Compound microbial fermentation bacterial fertilizer for antagonizing Amomum tsao-ko withering pathogens and application thereof

Abstract

The invention discloses a compound microbial fermentation bacterial fertilizer for antagonizing Amomum tsao-ko withering pathogenic bacteria and application thereof. The compound microbial fermentation bacterial fertilizer contains bacillus polymyxa and / or bacillus polymyxa fermentation liquor; sinorhizobium and / or sinorhizobium fermentation broth; and comamonas testosteroni and / or a comamonas testosteroni fermentation broth. The bacterial strains in the compound microbial fertilizer comprise bacillus polymyxa, sinorhizobium and comamonas testosteroni, the bacillus polymyxa, the sinorhizobium and the comamonas testosteroni have a synergistic inhibition effect on Amomum tsao-ko wilt related fusarium pathogenic fungi, and the sinorhizobium and the comamonas testosteroni are known functional bacteria with nitrogen fixation and phosphorus solubilization effects; therefore, the combined use of the three strains not only can achieve the effect of preventing and treating diseases, but also can improve the soil environment. In addition, the preparation method of the compound microbial fertilizer is easy to operate, low in cost and suitable for large-scale industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control microorganisms and biological fertilizers, and particularly relates to a composite microbial fermentation fertilizer for antagonizing grass fruit wilt pathogens and an application thereof. Background Art

[0002] In plant taxonomy, Amomum villosum is a perennial herb of the genus Cardamom in the ginger family. It can be used as a medicinal material to dry dampness and resolve phlegm, warm the middle and strengthen the stomach, and digest food and relieve gas. It is a good spicy condiment for both medicine and food. Fugong County was successfully selected as a characteristic county of "one county, one industry" in Yunnan Province for its development of the Amomum villosum industry. The Amomum villosum industry has become the pillar industry with the strongest driving force, the widest radiation, and the largest contribution rate. With the expansion of the planting area and the imperfect supporting management, its diseases have shown a trend of local spread, especially the wilt disease, which is a type of soil-borne disease caused by fungi mainly caused by mango deformity Fusarium. Amomum villosum is relatively likely to suffer from wilt diseases in its infancy, which is mainly manifested in the occasional wilting of individual plants from seedlings to flowering and fruiting. At present, the main prevention and control method of Amomum villosum wilt disease is chemical agents, but the use of chemical agents will cause environmental pollution and easily produce drug resistance. Therefore, it is urgent to develop safe, environmentally friendly and efficient biological control methods.

[0003] Microbial fertilizers can make ineffective nutrients in the soil effective, prevent and control crop diseases, and reduce the use of pesticides and fertilizers. They are the fundamental way to solve soil, water and food pollution. They are generally considered to be an environmentally friendly, cost-effective way to increase crop yields and are widely used in agricultural production. However, there is a lack of compound microbial fermentation fertilizers specifically for amomum villosum diseases on the market, and ordinary microbial fermentation fertilizers are not effective in preventing and controlling amomum villosum diseases, which makes it difficult to meet the actual needs of amomum villosum cultivation. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention proposes a composite microbial fermentation fertilizer for antagonizing the pathogenic bacteria of Amomum villosum wilt and its application, which has the functions of preventing and controlling diseases and improving the soil environment.

[0005] The technical solution of the present invention is as follows:

[0006] A composite microbial fermentation fertilizer for antagonizing the pathogenic bacteria of amomum villosum wilt, the composite microbial fermentation fertilizer containing the following components (1) to (3):

[0007] (1) Bacillus polymyxa and / or Bacillus polymyxa fermentation broth;

[0008] (2) Sinorhizobium and / or Sinorhizobium fermentation broth;

[0009] (3) Comamonas testosteroni and / or Comamonas testosteroni fermentation broth.

