A strain of Amycoticola tucumiensis KC 316 and its application

Abstract

The invention discloses a strain of Amycolatopsis tucumanensis KC 316 and its application. The microorganism deposit registration number of the strain is CGMCC No. 26012. The invention provides a method for preparing a fermentation product of Amycolatopsis tucumanensis KC 316. Experiments have shown that the fermentation product of Amycolatopsis tucumanensis KC 316 can promote crop growth and has high activity of killing root-knot nematodes. The strain screened by the invention is reported for the first time to be used for the prevention and treatment of nematode diseases, and also has the effect of promoting plant growth. Compared with the currently used chemical synthetic pesticides, it comes from the natural environment, has the advantages of high efficiency, low toxicity, no environmental pollution, and is not easy to produce drug resistance, and is of great significance to the sustainable development of agriculture.

Description

Technical Field

[0001] The invention relates to the technical field of microbial engineering, and in particular to a strain of Amycopsis tucumanicum KC 316 and an application thereof. Background Art

[0002] Amycolatopsis belongs to the Pseudonocardaceae family. The secondary products of many strains of this genus have novel biological activities, most of which are important antibiotics with strong antibacterial ability, and are therefore widely used in the industrial production of antibiotics. Amycolatopsis tucumanensis was reported by Albarracín et al. in 2010. Its cells can synthesize a low molecular weight cysteine-rich protein, which has a strong ability to bind copper and thus has a good ability to accumulate copper in the cell. It can effectively reduce the effective copper content of plants in copper-polluted areas and can be used to treat environmental copper pollution.

[0003] In recent years, long-term monoculture and the rapid development of intensive greenhouse vegetable cultivation have led to frequent root-knot nematodes. The direct economic losses caused by root-knot nematodes in my country are as high as 50 billion yuan each year, which seriously restricts agricultural development. Root-knot nematodes damage the epidermal cells of the roots through their stylets, causing pathological changes in the tissue cells of the host plant, producing giant cells and forming root knots. At the same time, the normal physiological metabolism of the plant is blocked, the plant absorbs insufficient water and inorganic salts, and the plant wilts, leaves turn yellow, the phenological period is delayed, and the yield decreases. In addition, the stylets of the root-knot nematodes cause mechanical damage to the epidermal cells of the host plant, providing an infection pathway for other pathogens in the soil, causing multiple pathogens to invade the host plant, forming complex diseases, and causing the plant to grow slowly, fall leaves, and even die.

[0004] At present, the prevention and control measures for root-knot nematodes mainly include agricultural prevention, physical prevention, chemical prevention and biological prevention. Agricultural prevention mainly prevents and controls root-knot nematodes through crop rotation, intercropping, soil tillage, field management of grafted resistant rootstocks, etc. However, agricultural prevention is limited by land resources and the number of rootstocks, and it is difficult to implement on a large scale. Physical prevention mainly eliminates nematode eggs in the soil through sunlight disinfection, hot water treatment of soil, etc., but it also eliminates some beneficial bacteria in the soil and destroys the soil aggregate structure and ecosystem. Chemical control is currently the most widely used nematode control method, mainly through methyl bromide, thiazophos, carbofuran, aldicarb and other synthetic chemical pesticides for prevention and control, but the long-term use of chemical agents is likely to cause nematode resistance and environmental pollution. Residual agents enter the food chain and cause poisoning to humans and animals. Therefore, many chemical nematicides such as carbofuran and methyl bromide fumigants have been explicitly banned by the state. Biological control is the use of plants, microorganisms or their metabolites to control root-knot nematodes. It not only reduces the toxic residues and environmental hazards of chemical control, but also achieves a control effect that is easy to degrade and not easy to develop resistance. Some functional microorganisms also show a promoting effect on crop growth and development. Therefore, screening new dual-functional strains with nematicidal activity and growth-promoting effects is of great significance to the sustainable development of agriculture.

[0005] After searching, no reports were found about the nematicidal activity and plant growth promoting function of Amycol. tucumana. Summary of the invention

[0006] In view of the above technical problems, the present invention provides a strain of Amycolatopsis tucumán KC 316 and applications thereof.

[0007] The present invention provides a strain of Amycolatopsis tucumanensis, whose classification name is (Amycolatopsis tucumanensis) KC 316, which has been deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on November 2, 2022, and its deposit registration number is CGMCC No. 26012.

