Complex microbial inoculant for degrading rice straws and application of complex microbial inoculant

Abstract

The invention discloses a complex microbial inoculant for degrading rice straws and application of the complex microbial inoculant. The bacterial strains of the complex microbial inoculant comprise Arophialophora sp. F35, Condenascus sp. F28, Streptomyces albogriseolus Y10F15 and Streptomyces sp. Y25F5, and the preparation method of the complex microbial inoculant comprises the following steps: respectively culturing the bacterial strains, and mixing the cultured bacterial solutions according to the same volume of the bacterial strains to form the complex microbial inoculant. The prepared complex microbial inoculant is inoculated to a rice straw degradation culture medium according to the dry weight of 1.25 mL / g, and the straw degradation rates reach 48.72% and 54.18% respectively after 15 days of inoculation and 18 days of inoculation. The rice straw degrading complex microbial inoculant disclosed by the invention is simple to prepare and easy to operate; when being applied to degradation of the rice straws, the rice straws are not required to be subjected to complex pretreatment and only need to be cut into small sections of 1-2 cm; the complex microbial inoculant has application value in the aspect of rapid decomposition of mechanically crushed and returned-to-field straws of early rice in South China.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a compound microbial inoculant for rice straw degradation and application thereof. BACKGROUND

[0002] In the double-cropping rice planting area in South China, the treatment of straw produced after the harvesting of early rice is an urgent and key agricultural link. The unique "double rush" (harvesting early rice and transplanting late rice) farming time requirement must be completed in a very short time. The treatment of early rice straw directly affects the planting and yield stability of late rice. At present, mechanical crushing and returning to the field has become the most important and widely promoted straw treatment method in this region. This measure can quickly clean the field and return the organic matter and nutrients such as nitrogen, phosphorus, potassium and silicon in the straw to the soil, which has long-term significance for maintaining and improving soil fertility.

[0003] However, the traditional straw returning mode faces severe challenges under the high-intensity farming system in South China. First, the interval between early rice and late rice is short, and the straw is slowly naturally decomposed in the soil, which competes with late rice seedlings for nitrogen, easily leading to yellowing and stunted seedlings. Second, the incompletely decomposed straw may produce harmful substances such as organic acids and hydrogen sulfide in the flooded environment, inhibiting root growth. In addition, if the crushing or plowing quality is not high, it will also affect the quality of transplanting and rice root growth. In order to reduce the adverse effects of incompletely decomposed straw, it is necessary to take measures to accelerate straw decomposition.

[0004] Applying microbial inoculants to promote decomposition is the most effective way to accelerate straw decomposition. Straw decomposition microorganisms can promote the rapid degradation and nutrient release of straw in a short time, thereby shortening the straw decomposition time and promoting the rapid biochemical decomposition and returning of straw to the field. Because polysaccharides and lignin in straw are cross-linked by ester bonds and ether bonds, the degradation and utilization of lignocellulose have high non-degradability, the accessibility of ligninase is poor, and the straw is difficult to degrade and utilize. Compared with single microorganisms, mixed microbial populations constructed by combining strains with high efficiency of degrading each component of straw often have more excellent degradation capacity. Exploring and developing microbial inoculants with high efficiency of degrading rice straw and shortening the degradation time are important measures to ensure the smooth progress of the "double rush" farming season and the stable yield of late rice in South China. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a synthetic bacterial colony for degrading rice straw based on functional complementation and its application.

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

[0007] A kind of composite microbial inoculant for rice straw degradation, including Acrophialophora sp. (F35), Condenascus sp. (F28), Streptomyces albogriseolus (Y10F15) and Streptomyces sp. (Y25F5);The preservation number of the Acrophialophora sp. (F35) is GDMCCNo: 67216, the preservation number of the Condenascus sp. (F28) is GDMCC No: 67215, the preservation number of the Streptomyces albogriseolus (Y10F15) is GDMCC No: 67213, and the preservation number of the Streptomyces sp. (Y25F5) is GDMCC No: 67214.

