Microbial agent and application thereof

Abstract

The invention provides a microbial agent and application thereof, the microbial agent contains burkholderia gladioli with the preservation number of CGMCC No.24934 and alcaligenes faecalis with the preservation number of CGMCC No.31941, and the burkholderia gladioli and the alcaligenes faecalis act together, so that the influence of low-temperature stress on crops can be relieved, the invasion of pathogenic bacteria on the crops can be effectively inhibited, and the growth of the crops can be promoted.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, and in particular, to a microbial agent and applications thereof. Background Art

[0002] During their growth and development, crops are often attacked by various pathogens. In severe cases, this attack can have disastrous consequences for crop yield and quality, leading to significant economic losses. Biological control of crop diseases generally relies on interspecific or intraspecific microbial resistance, competition, hyperparasitism, and lytic interactions, or on the use of microbial metabolites to induce disease resistance in crops. This approach inhibits the survival and activity of certain pathogens, thereby controlling the occurrence and development of crop diseases.

[0003] Compared to non-biological control methods like pesticides, biological control's most prominent advantage is its environmental friendliness, safety, and harmlessness to humans and animals. Biocontrol microorganisms primarily encompass bacteria, fungi, and actinomycetes. Bacteria are characterized by their diverse diversity, robust reproductive capacity, complex metabolic activity and abundant product output, diverse modes of action against pathogens, and ease of artificial cultivation. Antagonistic bacteria and their metabolites play a key role in both natural biological control and human-initiated biological control efforts.

[0004] However, single biocontrol agents have drawbacks such as unstable and persistent effects on some diseases. The combined use of multiple, compatible biocontrol agents can produce mutually reinforcing and complementary effects. Therefore, there is an urgent need to study the synergistic effects between microorganisms with different functionalities and develop composite microbial agents to effectively control crop diseases and improve crop performance. Summary of the Invention

[0005] The purpose of the present invention is to provide a microbial agent that can effectively prevent and control crop diseases and improve the cold resistance of crops.

[0006] In order to achieve the above-mentioned object, the present disclosure provides a microbial agent, which includes a first fermentation broth and a second fermentation broth; The first fermentation broth is Burkholderia gladiolus ( Burkholderia gladioli ) fermentation broth; The second fermentation broth is a strain of Alcaligenes faecalis ( Alcaligenes faecalis ) of the fermentation broth.

[0007] Optionally, in the microbial agent, the weight ratio of the first fermentation broth to the second fermentation broth is 1:(0.5-2), preferably 1:(0.5-1.5).

[0008] Optionally, in the microbial agent, the Burkholderia gladiolus with a deposit number of CGMCC No. 24934 ( Burkholderia gladioli ) is the concentration of OD 600 =0.8-1.2; The deposit number is CGMCC No. 31941 Alcaligenes faecalis ( Alcaligenes faecalis ) is the concentration of OD 600 =0.8-1.2.

[0009] Optionally, the microbial agent further includes an auxiliary agent, which includes at least one of a dispersant, a synergist, and a suspending agent.

[0010] Optionally, the dispersant includes at least one of sodium lignin sulfonate, sodium carboxymethyl cellulose and soapberry powder; the enhancer includes at least one of mineral potassium fulvic acid, chitosan and compound amino acid powder; the suspending agent includes at least one of Langti LT676, Langti EF96 and Langti LTQ661.

[0011] Optionally, in the microbial agent, the weight ratio of the first fermentation broth, the second fermentation broth, the dispersant, the synergist, and the suspending agent is 1: (0.5-2): (0.01-0.02): (0.01-0.02): (0.01-0.02); Optionally, the preparation process of the first fermentation broth comprises: separating and purifying the Burkholderia gladioli ( Burkholderia gladioli ) Streak inoculate on LB solid medium, culture at 24-30°C for 20-30 h, pick a single colony into LB liquid medium, and culture at 24-30°C in a shaking incubator for 20-30 h.

[0012] Optionally, the preparation process of the second fermentation broth comprises: separating and purifying the Alcaligenes faecalis with a preservation number of CGMCC No.31941 ( Alcaligenes faecalis ) Streak inoculate on LB solid medium, culture at 24-30°C for 20-30 h, pick a single colony into LB liquid medium, and culture at 24-30°C in a shaking incubator for 20-30 h.