[0008] The present invention also protects a method for preparing a fermentation product of Amycolatopsis tucumánensis, comprising the following steps:

[0009] Activation of S1 strain: The Amycoticola tucumiensis strain stored in a glycerol tube was streaked onto a solid medium of modified International Streptomyces 2 medium (ISP 2) and cultured for 3-5 days;

[0010] Preparation of S2 seed solution: inoculate a single colony of Amycota tucumanicum activated in S1 into a seed culture medium, culture on a shaker at 25-40°C and 80-200 rpm, and ferment for 3-5 days to prepare a seed solution;

[0011] S3 large-scale fermentation, take 5 mL of the seed solution prepared in S2 and inoculate it into 100 mL of fermentation medium, shake and culture at 25-40°C and 80-200 rpm, and ferment on a shaker for 5-9 days to obtain a fermentation liquid;

[0012] S4 centrifuges, centrifuges the fermentation liquid obtained in S3 at 4000 rpm for 10 min, and collects the fermentation liquid supernatant;

[0013] S5: Extraction of fermentation products: The fermentation liquid supernatant obtained in the above step S4 is extracted with an organic reagent, and the extract is subjected to reduced pressure distillation to remove the organic solvent, and the obtained product is the fermentation crude extract.

[0014] Furthermore, the specific method in S5 is: extracting with an equal volume of ethyl acetate, standing until the aqueous phase and the ethyl acetate phase are separated, and collecting the ethyl acetate phase; extracting the remaining aqueous phase with an equal volume of ethyl acetate again, standing until the aqueous phase and the ethyl acetate phase are separated, and collecting the ethyl acetate phase at the upper layer; extracting the remaining aqueous phase with an equal volume of ethyl acetate for the third time, standing until the aqueous phase and the ethyl acetate phase are separated, and collecting the ethyl acetate phase; combining the ethyl acetate phases obtained by the three extractions, and distilling under reduced pressure at 40-50°C to remove the organic solvent, and the obtained substance is the fermentation crude extract.

[0015] Furthermore, the preparation method of the fermentation medium in step S3 is as follows: 5g glucose, 5g yeast extract, 5g malt extract powder, dissolved in water, adjusted to pH 7.5, 18-20g agar, diluted to 1L with water, and sterilized at 121°C for 15 minutes.

[0016] Preferably, the preparation method of the seed culture medium in step S2 is: dissolve 5g yeast extract, 10g malt extract and 4g glucose in water, adjust the pH to 7.0-7.2, dilute to 1L with water, sterilize at 121°C for 30 minutes, and the seed solution OD 600 nm is 1.6-2.0.

[0017] Preferably, the preparation method of the improved international Streptomyces 2 medium (I SP 2) in step S1 is as follows: 5g glucose, 5g yeast extract, 5g malt extract powder, adjust the pH to 7.5, and dilute to 1L with water.

[0018] Preferably, the solid culture medium in step S1 is prepared as follows: 5 g glucose, 5 g yeast extract, and 5 g malt extract powder are dissolved in water and the pH is adjusted to 7.5, 18-20 g agar is diluted to 1 L with water, and sterilized at 121° C. for 15 minutes.

[0019] The present invention also protects the use of the fermentation product of Amycolatopsis tucumanicum in the preparation of nematicide fungicides.

[0020] The present invention also protects the use of the fermentation product of Amycolatopsis tucumanicum in the preparation of a microbial agent for promoting crop growth.

[0021] Preferably, the nematode is a root-knot nematode.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention provides a strain of Amycota tucumiensis KC 316, which is reported for the first time to be useful for the prevention and treatment of nematode diseases.

[0024] (2) The fermentation product of Amycol. tucumense KC 316 of the present invention not only kills nematodes but also promotes plant growth.

[0025] (3) Compared with the current use of chemical synthetic pesticides for prevention and control, the fermented bacterial liquid of the present invention comes from the natural environment and has the advantages of high efficiency, low toxicity, no pollution to the environment and not easy to produce drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a phylogenetic tree diagram;

[0027] Figure 2 This is the result diagram of the nematicidal activity of the crude extract;

[0028] Figure 3 This is the effect diagram of KC 316 fermentation liquid promoting root growth;

[0029] Figure 4 The effect of KC 316 fermentation liquid on promoting stem growth;

[0030] Figure 5 This is a comparison chart of the effects of KC 316 fermentation broth. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0032] The experimental methods in the following examples are conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. DMSO is the full name of dimethyl sulfoxide. Unless otherwise specified, the quantitative tests in the following examples are repeated three times, and the results are averaged.

[0033] Example 1 Isolation, identification and preservation of strains

[0034] 1.1 A strain was isolated from the rhizosphere soil of plants collected from Jiaozi Mountain and named strain KC 316.