[0008] The application also provides a method for preparing the above-mentioned composite microbial inoculant, comprising the following steps: inoculating Acrophialophora sp. (F35) and Condenascus sp. (F28) into PDA liquid medium respectively, and culturing at 25-28 DEG C and 150-200 rpm for 48-96 h; inoculating Streptomyces albogriseolus (Y10F15) and Streptomyces sp. (Y25F5) into LB liquid medium respectively, and culturing at 28-30 DEG C and 150-200 rpm for 48-72 h; mixing the cultured bacterial liquid of the above-mentioned strains in equal volume to form a composite microbial inoculant.

[0009] Preferably, the culture conditions of the Acrophialophora sp. (F35) and the Condenascus sp. (F28) are 28 DEG C, 200 rpm and 72 h.

[0010] Preferably, the culture conditions of the Streptomyces albogriseolus (Y10F15) and the Streptomyces sp. (Y25F5) are 30 DEG C, 200 rpm and 60 h.

[0011] Preferably, the preparation method of the above-mentioned composite microbial inoculant further comprises the step of adding carriers and adjuvants to the mixed bacterial liquid, and then spray drying or freeze drying to obtain the finished product.

[0012] Preferably, the finished product of the microbial inoculant comprises wettable powder, granules, suspension or freeze-dried preparation.

[0013] The application also provides the application of the above-mentioned composite microbial inoculant in rice straw decomposition.

[0014] Preferably, in the application, the mass-volume ratio of the rice straw to the inoculated compound microbial agent is 0.8-1 g / mL.

[0015] The application also provides the use of the compound microbial agent described above in the preparation of bio-organic fertilizer, straw compost or animal bedding degradation additives.

[0016] The application also provides a method for decomposing rice straw, comprising the step of mixing the compound microbial agent described above with the rice straw.

[0017] Preferably, the decomposition time is 2-3 weeks.

[0018] The application has the following beneficial effects:

[0019] The rice straw degradation compound microbial agent described in the application is simple to prepare and easy to operate; when applied to rice straw degradation, it does not require complex pretreatment of the rice straw, but only needs to be cut into small pieces of 1-2 cm; the compound microbial agent has application value in the rapid decomposition of mechanized pulverized rice straw in South China.

[0020] Acrophialophora sp. F35 was deposited with the Guangdong Microbial Culture Collection Center (GDMCC) on November 05, 2025, at address: 59 Building, 5th Floor, 100, Jiefang Road, Yuexiu District, Guangzhou, Guangdong, China, with postcode: 510070, and with deposit number: GDMCC No: 67216.

[0021] Condenascus sp. F28 was deposited with the Guangdong Microbial Culture Collection Center (GDMCC) on November 05, 2025, at address: 59 Building, 5th Floor, 100, Jiefang Road, Yuexiu District, Guangzhou, Guangdong, China, with postcode: 510070, and with deposit number: GDMCC No: 67215.

[0022] Streptomyces albogriseolus Y10F15 was deposited with the Guangdong Microbial Culture Collection Center (GDMCC) on November 05, 2025, at address: 59 Building, 5th Floor, 100, Jiefang Road, Yuexiu District, Guangzhou, Guangdong, China, with postcode: 510070, and with deposit number: GDMCC No: 67213.

[0023] Streptomyces sp. Y25F5 was deposited with the Guangdong Microbial Culture Collection Center (GDMCC) on November 05, 2025, at address: 59 Building, 5th Floor, 100, Jiefang Road, Yuexiu District, Guangzhou, Guangdong, China, with postcode: 510070, and with deposit number: GDMCC No: 67214. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a phylogenetic tree of 16S rRNA gene sequence constructed by Acrophialophora sp. F35 and its close type strain.

[0025] Figure 2 is a phylogenetic tree of 16S rRNA gene sequence constructed by Condenascus sp. F28 and its close type strain.

[0026] Figure 3 is a phylogenetic tree of 16S rRNA gene sequence constructed by Streptomyces albogriseolus Y10F15 and its close type strain.

