A straw low-temperature degradation bacterium with high efficiency in inhibiting crop pathogenic fungi and its application

Abstract

The invention discloses a straw low-temperature degradation bacterium capable of efficiently inhibiting crop pathogenic fungi and application thereof. The straw low-temperature degradation bacterium is Bacillus velezensis BD3, and the Bacillus velezensis BD3 can be used for returning straw to fields. The Bacillus velezensis BD3 of the invention is derived from winter paddy field soil, can adapt to low temperature environment, still has high enzyme production activity under 15 DEG C, and has a rice straw degradation rate of 28.15% under outdoor low temperature, effectively solving the problems of low enzyme production activity and low straw degradation efficiency of straw degradation bacteria under low temperature; at the same time, the strain has a significant inhibitory effect on various pathogenic fungi of rice and corn, and the inhibition rate of rice blast pathogen and rice false smut pathogen is more than 90%, and the inhibition rate of rice sheath blight pathogen, rice seedling pathogen, corn leaf spot pathogen, corn leaf spot pathogen and corn curved spore pathogen is also more than 70%, and the pathogenic fungi remaining in crop straw can be inhibited, effectively avoiding the problem of aggravation of diseases after straw is returned to the field.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a straw low-temperature degradation bacterium capable of efficiently inhibiting crop pathogenic fungi and application thereof. Background Art

[0002] Straw is a treasure when used and a hazard when abandoned. my country has achieved bumper harvests in grain production year after year, and crop straw resources are very rich, with an annual amount of about 700 million tons of resources that can be collected. As a renewable resource, the comprehensive utilization of crop straw has been valued by the world, and the utilization modes of feed, energy, base material, raw material, and fertilizer have been explored. Among them, returning straw to the field is the main way to use straw as fertilizer, which not only reduces the air pollution caused by straw burning, but also increases the organic carbon and nutrient content of the soil, effectively reduces the soil bulk density, and improves the soil structure and microbial community composition. At the same time, returning straw to the field promotes the recycling and efficient utilization of nutrients in farmland, replaces some chemical fertilizers, and plays an important role in protecting and utilizing black soil resources, ensuring food security, and promoting sustainable agricultural development. my country's straw returning technology is still imperfect and there are many problems.

[0003] The United States has abundant arable land resources, and can implement fallow to fully degrade the straw in the soil and avoid the adverse effects of returning straw to the field. However, my country has a large population base and limited arable land resources. It is impossible to implement fallow. The land is planted year after year, and the straw cannot be fully degraded in the field, causing a series of problems. For example, too many soil pores make it impossible for the soil to be in close contact with the seeds, which will reduce the crop emergence rate; the peak period of straw degradation coincides with the crop growth period, affecting crop growth and reducing crop yield and quality. The direct return of crop straw to the field is slow, in addition to the fact that the structure of the straw itself is complex (mainly composed of cellulose, hemicellulose and lignin), the structure is stable and not easy to decompose. This is mainly because the microorganisms in the soil that can degrade straw have low degrading enzyme activity, especially in low temperature areas, the activity of soil microorganisms is inhibited, and the straw is difficult to degrade naturally in the soil after being directly returned to the field. By screening low-temperature resistant straw-degrading bacteria and developing low-temperature degradation agents, the process of straw fertilizer in my country will be greatly promoted, which is an effective way to achieve rapid degradation of straw and efficient return to the field.

[0004] On the other hand, under the straw-returning planting model, diseases are exacerbated. Pathogens carried by straw enter the soil with the straw, increasing the number of pathogens in the soil as the primary infection source and increasing the risk of disease in the next crop. In particular, the straw is not crushed enough and is scattered and covered on the soil surface, providing a place for pathogens to overwinter, which is conducive to the overwintering and reproduction of pathogens, and aggravates the occurrence of diseases in the next crop. Meta-analysis shows that the incidence and disease index of diseases increased by 9.5% and 12.2% respectively after straw return to the field; the incidence and disease index of rice sheath blight, rice blast, corn and wheat soil-borne diseases increased significantly. This directly leads to huge losses in the yield of the next crop, affecting the effect of straw return to the field. At the same time, with the continuous increase in the amount of pesticide application, the risk of resistance and pesticide residues increases, the soil quality is damaged, and a vicious circle is formed, which seriously threatens my country's food security and sustainable agricultural development.

[0005] Therefore, those skilled in the art provide a straw low-temperature degrading bacterium that is highly effective in inhibiting crop pathogenic fungi to solve the problems raised in the above background technology. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a straw low-temperature degrading bacteria and application thereof for effectively inhibiting crop pathogenic fungi.