[0013] In another aspect, the present disclosure provides a use of the above-mentioned microbial agent in improving the low temperature tolerance of crops, wherein the crops include at least one of cotton, tobacco and tomato.

[0014] In another aspect, the present disclosure provides use of the above-mentioned microbial agent in promoting crop growth, wherein the crop comprises at least one of cotton, tobacco, and tomato.

[0015] In another aspect, the present disclosure provides use of the above-mentioned microbial agent in preventing and controlling Verticillium wilt in crops, wherein the crops include at least one of cotton, tobacco, and tomato.

[0016] Through the above technical solution, the present disclosure provides a microbial agent and its application. The microbial agent contains Burkholderia gladioli with a preservation number of CGMCC No. 24934 and Alcaligenes faecalis with a preservation number of CGMCC No. 31941. The two bacteria work together to alleviate the effects of low temperature stress on crops, effectively inhibit the invasion of pathogens on crops, and promote crop growth.

[0017] Other features and advantages of the present disclosure will be described in detail in the following detailed description.

[0018] Biomaterial deposit information Burkholderia, classified as Burkholderia gladioli ( Burkholderia gladioli ), deposited in the General Microbiology Center of China Culture Collection Administration, the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is May 19, 2022, and the deposit number is: CGMCC No. 24934.

[0019] Alcaligenes faecalis, classified as Alcaligenes faecalis ( Alcaligenes faecalis ), deposited in the General Microbiology Center of China Culture Collection Committee of Microorganisms, with the deposit address at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on September 11, 2024, with the deposit number: CGMCC No. 31941. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following detailed description, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 Schematic diagram of the compatibility test results between Burkholderia gladiolus KRS027 and Alcaligenes faecalis KRS268.

[0021] Figure 2 Schematic diagram of the growth promotion results of Burkholderia gladiolus KRS027 and Alcaligenes faecalis KRS268 single bacterial solution and mixed bacterial solution.

[0022] Figure 3This is a schematic diagram of the test results of the effects of single and mixed bacterial solutions of Burkholderia gladioli KRS027 and Alcaligenes faecalis KRS268 on the germination rate of cotton seeds.

[0023] Figure 4 This is a schematic diagram of the test results of the effects of single and mixed bacterial solutions of Burkholderia gladioli KRS027 and Alcaligenes faecalis KRS268 on low-temperature growth of cotton.

[0024] Figure 5 To determine the effectiveness of microbial agents in promoting cotton growth and controlling cotton Verticillium wilt. DETAILED DESCRIPTION

[0025] The following describes the specific embodiments of the present disclosure in detail. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure and are not intended to limit the present disclosure.

[0026] The present disclosure provides a microbial agent, which includes a first fermentation broth and a second fermentation broth, wherein the first fermentation broth is Burkholderia gladiolus ( Burkholderia gladioli ) fermentation broth; The second fermentation broth is a strain of Alcaligenes faecalis ( Alcaligenes faecalis ) of the fermentation broth.

[0027] The microbial agent provided herein contains both Burkholderia gladioli, deposited with CGMCC No. 24934, and Alcaligenes faecalis, deposited with CGMCC No. 31941. The two bacteria work together to regulate the metabolic processes of crops, thereby improving their tolerance to low temperatures, inhibiting pathogens, and promoting overall crop growth.

[0028] Optionally, in the microbial agent, the weight ratio of the first fermentation broth to the second fermentation broth is 1:(0.5-2), preferably 1:(0.5-1.5). In the microbial agent, the Burkholderia gladiolus with a deposit number of CGMCC No. 24934 ( Burkholderia gladioli ) is the concentration of OD 600 =0.8-1.2; the deposit number is CGMCC No. 31941 Alcaligenes faecalis ( Alcaligenes faecalis ) is the concentration of OD 600 =0.8-1.2.