[0010] Furthermore, the Latin name of the Bacillus polymyxa is Paenibacillus polymyxa, which is deposited in the Guangdong Provincial Microbiological Culture Collection Center, with a deposit number of GDMCC NO: 64948, a deposit date of April 19, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0011] Furthermore, the Latin name of the Chinese rhizobium is Sinorhizobium, which is deposited in the Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC NO: 64949, a deposit date of April 19, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0012] Furthermore, the Latin name of the Comamonas testosteroni is Comamonas testosteroni, which is deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 19, 2024, with a collection number of GDMCC NO: 64950, and a collection address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0013] Furthermore, the fermentation broth is obtained by inoculating the strain into a fermentation medium and culturing the fermentation medium. The formula of the fermentation medium is Formula 1 or Formula 2:

[0014] Formula 1: brown sugar 23.0g / L, soybean meal 24.0g / L, KH2PO4 1.0g / L, CaCO3 10.0g / L, ultrapure water 1.0L, 1wt% amino acid powder;

[0015] Formula 2: corn flour 20.0g / L, soybean meal 20.0g / L, K2HPO4 5.0g / L, CaCl2 5g / L, MgSO4·7H2O0.5g / L, ultrapure water 1.0L, 1wt% amino acid powder.

[0016] Furthermore, the method for preparing the fermentation broth comprises the following steps:

[0017] The seed liquid of Bacillus polymyxa, Sinorhizobium and Comamonas testosteroni strains is prepared; the Bacillus polymyxa seed liquid is inoculated into a fermentation medium, cultured in a shaking flask, and then inoculated with Sinorhizobium seed liquid and Comamonas testosteroni seed liquid, and the shaking flask culture is continued to obtain a fermentation liquid.

[0018] Furthermore, the method for preparing the fermentation broth comprises the following steps:

[0019] The seed liquid of Bacillus polymyxa, Sinorhizobium and Comamonas testosteroni strains is prepared; the seed liquid of Bacillus polymyxa is inoculated into a fermentation medium, and the culture is carried out in a shake flask for at least 2 days, and then the seed liquid of Sinorhizobium and Comamonas testosteroni is inoculated, and the shake flask culture is continued for at least 7 days to obtain a fermentation liquid.

[0020] The second purpose of the present invention is to provide the application of the composite microbial fermentation fertilizer in the cultivation of tsaoko.

[0021] The third object of the present invention is to provide the application of the composite microbial fermentation fertilizer in inhibiting plant pathogens.

[0022] Furthermore, the plant pathogen is Fusarium mangiferae.

[0023] Beneficial effects of the present invention:

[0024] The strains in this composite microorganism are all isolated from the rhizosphere soil of the Amomum villosum in Longyuan Village, Dulongjiang Township, Gongshan County, Nujiang Prefecture, Yunnan Province, China, ensuring that the strains can effectively colonize and survive after the bacterial fertilizer is returned to the Amomum villosum field, laying the foundation for the practicality of the future development of bacterial fertilizers.

[0025] The strains in the composite microbial fertilizer of the present invention include Bacillus polymyxa, Sinorhizobium and Comamonas testosteroni, which have a synergistic inhibitory effect on Fusarium pathogenic fungi associated with grass fruit wilt disease. Sinorhizobium and Comamonas testosteroni are known functional bacteria with nitrogen fixation and phosphorus solubilization effects. Therefore, the combined use of the three strains can not only achieve the effect of preventing and controlling diseases but also improve the soil environment.

[0026] The fermentation culture medium of the composite microbial fertilizer can be used directly without sterilization, and the materials in the formula are common, so the composite fertilizer has the characteristics of easy operation, cost saving, and suitability for mass production of fertilizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : The culture effects of strains cao48, caoK01 and caoP03 on plates.

[0028] Figure 2 : Colony images of three bacterial strains: cao48, caoK01, and caoP03.

[0029] Figure 3 : Evaluation diagram of the activity of each treatment against Fusarium solani causing mango malformation disease.

[0030] Figure 4 : Three-line cross diagram of the three bacterial strains cao48, caoK01 and caoP03.

[0031] Figure 5: Microscope image of composite microbial fermentation broth diluted 40 times, white arrows: live bacterial cells, black arrows: impurities. DETAILED DESCRIPTION

[0032] In order to better understand the technical content of the present invention, the present invention is further described below through specific embodiments.