[0007] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0008] One of the purposes of the present invention is to provide a straw low-temperature degradation bacterium that efficiently inhibits crop pathogenic fungi. The straw low-temperature degradation bacterium is Bacillus velezensis BD3, which is preserved in Guangdong Microbiological Culture Collection Center with a preservation number of GDMCC No: 64386, a preservation date of 2024.03.04, and a preservation address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] The second object of the present invention is to provide an application of straw low-temperature degradation bacteria with high efficiency in inhibiting crop pathogenic fungi in straw return to the field, specifically: at an outdoor ambient temperature of 15°C, the straw sprayed with Bacillus velezensis BD3 is directly plowed and returned to the field; the number of live bacteria of Bacillus velezensis BD3 is not less than 1×10 8 CFU / mL, the usage of Bacillus velezensis BD3 is: 100mL of straw low-temperature degrading bacteria BD3 fermentation liquid is applied for every kilogram of straw.

[0010] Compared with the prior art, the Bacillus velezensis BD3 of the invention is derived from winter paddy field soil, can adapt to low temperature environment, still has high enzyme production activity under 15°C condition, and has a rice straw degradation rate of 28.15% under low outdoor temperature, effectively solving the problems of low enzyme production activity and low straw degradation efficiency of straw degradation bacteria under low temperature; at the same time, the strain has a significant inhibitory effect on various pathogenic fungi of rice and corn, and the inhibition rate of rice blast pathogen and rice false smut pathogen is more than 90%, and the inhibition rate of rice sheath blight pathogen, rice seedling pathogen, corn leaf spot pathogen, corn leaf spot pathogen and corn curved spore pathogen is also more than 70%, which can inhibit the pathogenic fungi remaining in the crop straw, and effectively avoid the problem of aggravation of diseases after the straw is returned to the field. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is the transparent zone produced by strain BD3 on the screening medium.

[0012] Figure 2 The effect of strain BD3 in degrading rice straw.

[0013] Figure 3 This is the inhibitory effect of strain BD3 on rice and corn pathogenic fungi.

[0014] Figure 4 This is the phylogenetic tree of strain BD3.

[0015] Figure 5 This is the inhibitory effect of strain BD3 on pathogens. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0017] Isolation and Identification of Bacillus velezensis BD3

[0018] 1. Isolation of Bacteria from Paddy Field Soil

[0019] Five paddy field soil samples were obtained from the paddy fields in winter at 0-20 cm below the soil surface by the five-point sampling method. Each sample weighed 200-300 g and was mixed evenly. 20 g of each soil sample was weighed and placed in a conical flask containing 50 mL of sterile water. The soil samples were shaken and cultured at 15 °C and 180 r / min for 1 h, then allowed to settle and the supernatant was aspirated. 10 μL of the diluted 10 3 , 10 4 , 10 5 , 106 and 10 7 The supernatant was evenly spread on LB medium (including the following ingredients: 6 g yeast extract, 10 g tryptone, 10 g sodium chloride, 17 g agar, 1 L deionized water, 20 g agar powder), cultured at 15°C for 24 h, single colonies were picked for purification, and after purification, stored in a -76°C refrigerator with 30% glycerol.

[0020] 2. Preliminary screening of low-temperature and high-efficiency straw degradation strains

[0021] Cellulose-Congo red medium (including the following ingredients: CMC-Na 2g / L, (NH4)2SO42g / L, MgSO4·7H2O 0.5g / L, K2HPO41.0g / L, NaCl 0.5g, Congo red 0.4g / L, agar powder 20g / L, deionized water 1L) and aniline blue medium (including the following ingredients: tryptone 10g, yeast extract 5g, NaCl 10g, agar powder 20g, aniline blue 0.4g, deionized water 1L) were used for primary screening. The strains were inoculated in the center of the plate and cultured at 15℃ for 7 days. The diameters of the transparent zone and the strain diameters were investigated. 20 bacterial strains with high cellulase and peroxidase activities were screened out, among which the strain with the highest enzyme activity was BD3. The results are shown in Table 1. Figure 1 .

[0022] Table 1 Diameters of transparent zones produced by strain BD3 on screening medium

[0023]

[0024] 3. Rescreening of low-temperature and high-efficiency straw degradation strains

[0025] The strain obtained in the initial screening was activated on a plate, and the bacterial cake was placed in a liquid LB medium, cultured at 15°C, 150r / min for 48h, and prepared into a bacterial suspension with a concentration of 1×10CFU / mL. The rice straw was cleaned and cut into small pieces of about 3cm, and dried at 80°C to constant weight. Weigh 10g of dry straw into a sterile conical flask (250mL), add 100mL of sterile water and 10mL of bacterial suspension, and add 10mL of sterile water to the blank control. After culturing for 15 days at 15°C and 150r / min, rinse the straw with clean water 5 times, dry it at 80°C to constant weight, weigh the remaining straw mass, and calculate the straw degradation rate by weight loss method. The strain BD3 with the highest straw degradation efficiency was determined, with a degradation rate of 24.70%, which was 59.35% higher than the blank control degradation rate. The degradation effect is as follows Figure 2 .