[0029] Optionally, the microbial agent further includes an auxiliary agent, which includes at least one of a dispersant, a synergist, and a suspending agent; the dispersant includes at least one of sodium lignin sulfonate, sodium carboxymethyl cellulose, and saponin powder; the synergist includes at least one of mineral potassium fulvic acid, chitosan, and compound amino acid powder; the suspending agent includes at least one of Langti LT676, Langti EF96, and Langti LTQ661; In the microbial agent, the weight ratio of the first fermentation broth, the second fermentation broth, the dispersant, the synergist, and the suspending agent is 1: (0.5-2): (0.01-0.02): (0.01-0.02): (0.01-0.02); Within the above-defined range, the microbial agents can fully exert their effects in promoting crop growth, inhibiting pathogens and alleviating low temperature stress; within this preferred range, crops can be protected from damage to the greatest extent.

[0030] Optionally, the preparation process of the first fermentation broth comprises: separating and purifying the Burkholderia gladioli ( Burkholderia gladioli ) was streaked onto a LB solid medium, cultured at 24-30°C for 20-30h, single colonies were picked and placed in LB liquid medium, and cultured on a shaker at 24-30°C for 20-30h; the preparation process of the second fermentation broth comprises: separating and purifying the isolated and purified Alcaligenes faecalis ( Alcaligenes faecalis ) Streak inoculate on LB solid medium, culture at 24-30°C for 20-30 h, pick a single colony into LB liquid medium, and culture at 24-30°C in a shaking incubator for 20-30 h.

[0031] In another aspect, the present disclosure provides a use of the above-mentioned microbial agent in improving the low temperature tolerance of crops, wherein the crops include at least one of cotton, tobacco and tomato.

[0032] In another aspect, the present disclosure provides use of the above-mentioned microbial agent in promoting crop growth, wherein the crop comprises at least one of cotton, tobacco, and tomato.

[0033] In another aspect, the present disclosure provides use of the above-mentioned microbial agent in preventing and controlling Verticillium wilt in crops, wherein the crops include at least one of cotton, tobacco, and tomato.

[0034] The present invention is further described in detail below through examples.

[0035] The Burkholderia gladiolus with the deposit number CGMCC No. 24934 in the present disclosure ( Burkholderia gladioli) and Alcaligenes faecalis with the deposit number CGMCC No. 31941 both come from the plant rhizosphere.

[0036] Example 1 This example is used to determine the compatibility of Burkholderia gladioli KRS027 and Alcaligenes faecalis KRS268. The specific process is as follows: Gladiolus Burkholderia KRS027 and Alcaligenes faecalis KRS268 were cultured separately, and the bacterial concentration was adjusted to OD600 = 1.0. One strain was spotted on both sides of the plate, and the other was placed in a sterile spray bottle with evenly sprayed liquid. After 12 hours of spotting, the plates were sprayed, sealed and placed in a 28°C constant temperature box. Cultured in the dark for 24 hours, the presence of an inhibition zone was observed. The presence of an inhibition zone indicated that the strains could not grow together, and the absence of an inhibition zone indicated that the two strains were compatible and could grow together. The two-way verification was repeated for 3 groups. The results showed that Gladiolus Burkholderia KRS027 and Alcaligenes faecalis KRS268 were compatible with each other and did not inhibit each other. Specifically, Figure 1 shown.

[0037] Example 2 This example is used to determine the biocontrol effect of a composite bacterial solution of Burkholderia gladioli KRS027 and Alcaligenes faecalis KRS268 on cotton Verticillium wilt. The specific process is as follows: Verticillium dahliae V.dahliae Preparation of conidia suspension: V.dahliae The 991 strain was inoculated on a PDA plate and cultured at 25°C for 7 days. The colony was cut into small pieces (1×1 mm) with a scalpel and placed in a 500 mL conical flask containing 200 mL of CM culture medium. The culture was kept at 25°C and shaken at 180 rpm for 4 days, and then filtered with sterile gauze. V.dahliae 991 mycelium, get V.dahliae The conidia suspension was counted using a hemocytometer to calculate its concentration.

[0038] Preparation of Burkholderia gladioli KRS027 fermentation broth: The Burkholderia gladioli KRS027 strain was inoculated onto an LB medium plate and cultured at 28°C for 24 hours. A single colony was inoculated into a 500-mL conical flask containing 200 mL of liquid LB medium and cultured at a constant temperature of 28°C and 200 rpm for 12-24 hours. The concentration was determined using a visible spectrophotometer at a wavelength of 600 nm. The fermentation broth of Alcaligenes faecalis KRS268 was prepared using the same method.