[0033] The amino acid powder described in the following examples contains the following ingredients (in parts by mass): 9.2 parts of aspartic acid, 2.8 parts of threonine, 4.0 parts of serine, 14.7 parts of glutamic acid, 5.6 parts of proline, 4.88 parts of glycine, 5.9 parts of alanine, 0.9 parts of cystine, 4.2 parts of valine, 0.9 parts of methionine, 3.0 parts of isoleucine, 6.1 parts of leucine, 2.1 parts of tyrosine, 4.6 parts of phenylalanine, 5.6 parts of lysine, 2.5 parts of histidine, and 3.84 parts of arginine.

[0034] Example 1 Isolation of bacteria with antagonistic, phosphate-solubilizing and potassium-solubilizing effects

[0035] (1) Preparation of culture medium

[0036] Preparation of LB solid medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, sodium chloride 10.0 g / L, agar 20.0 g / L, ultrapure water 1.0 L, pH 7.2±0.2.

[0037] Preparation of phosphate-solubilizing bacteria culture medium (inorganic phosphorus bacteria culture medium): glucose 10.0 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, manganese sulfate 0.03 g / L, potassium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, calcium phosphate 5.0 g / L, agar 15.0 g / L, ultrapure water 1.0 L, pH = 7.0-7.5.

[0038] The nitrogen-fixing bacteria were prepared with Ashurberg medium: 0.2 g / L potassium dihydrogen phosphate, 0.2 g / L magnesium sulfate, 0.2 g / L sodium chloride, 5.0 g / L calcium carbonate, 10.0 g / L mannitol, 0.1 g / L calcium sulfate, 15.0 g / L agar, pH = 7.0 ± 0.1.

[0039] Sample collection and processing The collection site was an Amomum vitis tsaoko field in Longyuan Village, Dulongjiang Township, Gongshan County, Nujiang Prefecture, Yunnan Province, China, and the rhizosphere soil of healthy Amomum vitis tsaoko plants was collected.

[0040] (2) Strain isolation and purification

[0041] LB solid medium was used as the separation plate. 1 g of soil sample was weighed into a sterile test tube, 10 ml of sterile water was added, and the tube was shaken at 28°C and 200 rpm for 30 minutes. Then, the soil sample was diluted with sterile water in multiple ratios to obtain 10 -1, 10 -2 , 10 -3 , 10 -4 Different concentrations of diluted soil sample solutions, due to the large number of bacterial cells, only 10 -2 , 10 -3 , 10 -4 100 μl of each sample solution was coated on the plate in triplicate. After 3 days of constant temperature culture at 28°C, the LB plate was observed and the bacterial strains were picked, purified and subcultured; after 10 days of constant temperature culture at 28°C, the phosphate-solubilizing and nitrogen-fixing plates were observed and the effective bacterial strains were picked and purified.

[0042] (3) Screening of strains

[0043] Through the plate confrontation method, cao48 with antagonistic effect, caoK01 with strong nitrogen fixation and caoP03 with good phosphorus solubilization effect were screened out. Figure 1 After purification, freeze in 25% glycerol tubes at -80°C.

[0044] ① The morphological characteristics of cao48 strain are shown in Table 1, caoK01 are shown in Table 2, and caoP03 are shown in Table 3:

[0045] Table 1 Morphological characteristics of strains

[0046]

[0047] Table 2 Morphological characteristics of strains

[0048]

[0049] Table 3 Morphological characteristics of strains

[0050]

[0051] ②After purification, the three strains were inoculated on LB plates and cultured at 28°C for 4 days. The bacterial plates were directly handed over to the Wuhan Branch of Beijing Liuhe BGI Technology Co., Ltd. for sequencing and strain identification. The results showed that cao48 was Paenibacillus polymyxa, caoK01 was Sinorhizobium, and caoP03 was Comamonas testosteroni. The three strains were deposited in the Guangdong Provincial Microbiological Culture Collection Center, with the deposit numbers GDMCC NO: 64948, GDMCC NO: 64949, and GDMCC NO: 64950, respectively, and the nucleotide sequences are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively.