[0035] 1.2 Identification of strain KC 316

[0036] The colonies of strain KC 316 on the modified medium of "International Streptomyces Medium No. 2" (I SP 2) were light yellow, dry, opaque, and had a dry powdery surface. The colonies were tightly connected to the medium, with irregular edges and were not easy to pick up.

[0037] The phylogenetic tree constructed from the 16S rRNA gene sequence of strain KC 316 is shown in Figure 1 strain KC 316 and Amycolatopsis tucumanensis ABO T The similarity of the two strains was 99.72%. Combined with physiological and biochemical tests, KC 316 was identified as Amycolatopsis tucumanensis.

[0038] 1.3 Deposit of strain KC 316

[0039] The above-mentioned strain KC 316 was identified as Amycolatopsis tucumanensis, and was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on November 2, 2022, with the deposit registration number CGMCC No. 26012.

[0040] Example 2 Preparation of Amycolatopsis tucumanicum fermentation product

[0041] 2.1 Activation of strains: Take the KC 316 strain stored in a glycerol tube, streak it onto a modified International Streptomyces 2 (I SP 2) solid medium, and culture it for 3-5 days. The preparation method of the modified I SP 2 medium is as follows: 5g glucose, 5g yeast extract, 5g malt extract powder, adjust the pH to 7.5, add 18-20g agar, dilute to 1L with water, and sterilize at 121℃ for 15 minutes;

[0042] 2.2 Preparation of seed solution: Inoculate the activated single colony in 2.1 into the seed culture medium, and ferment in a shaking incubator at 25-40°C and 80-200 rpm for 3-5 days to prepare seed solution; the seed solution is OD 600 nm is 1.6-2.0;

[0043] 2.3 Take 5 mL of the seed solution in 2.2 and inoculate it into 100 mL of fermentation medium, and ferment it on a shaker at 25-40°C and 80-200 rpm for 5-9 days. The resulting product is the fermentation liquid; the fermentation medium: 5 g of glucose, 5 g of yeast extract, and 5 g of malt extract are dissolved in water, adjusted to pH 7.5, diluted to 1 L with water, and sterilized at 121°C for 15 minutes;

[0044] 2.4 The fermentation broth obtained in the above step is centrifuged at 4000rpm for 10min, the supernatant is collected, and extracted with an equal volume of ethyl acetate. After the aqueous phase and the ethyl acetate phase are allowed to stand for separation, the ethyl acetate phase is collected; the remaining aqueous phase is extracted again with an equal volume of ethyl acetate, after the aqueous phase and the ethyl acetate phase are allowed to stand for separation, the ethyl acetate phase is collected; the remaining aqueous phase is extracted for the third time with an equal volume of ethyl acetate, after the aqueous phase and the ethyl acetate phase are allowed to stand for separation, the ethyl acetate phase is collected; the ethyl acetate phases obtained by the three extractions are combined, and distilled under reduced pressure at 40-50°C to remove the organic solvent, and the obtained product is the crude extract.

[0045] Example 3 The lethal effect of the fermentation product of Amycota tucumanicum KC 316 on root-knot nematodes

[0046] Take the crude extract prepared in Example 2, dissolve it with DMSO to make the crude extract concentration 500μg / μL; take a 96-well plate, add 25μL of Java root-knot nematode suspension (60-80), 100μL of crude extract solution and 75μL of sterile water to each well, gently blow and mix with a pipette, let stand at 20°C for 12h, and then count the nematode mortality rate. Use an equal volume of DMSO instead of the crude extract solution as a negative control. Carry out three repeated experiments, set 3 repeated treatments for each repeated experiment, and take the average value of the results. As a result, the nematode mortality rate of the negative control treatment was 6.5%, and the nematode mortality rate of the crude extract treatment group was 86.3% (see Figure 2 ).

[0047] Example 4 Effect of the fermentation liquid of Amycolatopsis tucumanicum KC 316 on crop growth

[0048] Take the fermentation broth obtained in step 2.3 of Example 2 and dilute the fermentation broth to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5, 10 -6 6 concentration gradients. Sterile distilled water was used as the negative control. 100 mL of each of the above 6 concentration gradients and fermentation stock solution were taken to detect the effect of different concentrations of fermentation solution on cucumber seed germination: the cucumber seeds were soaked in 40-50°C warm water for 12-24 hours, then taken out and placed in a 9 cm culture dish covered with sterilized filter paper (5 seeds / dish × 5). 3 mL of fermentation solution corresponding to the concentration gradient was added to soak the filter paper, and then placed in a light incubator with a temperature of 28°C and a humidity of 70%, and cultured with 12 hours of darkness and 12 hours of light alternately. 2 mL of fermentation solution was added every 2 days, and observations were made every day. The results were recorded continuously for 14 days, and the main root length and bud length were recorded. The experiment was repeated 3 times, and at least 15 repeated treatments were set for each repeated experiment. The results were averaged. The root length of cucumbers treated with the negative control was 10.93 cm, and the stem length was 2.74 cm. The root length of cucumbers treated with the fermentation solution was 15.73 cm, and the stem length was 4.17 cm (see for details). Figure 3-Figure 5 ), indicating that KC 316 fermentation liquid can promote root growth and stem growth.