[0039] When the cotton was 3 weeks old, KRS027 and KRS268 bacterial solutions were fermented separately or mixed in a ratio of 1:1. 600 =1.0 for the root irrigation treatment, and the plants irrigated with clean water were used as the control. 2 days later, they were inoculated with Verticillium dahliae. V.dahliae991 (1×10 7 cfu), 30 mL was inoculated for each treatment, and 4 replicates were performed. Watering was carried out regularly and uniformly, and the disease condition of the plants was observed after 30 days. The results showed that compared with the single bacterial solution of KRS027 or KRS268, the 1:1 mixed bacterial solution of KRS027 and KRS268 after fermentation alone, and the mixed bacterial solution of KRS027 and KRS268 could significantly inhibit the fungus Verticillium wilt. V.dahliae 991 has a stronger biocontrol effect and growth-promoting effect on cotton. Figure 2 shown.

[0040] Example 3 This example is used to determine the effect of a composite bacterial solution of Burkholderia gladioli KRS027 and Alcaligenes faecalis KRS268 on cotton seed germination. The specific process is as follows: Using nutrient soil and vermiculite mixed in equal proportions as the culture medium, cotton seeds of uniform size and full grains were selected, and 5 experimental groups were set up, with 6 replicates in each group and 7 seeds in each replicate. Group 1: only watered as a control; Group 2: only watered with KRS027 fermentation liquid (OD600=1.0); Group 3: only watered with KRS268 fermentation liquid (OD600=1.0); Group 4: KRS027 and KRS268 were fermented separately, and the concentration of the fermentation liquid was adjusted to OD600=1.0. After mixing in equal proportions, watering was performed; Group 5: equal amounts of KRS027 and KRS268 were mixed and fermented, and the mixed fermentation liquid (OD600=1.0) was watered. Culture was carried out at 25℃, 16h light / 8h dark conditions, and the seed germination amount was recorded at 5d, 7d, 9d, and 11d respectively. The results are as follows Figure 3 As shown in the results, compared with the single bacterial solution of KRS027 or KRS268, the 1:1 mixed bacterial solution of KRS027 and KRS268 after fermentation alone can significantly improve the germination rate of cotton seeds.

[0041] Example 4 This example is used to determine the effect of a composite bacterial solution of Burkholderia gladioli KRS027 and Alcaligenes faecalis KRS268 on cotton growth under low temperatures. The specific process is as follows: Plant cotton and irrigate the roots with the corresponding bacterial solution when the cotton grows the first true leaf; place it in a light incubator and culture it at 15℃, 16h light / 8h dark conditions. Rinse it again after 7 days and water it regularly; after 1 month, record the phenotype of each experimental group and the plant height, stem diameter, chlorophyll content, nitrogen content, dry weight, fresh weight, and collect growth promotion data.

[0042] The results showed that compared with the single bacterial solution of KRS027 or KRS268, the bacterial solution of KRS027 and KRS268 fermented separately in a 1:1 ratio, and the bacterial solution of KRS027 and KRS268 mixed fermented at low temperature (15°C) also had a better effect on promoting cotton growth, which can be used to alleviate the adverse effects of low temperature stress on crops. Figure 4 shown.

[0043] As shown in Examples 1-4, the combined use of Burkholderia gladioli with a deposit number of CGMCC No. 24934 and Alcaligenes faecalis with a deposit number of CGMCC No. 31941 can effectively alleviate the adverse effects of low temperature stress on crops, inhibit crop pathogens, and promote crop growth.