[0027] Figure 4 is a phylogenetic tree of 16S rRNA gene sequence constructed by Streptomyces sp. Y25F5 and its close type strain.

[0028] Figure 5 is the degradation rate of rice straw after 15 days (left) and 18 days (right) of inoculation of the complex microbial agent (FB) and single strain. DETAILED DESCRIPTION

[0029] The following examples are further illustrations of the present application and do not limit the present application.

[0030] Example 1: Isolation and purification of strains

[0031] (1) Isolation of rice straw degradation microorganisms

[0032] Into 150 g of air-dried paddy field soil, 6 g of dry rice straw segments of 1-2 cm in length were added, and after mixing, the moisture content was adjusted to 20%, and the moisture content was adjusted every 2 days by weighing method. After 120 days of cultivation under natural conditions, the rice straw decomposition residues and soil were collected for microbial isolation.

[0033] Take 10 g of rice straw decomposition residues and soil mixture into a triangular flask containing 100 mL of sterile normal saline, shake at room temperature for 30 min, stand and take the supernatant for microbial isolation after dilution. R2A (yeast extract powder 0.5 g / L, protein peptone 3 g / L, casein 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, potassium phosphate dibasic 0.3 g / L, magnesium sulfate 0.024 g / L, sodium pyruvate 0.3 g / L, agar 15 g / L, pH 7.2, solvent water) and PDA medium (peeled potato 200 g / L, glucose 20 g / L, agar 15 g / L, pH 7.0, solvent water) were used to isolate bacteria and fungi, respectively.

[0034] (2) Species identification based on 16S rRNA gene sequence

[0035] Scrape fresh bacterial cells growing well on the medium, add 10 μL of alkaline lysis solution (25 mM NaOH, 0.2 mM Na2-EDTA, pH 12, solvent water), and treat at 95℃ for 15 min. After cooling, add 10 μL of neutralizing solution (40 mM Tris-HCl, pH 7.5, solvent water) and mix well, then centrifuge and take the supernatant as the template for PCR reaction. PCR reaction system (25 μL): buffer 12.5 μL, each 1 μL of upstream and downstream primers (27F / 1492R), template 1 μL, sterile deionized water 9.5 μL. Reaction program: 95℃ 5 min, 95℃ 30 s, 56℃ 30 s, 72℃ 90 s, 30 cycles, 72℃ 5 min. After agarose electrophoresis to determine the correctness of the PCR product, it is sent to Jinweizhi Biotechnology Co., Ltd. for sequencing.

[0036] BLAST comparison of bacterial 16S rRNA gene sequence and fungal ITS sequence was performed on EzBioCloud (https: / / www.ezbiocloud.net / ) and NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), respectively. The results of fungal ITS sequence comparison showed that strain F35 had the highest similarity with Acrophialophora nainiana CBS100.60, and the ITS sequence similarity between the two was 99.81%. Phylogenetic analysis of ITS sequences of the strain and its close species showed that F35 was a species of Acrophialophora (Fig. 2a); strain F28 had 100% ITS sequence similarity with Condenascus tortuosus, and phylogenetic analysis of ITS sequences of the strain and its close species showed that F28 was a species of Condenascus (Fig. 2b). Figure 1 )Figure 2 ). The bacterial 16S rRNA gene sequence alignment results showed that strain Y10F15 had 100% sequence similarity with the type strain Streptomyces albogriseolus NRRL B-1305 T , and the 16S rRNA gene sequence phylogenetic analysis of the strain and its close species indicated that Y10F15 was identified as Streptomyces albogriseolus Figure 3 ); strain Y25F5 had 99.25% sequence similarity with the type strain Streptomyces lusitanus NBRC 13464 T , and the 16S rRNA gene sequence phylogenetic analysis of the strain and its close species indicated that Y10F15 was a species of Streptomyces Figure 4 ). Strain F35 was named as Acrophialophora sp. F35; strain F28 was named as Condenascus sp. F28; strain Y10F15 was named as Streptomyces albogriseolus Y10F15; strain Y25F5 was named as Streptomyces sp. Y25F5. The above strains were preserved in Guangdong Microbial Culture Collection Center (GDMCC), address: 59 Building, 5th Floor, 100 Middle Xianlie Road, Guangzhou, China, postcode: 510070. The preservation number of the Acrophialophora F35 was GDMCC No: 67216, the preservation number of the Condenascus F28 was GDMCC No: 67215, the preservation number of the Streptomyces albogriseolus Y10F15 was GDMCC No: 67213, and the preservation number of the Streptomyces Y25F5 was GDMCC No: 67214.