Claims

1. A strain of Amycota tucumana, characterized in that: The classification name is ( Amycolatopsis tucumanensis ) KC 316 was deposited on November 2, 2022 in the General Microbiology Center of China Microorganism Culture Collection (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), and its deposit registration number is CGMCC No. 26012.

2. A fermentation product of Amycolatopsis tucumanensis as claimed in claim 1, wherein the preparation method comprises the following steps: S1 strain activation: the Amycoticola tucumiensis strain stored in a glycerol tube was streaked onto a solid medium of modified International Streptomyces 2 (ISP 2) and cultured for 3-5 days; Preparation of S2 seed solution: inoculate a single colony of Amycota tucumanicum activated in S1 into the seed culture medium, culture in a shaking incubator at 25-40°C and 80-200 rpm, and ferment for 3-5 days to prepare the seed solution; S3 large-scale fermentation, take 5 mL of seed solution prepared in S2 and inoculate it into 100 mL of fermentation medium, culture it at 25-40 °C and 80-200 rpm, and ferment it on a shaker for 5-9 days to obtain fermentation liquid; S4 centrifugation: centrifuge the fermentation broth obtained in S3 at 4000 rpm for 10 min, and collect the fermentation broth supernatant; S5 Extraction of fermentation products: The fermentation supernatant obtained in the above step S4 is extracted with an organic reagent, and the extract is subjected to reduced pressure distillation to remove the organic solvent. The obtained product is the fermentation crude extract.

3. The method for preparing the fermentation product of Amycolatopsis tucumanensis according to claim 2, characterized in that: The specific method in S5 is: extracting with an equal volume of ethyl acetate, standing until the aqueous phase and the ethyl acetate phase are separated, and collecting the ethyl acetate phase; extracting the remaining aqueous phase with an equal volume of ethyl acetate again, standing until the aqueous phase and the ethyl acetate phase are separated, and collecting the ethyl acetate phase at the upper layer; extracting the remaining aqueous phase with an equal volume of ethyl acetate for the third time, standing until the aqueous phase and the ethyl acetate phase are separated, and collecting the ethyl acetate phase; combining the ethyl acetate phases obtained by the three extractions, and distilling under reduced pressure at 40-50° C. to remove the organic solvent, and the obtained substance is the fermentation crude extract.

4. The method for preparing the fermentation product of Amycolatopsis tucumanensis according to claim 2, characterized in that: The preparation method of the fermentation medium in step S3 is as follows: 5 g glucose, 5 g yeast extract, and 5 g malt extract powder are dissolved in water and the pH is adjusted to 7.5, 18-20 g agar is diluted to 1 L with water, and sterilized at 121°C for 15 minutes.

5. The method for preparing the fermentation product of Amycolatopsis tucumanensis according to claim 2, characterized in that: The preparation method of the seed culture medium in step S2 is: dissolve 5 g yeast extract, 10 g malt extract and 4 g glucose in water, adjust the pH to 7.0-7.2, dilute to 1 L with water, sterilize at 121°C for 30 minutes, and the OD600nm of the seed solution is 1.6-2.

0.

6. The method for preparing the fermentation product of Amycolatopsis tucumanensis according to claim 2, characterized in that: The preparation method of the improved International Streptomyces 2 medium (ISP 2) described in step S1 is as follows: 5 g glucose, 5 g yeast extract, 5 g malt extract powder, adjust the pH to 7.5, and dilute to 1 L with water.

7. The method for preparing the fermentation product of Amycolatopsis tucumanensis according to claim 2, characterized in that: The solid culture medium in step S1 is prepared as follows: 5 g glucose, 5 g yeast extract, and 5 g malt extract powder are dissolved in water and the pH is adjusted to 7.5; 18-20 g agar is diluted to 1 L with water and sterilized at 121°C for 15 minutes.

8. Use of the fermentation product of Amycolatopsis tucumanicum according to any one of claims 2 to 7 in the preparation of a microbial agent for killing root-knot nematodes.

9. Use of the fermentation product of Amycolatopsis tucumanensis according to any one of claims 2 to 7 in the preparation of a microbial agent for promoting cucumber seed germination and rhizome growth.

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