[0044] Example 5 The microbial agent includes the fermentation broth of Burkholderia gladioli with a preservation number of CGMCC No. 24934, the fermentation broth of Alcaligenes faecalis with a preservation number of CGMCC No. 31941, sodium lignin sulfonate as a dispersant, potassium fulvic acid from mineral source as a synergist, and Langtai EF96 as a suspending agent, in a weight ratio of 1:1:0.015:0.015:0.015; the fermentation broth preparation method is the same as that in Example 2. The viable cell counts of the two strains in the agent are respectively 1×10 9 CFU / mL or more, Burkholderia ( Burkholderia gladioli ) is the concentration of OD 600 = 1.0 or above, Alcaligenes faecalis ( Alcaligenes faecalis ) is the concentration of OD 600 = 1.0 or above, the microbial agent was diluted 1 / 200 and applied to plants in the same manner as in Examples 2 and 3, and its effects on promoting crop growth, inhibiting pathogens and resisting low temperatures were determined.

[0045] The test results showed that the microbial agent prepared by gladiolus Burkholderia KRS027, Alcaligenes faecalis KRS268 and a series of adjuvants had good effects in promoting cotton growth and preventing and controlling cotton Verticillium wilt ( Figure ⑤ A, CK represents the untreated group, and Vd represents the group inoculated with Verticillium dahliae, the pathogen of cotton wilt). At the same time, it can still promote cotton seed germination under low temperature conditions ( Figure ⑤ B).

[0046] The preferred embodiments of the present disclosure are described in detail above. However, the present disclosure is not limited to the specific details of the above embodiments. Within the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all fall within the scope of protection of the present disclosure. It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0047] In addition, the various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. A microbial agent, characterized in that: The microbial agent includes a first fermentation broth and a second fermentation broth; The first fermentation broth is Burkholderia gladiolus ( Burkholderia gladioli ) fermentation broth; The second fermentation broth is a strain of Alcaligenes faecalis ( Alcaligenes faecalis ) of the fermentation broth.

2. The microbial agent according to claim 1, wherein In the microbial agent, the weight ratio of the first fermentation broth to the second fermentation broth is 1:(0.5-2), preferably 1:(0.5-1.5).

3. The microbial agent according to claim 2, wherein Among the microbial agents, the Burkholderia gladiolus with the deposit number CGMCC No. 24934 ( Burkholderia gladioli ) is the concentration of OD 600 =0.8-1.2; The deposit number is CGMCC No. 31941 Alcaligenes faecalis ( Alcaligenes faecalis ) is the concentration of OD 600 =0.8-1.

2.

4. The microbial agent according to claim 1, wherein The microbial agent also includes an auxiliary agent, which includes at least one of a dispersant, a synergist, and a suspending agent.

5. The microbial agent according to claim 4, wherein In the microbial agent, the weight ratio of the first fermentation broth, the second fermentation broth, the dispersant, the synergist, and the suspending agent is 1: (0.5-2): (0.01-0.02): (0.01-0.02): (0.01-0.02); The dispersant comprises at least one of sodium lignin sulfonate, sodium carboxymethyl cellulose and soapberry powder; The synergist comprises at least one of mineral-source potassium fulvic acid, chitosan and composite amino acid powder; The suspending agent includes at least one of Langti LT676, Langti EF96 and Langti LTQ661.

6. The microbial agent according to claim 1, wherein The preparation process of the first fermentation liquid comprises: separating and purifying the Burkholderia gladioli (CGMCC No. 24934) Burkholderia gladioli ) Streak inoculate on LB solid medium, culture at 24-30°C for 20-30 h, pick a single colony into LB liquid medium, and culture at 24-30°C in a shaking incubator for 20-30 h.

7. The microbial agent according to claim 1, wherein The preparation process of the second fermentation liquid comprises: separating and purifying the Alcaligenes faecalis (CGMCC No. 31941) Alcaligenes faecalis ) Streak inoculate on LB solid medium, culture at 24-30°C for 20-30 h, pick a single colony into LB liquid medium, and culture at 24-30°C in a shaking incubator for 20-30 h.

8. Use of the microbial agent according to any one of claims 1 to 7 in improving the low temperature resistance of crops, wherein: The crops include at least one of cotton, tobacco and tomato.

9. Use of the microbial agent according to any one of claims 1 to 7 in promoting crop growth, wherein: The crops include at least one of cotton, tobacco and tomato.

10. Use of the microbial agent according to any one of claims 1 to 7 in preventing and controlling Verticillium wilt in crops, wherein: The crops include at least one of cotton, tobacco and tomato.

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