[0026]

[0027] 4. Screening of antagonistic bacteria against crop pathogenic fungi

[0028] The plate confrontation culture method was used to inoculate the cakes of rice blast pathogen, rice false smut pathogen, sheath blight pathogen, seedling rust pathogen, corn leaf spot pathogen, small leaf spot pathogen and corn curvature leaf spot pathogen into PDA medium (including the following components: 200g potato, 20g glucose, 20g agar, 1L deionized water), and the strains obtained by repeated screening were inoculated 3cm away from the center of the cake, and inverted for culture at 28℃. The growth inhibition rate of the strains was investigated after the sheath blight pathogen was cultured for 3d, the seedling rust pathogen was cultured for 5d, the rice blast pathogen, corn leaf spot pathogen, small leaf spot pathogen and corn curvature leaf spot pathogen were cultured for 7d, and the rice false smut pathogen was cultured for 50d. The results showed that BD3 had a significant inhibitory effect on the above-mentioned crop pathogenic fungi. The results are shown in Table 3. Figure 3 .

[0029] Table 3 Inhibition rate of strain BD3 on rice and corn pathogenic fungi

[0030]

[0031] 5. Enzyme Activity Assay

[0032] A single colony of BD3 was inoculated into a liquid enzyme-producing culture medium (including the following ingredients: peptone 10g, NaCl 10g, yeast powder 5g, sodium carboxymethyl cellulose 10g, deionized water 1L), and cultured in a shaking flask at 15°C and 150r / min for 3 days. The enzyme activity of the aseptic fermentation broth of strain BD3 was determined by the kit method. The carboxymethyl cellulase activity, lignin peroxidase and laccase activities of strain BD3 were 139.81U / mL, 4405.33U / L and 246.93U / L, respectively.

[0033] 6. Strain Identification

[0034] Through morphological, physiological, biochemical and molecular identification, it was confirmed that the low-temperature degradation bacteria used in the application was Bacillus velezensis;

[0035] Colony morphology of strain BD3: A single colony of strain BD3 was inoculated into solid LB medium and cultured at 35°C to observe the colonies. After 24 hours, the colonies were milky white, round or oval, with a smooth, moist, slightly convex surface, neat edges, and full of mucus. After 3 days of culture, wrinkles formed on the surface of the colonies.

[0036] Physiological and biochemical identification:

[0037] The colony morphology and physiological and biochemical identification were referred to the Manual of Identification of Common Bacterial Systems, and strain BD3 was identified as Bacillus. The specific results are shown in Table 4.

[0038] Table 4 Physiological and biochemical characteristics of strain BD3

[0039] Measurement items result Measurement items result Gram stain + Methyl red test - Starch hydrolysis test + Indole test - Gelatin liquefaction test + Catalase reaction + Oxidase reaction + Nitric acid reduction reaction + VP reaction + Urease +

[0040] Molecular identification:

[0041] The genomic DNA of strain BD3 was extracted using a bacterial rapid extraction kit;

[0042] PCR amplification and sequence determination of 16S rDNA sequences: The bacterial universal primers 27F and 1492R were used for PCR amplification of 16S rDNA. The PCR amplification conditions were: 95°C for 5 min, 95°C for 1 min, 56°C for 2 min, 72°C for 2 min, 30 cycles, and 72°C for 10 min. The samples were sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for sequencing;

[0043] Phylogenetic analysis: The 16S rDNA gene sequences obtained by sequencing were subjected to BLAST analysis with the nucleic acid data in GenBank. Gene sequences with high similarity were selected and homology was compared using the neighbor-joining method (NJ) of MEGA6 software to construct a phylogenetic tree.

[0044] Identification results:

[0045] After sequencing, a 1411 bp sequence was obtained and compared with the data in the NCBI database. A phylogenetic tree was constructed using MEGA6 (see Figure 4 ), the results showed that BD3 and Bacillus velezensis FZB42 (GenBank accession number NR07500) in the database were clustered into one branch, with the highest homology. Based on the results of physiological, biochemical and molecular identification, strain BD3 was identified as Bacillus velezensis and named Bacillus velezensis BD3. Its 16S rDNA sequence has been submitted to the GenBank database with accession number PP033752. The strain has been deposited in the Guangdong Microbial Culture Collection Center with the deposit number GDMCC No: 64386 and the deposit date 2024.03.04. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0046] In order to verify that the Bacillus velezensis BD3 isolated and screened in the above example can be used for returning straw to the field, the following test was conducted:

[0047] Study on the effect of Bacillus velezensis BD3 on straw rot and antibacterial effect of rice blast fungus Experimental method: The outdoor experiment of Bacillus velezensis BD3 on straw rot was completed from September 26, 2023 to November 25, 2023, and three treatments were set: ① rice straw; ② rice straw + rice blast fungus; ③ rice straw + rice blast fungus + fungal agent. The rice straw was cleaned and cut into about 3-5 cm, and dried at 80℃ to constant weight. Take 40g of straw in a mesh bag and inoculate rice blast fungus with an inoculation concentration of 1×10 5 CFU / mL, the inoculation volume was 20 mL, and the number of pathogens in the straw was detected by qRT-PCR. Then 40 mL of BD3 strain fermentation liquid was added, and the bacterial liquid concentration was 1×10 7 CFU / mL, 36 replicates for each treatment, buried in soil, 6 replicates were taken on the 10th, 20th, 30th, 40th, 50th, and 60th days, and the sample samples were divided into two parts. In the first part, the straws of treatments 1 and 3 were washed and dried at 80°C to constant weight, and the decomposition rate and decomposition rate were calculated by weight loss method;

[0048]

[0049]

[0050] The second part, treatment 2 and 3, qRT-PCR detection of pathogen content in straw, calculation of inhibition rate, rice blast fungus specific primers: 28SMF / 28SMR: 28SMF (5'-ACCCTACTGATGACCTCG-3') and 28SMR (5'-GTGTCAAAATTACAATACGC-3'). Reaction system: template DNA 2μL, 2×SYBR Mix 10μL, primers 0.25μL each, ddH2O 7.5μL. PCR protocol includes 95℃ initial denaturation for 3min, 95℃ 30s, 56℃ 30s, 72℃ 40s for a total of 35 cycles, repeated three times. Data analysis was performed using the comparative Ct value method (2 -△△CT The initial expression level of rice blast fungus in straw was set as 1, i.e. the control group, and the others were relative expression levels, reflecting the changes in the content of rice blast fungus.

[0051] Test results: In the outdoor pot test, Bacillus velezensis BD3 can significantly improve the degradation rate and degradation rate of rice straw (Table 5). After 60 days of decomposition at low temperature, the degradation rate of rice straw by Bacillus velezensis BD3 reached 28.15%, while the degradation rate of the control was 22.83%, and the degradation rate was increased by 23.33%. Figure 5The results showed that there was no significant difference in the pathogen base number between the two treatment groups. As the degradation days increased, the number of straw pathogens treated with BD3 was significantly lower than that in the control group. After 60 days of degradation, the inhibition rate of B. velezensis BD3 against rice blast fungus reached 70.67%, indicating that B. velezensis BD3 significantly inhibited the reproduction of rice blast fungus in straw.

[0052] Table 5 Effect of BD3 on rice straw degradation under outdoor low temperature conditions

[0053]

[0054] In summary, the Bacillus velezensis BD3 of the present invention can adapt to low temperature environment, still has high enzyme production activity under 15°C, and the degradation rate of rice straw at low temperature reaches 28.15%, which effectively solves the problems of low enzyme production activity and low straw degradation efficiency of straw degrading bacteria at low temperature; at the same time, the strain has a significant inhibitory effect on various pathogenic fungi of rice and corn, and the inhibition rate of rice blast fungus and rice false smut fungus reaches more than 90%, and the inhibition rate of rice sheath blight fungus, rice seedling pathogen, corn leaf spot fungus, small leaf spot fungus and corn leaf spot fungus also reaches more than 70%, which can inhibit the pathogenic fungi remaining in the crop straw, and effectively avoid the problem of aggravation of diseases after the straw is returned to the field.

[0055] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A straw low-temperature degradation bacterium that efficiently inhibits crop pathogenic fungi, characterized in that: The straw low-temperature degrading bacteria is Bacillus velezensis BD3, which is preserved in the Guangdong Provincial Microbial Culture Collection Center with the preservation number GDMCC No: 64386, the preservation date is 2024.03.04, and the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. Use of the straw low-temperature degrading bacteria with high efficiency in inhibiting crop pathogenic fungi as claimed in claim 1 in returning straw to the field.

3. The use of the straw low temperature degrading bacteria with high efficiency in inhibiting crop pathogenic fungi in straw return to the field according to claim 2, characterized in that: At an outdoor ambient temperature of 15°C, the straw sprayed with Bacillus velezensis BD3 was directly plowed and returned to the field; the number of viable bacteria of Bacillus velezensis BD3 was not less than 1×10 8 CFU / mL, the usage of Bacillus velezensis BD3 is: spray 100mL of straw low-temperature degrading bacteria BD3 fermentation liquid for every kilogram of straw.

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