[0052] Example 2: Verification of the activity of the fermentation broth of the unsterilized fermentation formula of the cao48 strain

[0053] (1) Preparation of culture medium

[0054] Preparation of LB liquid culture medium: 5.0 g / L yeast powder, 10.0 g / L tryptone, 10.0 g / L sodium chloride, 1.0 L ultrapure water, pH 7.2 ± 0.2. (sterilized)

[0055] Bacillus polymyxa formula 1: brown sugar 23.0g / L, soybean meal 24.0g / L, KH2PO4 1.0g / L, CaCO3 10.0g / L, ultrapure water 1.0L, 1% amino acid powder. (No need to sterilize)

[0056] Bacillus polymyxa formula 2: corn flour 20.0g / L, soybean meal 20.0g / L, K2HPO4 5.0g / L, CaCl2 5g / L, MgSO4·7H2O 0.5g / L, ultrapure water 1.0L, 1% amino acid powder. (No need to sterilize)

[0057] (2) Activity verification of bacterial cao48 fermentation broth

[0058] Pathogenic fungi of tested amomum villosum: Fusarium mangiferae

[0059] First, prepare the seed solution, take the activated cao48 strain block and put it into a shake flask containing 200 ml of LB liquid culture medium, and shake it at 28°C and 200 rpm for 2 days.

[0060] The shaken cao48 seed liquid was inoculated into the culture medium shake flask of Bacillus polymyxa formula 1 and 2 at an inoculation ratio of 2%, and shaken at 30°C and 200 rpm for 7 days to obtain the fermentation liquid.

[0061] Each formula has two treatments, namely, filtration (filtered with a 0.22um sterile filter) and no filtration. Take the activated mango deformity disease Fusarium plate, use a sterile puncher (Φ5mm) to make a bacterial cake for use, inoculate it on the center point of the PDA medium plate, and use a sterile puncher (Φ5mm) to punch holes at a spacing of 2.0cm. Take the same fermentation liquid from each plate, take 110ul from each hole, and use sterile water as the control group, repeat three times. Culture at a constant temperature of 28℃ for 6 days.

[0062] Mycelial growth inhibition rate = (control colony growth diameter - treated colony growth diameter) / control colony growth diameter × 100%.

[0063] (3) Test results

[0064] The filtered fermentation broth had no antibacterial effect, while the unfiltered fermentation broth of Formula 1 had an antibacterial rate of 28.6%, and the fermentation broth of Formula 2 had an antibacterial rate of 21.4%. Formula 1 was better than Formula 2, so the composite microbial fertilizer was fermented with Bacillus polymyxa Formula 1. Figure 3 .

[0065] Example 3 Preparation of composite microbial fertilizer

[0066] (1) Verify whether the three strains cao48, caoK01, and caoP03 have inhibitory activity

[0067] The three activated strains were streaked on the LB plate using the three-line cross method and cultured at 28°C for 4 days to observe the results. It was found that the three strains grew normally, that is, there was no mutual inhibition. Figure 4 .

[0068] (2) Pre-preparation process of composite microbial fertilizer

[0069] Prepare seed liquid of cao48, caoK01 and caoP03 strains: take activated bacterial blocks of cao48, caoK01 and caoP03 strains and place them in shake flasks containing 200 ml of LB liquid culture medium, and shake them at 30°C and 200 rpm for 2 days.

[0070] First, 1% of cao48 strain seed solution was inoculated into a shake flask containing 200 ml of Bacillus polymyxa formula 1 (see Example 2 for details), and the shaker was shaken at 30°C and 200 rpm for 2 days. Then, 1% of caoK01 and caoP03 seed solution was inoculated into the shake flask, and the shaker was shaken at 30°C and 200 rpm for 7 days.

