liquid medium and cultured at 25 °C and 170 rpm for 2 d to obtain a fermentation broth, and then the cell concentration of the fermentation broth was adjusted to 1×10 9 CFU / mL (dilution coating to count the number of cells) to obtain the microbial agent.

[0060] Fungal single agent: The activated fungal strains (strain 1-1322, strain 1-3221, strain 2-4316, and strain 2-4311) were inoculated onto PDA medium plates and cultured at 28 °C for 3 days to obtain fungal strain plates. 40 ml of sterile water was used to collect the spores and hyphae on the surface of the fungal strain plates to obtain the agent. The number of fungal spores in the agent was determined by the hemocytometer method to be 1-5×10 8 cells / ml (this method was used in this example). The activated fungal strains can also be inoculated into PDA liquid medium for liquid fermentation: the fermentation temperature is 28 °C, the rotation speed is 170 r / min, and the fermentation time is 3 days to obtain the agent.

[0061] Compound agent: The corresponding single agents were mixed at a volume ratio of 1:1 or 1:1:1:1 to obtain the compound agent.

[0062] The rice variety is Huanghuazhan. The bottom of the sterilized petri dish was covered with filter paper, and the disinfected rice seeds were placed in it, soaked with sterilized water and submerged to half the height of the rice seeds, and cultured at 28 °C for 2 days until germination for later use.

[0063] First, about 2 cm deep of soil was filled into each pot, and then 3 bags of rice straw (the rice straw bags were equipped with traction ropes) were added. Each bag of rice straw was 4 g. Sterile water / agent was poured onto the surface of the rice straw according to the treatment, and the inoculation amount was 1 ml / g of rice straw, that is, 12 ml of sterile water / agent was poured into each pot. Then, soil was continuously filled, and the total depth of the soil was about 12 cm. Each pot contained about 2000 g of soil in total. Each pot was placed in the greenhouse. After 2 days, an equal amount of sterile water was added to each pot until it was completely soaked. Rice seeds with the same bud length were selected and sown into the pots, and the sowing depth was 1-2 cm. 5 seeds were sown in each pot. During the planting period, the light irradiation time was from 8:00 to 20:00. The temperature was controlled at 20 °C - 25 °C in the first week, and then at 25 °C - 30 °C. Water was replenished in equal amounts 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 each watering made the soil keep flooded by 2-3 cm. 40 days after sowing, the rice straw bags were carefully taken out from the periphery of the soil using the traction ropes of the rice straw bags, and the degradation rate of the rice straw was measured. The method for measuring the degradation rate of the rice straw is as follows: After the rice straw was taken out, the soil on the surface of the rice straw was carefully rinsed off with clean water, dried to a constant weight at 105 °C and weighed to obtain the remaining weight. The difference between the initial dry weight and the remaining weight divided by the initial dry weight is the degradation rate of the rice straw. As Figure 2 shown, the straw degradation rate of the CK treatment without adding bacteria was the lowest, only 2.83%. The straw degradation rates of the treatments with different bacteria added were about 40%, all significantly better than the control treatment. Among them, the treatment with 4 facultative anaerobic bacteria added simultaneously (ABCD) had the highest degradation rate, reaching 45.9%.

[0064] Effect of Different Bacterial Communities on the Plant Height of Rice at the 50th Day of Growth

[0065] Following Example 4, 50 days after sowing, the plant height of rice was measured. The specific measurement method was as follows: The height from the root of the rice to the tip of the highest leaf was measured using a ruler. As Figure 3 shown, the plant height of the CK treatment without adding bacterial communities was the lowest, only 51.2 cm. The treatments with different added bacterial communities were all significantly better than the control treatment, and the average plant height was about 60 cm. Among them, the treatment with 4 facultative anaerobic bacteria (ABCD) had the highest plant height, reaching 65 cm.

[0066] In summary, the 4 facultative anaerobic bacterial communities obtained by anaerobic enrichment-aerobic separation had the best effects on both the degradation of rice straw and the promotion of rice growth. The present invention obtained a facultative anaerobic bacterial community that has both the effects of promoting the decay of directly returned straw and promoting the growth of rice.

Claims

1. A facultative anaerobic bacterial community that can directly return straw to the field to promote decomposition and promote rice growth, characterized in that: The bacterial flora includes any one of the following (1) to (5): (1) Bacillus siamense 2-3C47 and Bacillus siamense 2-3C48; (2) bacterial colonies formed by Bacillus siamensis 2-3C47 and Bacillus siamensis 2-4A51; (3) bacterial colonies formed by Bacillus siamense 2-3C48 and Bacillus nanhaiense 2-3C312; (4) bacterial colonies formed by Bacillus nanhaiensis 2-3C312 and Bacillus siamensis 2-4A51; (5) Bacillus siamense 2-3C47, Bacillus siamense 2-3C48, Bacillus siamense 2-4A51 and Bacillus nanhaiense 2-3C312; Among them, Bacillus siamensis 2-3C47 is classified and named as Bacillus siamensis, with a preservation number of CGMCC NO.29562; Bacillus siamensis 2-3C48 is classified and named as Bacillus siamensis, with a preservation number of CGMCC NO.29563; Bacillus siamensis 2-4A51 is classified and named as Bacillus siamensis, with a preservation number of CGMCC NO.29564; Bacillus australimaris 2-3C312 is classified and named as Bacillus australimaris, with a preservation number of CGMCC NO.29561.

2. The bacterial colony according to claim 1, characterized in that The addition ratio of different bacteria in groups (1 to (4) was 1:1; the addition ratio of Bacillus siamense 2-3C47, Bacillus siamense 2-3C48, Bacillus siamense 2-4A51 and Bacillus nanhaiense 2-3C312 in group (5) was 1:1:1:

1.

3. Use of the bacterial community described in claim 1 or 2 in the degradation of returned straw.

4. Application of the bacterial flora described in claim 1 or 2 in promoting the decomposition of double-season rice straw in the middle and lower reaches of the Yangtze River.

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

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

7. Use of the bacterial flora according to claim 1 or 2 in promoting rice growth.

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