[0037] Example 2: Construction of complex microbial community

[0038] (1) Characterization of strain degradation function

[0039] A mineral salt solution ((NH4)2SO41.0 g / L, K2HPO41.0 g / L, MgSO4·7H4O 0.5 g / L, KCl 0.5 g / L, FeSO4·7H2O 0.01 g / L, solvent is water) was prepared, and 10.0 g / L sodium carboxymethyl cellulose (agar 15 g / L, pH 7.0) or xylan (agar 15 g / L, pH 7.0) was added as a sole carbon source to prepare a solid culture medium, respectively. Then, the bacterial cake was inoculated on the culture medium, and cultured at 30°C for 3 days. After washing the bacterial colonies on the culture dish with 75% alcohol, 10 mL of 1 mg / mL Congo red solution was added for staining for 30 min, and then discarded. Then, 10 mL of 1 mol / L NaCl solution was added for decolorization for 30 min, and then discarded. Whether a transparent ring was generated outside the colonies after decolorization was used to determine the cellulose or hemicellulose degradation ability of the strains.

[0040] The lignin degradation ability was characterized by laccase and lignin peroxidase activities. The bacterial cake was inoculated on a culture medium with 0.04% guaiacol or 1% alkali lignin as a substrate (other inorganic salt components of the culture medium were the same as above) and cultured at 30°C for 5 days. Whether a red-brown color was generated at the edge of the colonies in the guaiacol culture medium was used to determine the laccase activity of the strains, and the transparent ring around the colonies on the alkali lignin plate was used to determine the lignin peroxidase activity of the strains.

[0041] It was found that strain F35 had strong laccase activity, strain F28 had strong lignin peroxidase activity, strain Y10F15 had strong cellulose degradation activity, and strain Y25F5 had strong hemicellulose degradation activity. In addition, strain F35 also had lignin peroxidase and hemicellulose degradation activities, and weak cellulose degradation activity; strain F28 also had hemicellulose degradation activity, and weak laccase and cellulose degradation activities; and strain Y25F5 also had cellulose degradation activity (Table 1). In summary, the construction of a complex bacterial population with the four strains is expected to achieve high-efficiency degradation activity on different components of rice straw.

[0042] Table 1. Activity characterization of strains in degrading different components of straw

[0043] +, active; -, inactive; w, weak activity; ++, strong activity

[0044] (2) Strain affinity test

[0045] The strains were inoculated in PDA solid medium (fungi-fungi), LB solid medium (bacteria-bacteria) or PDA solid medium added with 1% trypsin peptone (fungi-bacteria) to test the affinity between the members of the pseudo-complex microbial community. It was found that any two of the strains F35, F28, Y10F15 and Y25F5 could grow together without obvious growth inhibition between the colonies. Therefore, it was determined that it was feasible to construct a complex microbial community with the four strains.

[0046] (3) Construction of complex microbial community

[0047] The preparation method of the rice straw degradation complex microbial agent is as follows: the endoconidiogenous fungus F35 and the conidiogenous fungus F28 are inoculated in PDA liquid medium and cultured at 28°C with 200 rpm shaking for 72 h; the streptomycete Y10F15 and the streptomycete Y25F5 are inoculated in LB liquid medium and cultured at 30°C with 200 rpm shaking for 60 h; and the cultured bacterial liquid is mixed in equal volume to form the complex microbial agent.