[0071] After the fermentation, the fermentation broth was filtered and not filtered. The antibacterial activity of the composite fertilizer was verified by the plate punching method (specific reference to Example 2), and the culture was carried out at a constant temperature of 28° C. for 6 days; the cells of the unfiltered fermentation broth were counted using a 96-well plate.

[0072] Mycelial growth inhibition rate = (control colony growth diameter - treated colony growth diameter) / control colony growth diameter × 100%.

[0073] Total number of cells = total number of counts / 80*400*10000*dilution factor

[0074] The test results showed that the fermented composite microbial fertilizer was inactive after filtration, while the unfiltered fermentation liquid had strong antagonism to the pathogenic fungi of Amomum villosum, with an inhibition rate of 57.1%. The number of live bacteria in the fermentation liquid after 7 days of shaking was 704 million.

[0075] (3) Preparation of composite microbial fertilizer

[0076] A large fermentation tank is used as a fermentation container, and the composite microbial fertilizer is produced in large quantities by referring to the above step (2).

[0077] The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. A composite microbial fermentation fertilizer for antagonizing the pathogenic bacteria of Amomum villosum wilt, characterized in that: The composite microbial fermentation fertilizer contains the following components (1) to (3): (1) Bacillus polymyxa and / or Bacillus polymyxa fermentation broth; (2) Sinorhizobium and / or Sinorhizobium fermentation broth; (3) Comamonas testosteroni and / or Comamonas testosteroni fermentation broth.

2. The composite microbial fermentation fertilizer according to claim 1, characterized in that: The Latin name of the polymyxa bacillus is Paenibacillus polymyxa, which is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCCNO:64948.

3. The composite microbial fermentation fertilizer according to claim 1, characterized in that: The Latin name of the Sinorhizobium is Sinorhizobium, which is deposited in Guangdong Microbial Culture Collection Center with a deposit number of GDMCC NO:64949.

4. The composite microbial fermentation fertilizer according to claim 1, characterized in that: The Latin name of the Comamonas testosteroni is Comamonas testosteroni, which is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCCNO:64950.

5. The composite microbial fermentation fertilizer according to claim 1, characterized in that: The fermentation broth is obtained by inoculating the strain into a fermentation medium and culturing the fermentation medium. The formula of the fermentation medium is Formula 1 or Formula 2: Formula 1: brown sugar 23.0g / L, soybean meal 24.0g / L, KH2PO4 1.0g / L, CaCO3 10.0g / L, ultrapure water 1.0L, 1wt% amino acid powder; Formula 2: corn flour 20.0g / L, soybean meal 20.0g / L, K2HPO4 5.0g / L, CaCl2 5g / L, MgSO4·7H2O 0.5g / L, ultrapure water 1.0L, 1wt% amino acid powder.

6. The composite microbial fermentation fertilizer according to claim 1, characterized in that: The method for preparing the fermentation broth comprises the following steps: The seed liquid of Bacillus polymyxa, Sinorhizobium and Comamonas testosteroni strains is prepared; the Bacillus polymyxa seed liquid is inoculated into a fermentation medium, cultured in a shaking flask, and then inoculated with Sinorhizobium seed liquid and Comamonas testosteroni seed liquid, and the shaking flask culture is continued to obtain a fermentation liquid.

7. The composite microbial fermentation fertilizer according to claim 6, characterized in that: The method for preparing the fermentation broth comprises the following steps: The seed liquid of Bacillus polymyxa, Sinorhizobium and Comamonas testosteroni strains is prepared; the seed liquid of Bacillus polymyxa is inoculated into a fermentation medium, and the culture is carried out in a shake flask for at least 2 days, and then the seed liquid of Sinorhizobium and Comamonas testosteroni is inoculated, and the shake flask culture is continued for at least 7 days to obtain a fermentation liquid.

8. Use of the composite microbial fermentation fertilizer according to any one of claims 1 to 7 in the cultivation of tsaoko.

9. Use of the composite microbial fermentation fertilizer according to any one of claims 1 to 7 in inhibiting plant pathogens.

10. The use according to claim 9, characterized in that: The plant pathogen is Fusarium mangiferae.

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