[0048] Example 3: Application of complex microbial agent in rice straw degradation

[0049] The rice straw is washed with water for 2-3 times to remove the surface impurities and dust, and is cut into small pieces of 1-2 cm and dried at 85°C. The inorganic salt solution is prepared according to the formula of Example 2, and the preparation solution is prepared at 2 g of dried rice straw per 100 mL of inorganic salt solution, and then the complex microbial agent or the bacterial liquid of single strain prepared in Example 2 is inoculated in the preparation solution at 2.5% (v / v). The samples are taken at the 15th day and the 18th day after inoculation. The preparation solution without inoculation is set as a control. The rice straw residues are collected by gauze filtration, and the straw residues are dried at 85°C until constant weight. The straw degradation efficiency at each sampling period is calculated by the following formula: degradation efficiency = [(control group straw weight - treatment group straw weight) / control group straw weight] x 100%. The results show that the straw without inoculation of the microbial agent is almost not decomposed, and the degradation efficiency is almost 0; the inoculation of the complex microbial agent promotes the degradation of the rice straw, and the degradation rates after 15 days and 18 days of inoculation are 48.72% and 54.18%, respectively, and the straw degradation rates of each strain in the complex microbial agent when inoculated alone are lower than those of the complex microbial agent. Figure 5 Therefore, the complex microbial agent provided by the present application can accelerate the decomposition of the straw after the early rice harvest in the South China region, so as to avoid the influence of the straw on the late rice planting.

[0050] The above merely describes the preferred embodiments of the present application, and it should be pointed out that the above preferred embodiments should not be regarded as a limitation to the present application, and the protection scope of the present application should be defined by the scope of the claims. For those skilled in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A compound microbial agent for the degradation of rice straw, characterized in that, The funnel includes *Acrophialophorasp.* F35, *Condenascus sp.* F28, *Streptomyces albogriseolus* Y10F15, and *Streptomyces sp.* Y25F5; the accession number of *Acrophialophorasp.* F35 is GDMCC No: 67216, the accession number of *Condenascus sp.* F28 is GDMCC No: 67215, the accession number of *Streptomyces albogriseolus* Y10F15 is GDMCC No: 67213, and the accession number of *Streptomyces sp.* Y25F5 is GDMCC No: 67214.

2. A method for preparing the compound microbial agent as described in claim 1, characterized in that, Includes the following steps: Acrophialophora sp. F35 and Condenascus sp. F28 were inoculated into PDA liquid medium and cultured at 25-28℃ with shaking at 150-200 rpm for 48-96 h. Streptomyces albogriseolus Y10F15 and Streptomyces sp. Y25F5 were inoculated into LB liquid medium and cultured at 28-30℃ with shaking at 150-200 rpm for 48-72 h. The cultured bacterial solutions were then mixed in equal volumes according to the strains to form a compound bacterial agent.

3. The preparation method according to claim 2, characterized in that, The culture conditions for *Terminus terrestris* F35 and *Commelina konjac* F28 were: 28℃, 200 rpm shaking culture for 72 h; the culture conditions for *Streptomyces leucosus* Y10F15 and *Streptomyces oryzae* Y25F5 were: 30℃, 200 rpm shaking culture for 60 h.

4. The preparation method according to claim 2, characterized in that, It also includes the step of adding carriers and excipients to the mixed bacterial solution, and then spray drying or freeze drying to produce the finished bacterial agent.

5. The preparation method according to claim 4, characterized in that, The finished microbial agent includes wettable powder, granules, suspension, or freeze-dried formulation.

6. The application of the compound microbial agent according to claim 1 in the decomposition of rice straw.

7. The application according to claim 6, characterized in that, The mass-to-volume ratio of rice straw to inoculated compound microbial agent is 0.8-1 g / mL.

8. The application of the compound microbial agent according to claim 1 in the preparation of bio-organic fertilizer, straw compost or animal bedding degradation additive.

9. A method for decomposing rice straw, characterized in that, The method includes the step of mixing the compound microbial agent described in claim 1 with rice straw.

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