Sea red bacteria strain, biocontrol agent and fermentation liquid, and preparation method and application thereof

Abstract

The invention discloses a sea red bacteria strain, a biocontrol agent and a fermentation liquid, as well as a preparation method and application thereof. The sea red bacteria strain is sea red bacteria RH-AZ ( Rhodobium marinum RH‑AZ), with a deposit number of CCTCC NO: M 20221252 and a deposit date of August 8, 2022. The biocontrol agent is obtained by seed culture and production culture of the sea red bacteria RH‑AZ. The fermentation broth is obtained by fermenting and culturing the biocontrol agent and centrifuging the supernatant. The sea red bacteria strain, the biocontrol agent and the fermentation broth of the present invention have the advantages of low production cost, good effect, high efficiency, non-toxicity and environmental protection, can significantly improve the salt tolerance of rice and promote its growth, and can be used to improve the growth and development of rice seedlings under salt stress.

Description

Technical Field

[0001] The invention belongs to the field of agricultural products and relates to a sea red mushroom strain, a biocontrol agent and a fermentation liquid as well as a preparation method and application thereof. Background Art

[0002] Improving the salt tolerance of rice can minimize the adverse impact on the environment while ensuring stable yields. This has very important research significance for the development and utilization of saline-alkali land, the increase in food crop yields, and the promotion of economic development.

[0003] The salt tolerance of rice is relatively weak during the seedling and reproductive growth stages. The salt tolerance concentration of rice seedlings is generally 0.5%, which is also the key period for evaluating whether rice can tolerate salt. Under high salt stress, rice seedlings gradually dry up and turn yellow from leaf tips to petioles, from lower leaves to new leaves, plant height decreases, new leaves curl, dead leaves increase, root growth is significantly inhibited, and finally the whole seedling appears scorched, eventually leading to the whole seedling withering and death. High salt stress during the reproductive growth period will directly affect many traits related to grain quality and quantity. The number of rice tillers, panicle weight, panicle length, number of primary branches and stalks, number of secondary branches and stalks, number of grains per panicle, and thousand-grain weight are all affected to varying degrees, ultimately leading to a significant decrease in yield. Therefore, improving the salt tolerance mechanism of rice plants during the reproductive growth period is of great significance to improving rice salt tolerance and high yield.

[0004] At present, rice seeds in coastal areas and other high-salinity areas are mainly irrigated with freshwater sources to reduce the inhibitory effect of high salinity on rice growth. The corresponding agricultural measures are complex, with large economic investment and low input-output ratio. The use of microbial agents to improve the salt tolerance of rice has good ecological, environmental and social benefits. Due to its advantages in environmental, ecological and human health safety, it is in line with the sustainable development of agriculture and is the current and future development trend for improving the salt tolerance of rice. In recent years, domestic and foreign scholars have made great progress in basic research and applied research on using microorganisms to improve the salt tolerance of rice. Many research results have shown that microbial preparations are rich in a variety of physiologically active substances and growth-promoting and stress-resistant factors. The exocrine compounds of the bacteria also have a stimulating effect on the salt-tolerant growth of rice, which can significantly improve the salt tolerance of rice. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the technical problems in the prior art and provide a sea red bacteria strain, a biocontrol agent and a fermentation liquid with low production cost, good effect, high efficiency, non-toxicity and environmental protection, as well as a preparation method and application, so as to solve the technical problems of great production difficulty, low yield and high cost that currently occur during the growth of rice under high salt stress.

[0006] In order to solve the above technical problems, the present invention provides a strain of Rhizopus spp., wherein the strain is Rhizopus spp. RH-AZ (Rhodobium marinum RH-AZ), the deposit number is CCTCC NO: M 20221252, and the deposit date is August 8, 2022.

[0007] The above-mentioned Hydatid strain, further, the DNA sequence of Hydatid RH-AZ is shown in SEQ ID NO.1.

[0008] Based on a general technical concept, the present invention also provides a biocontrol agent, which is obtained by seed culture and production culture of the Rhizoctonia solani RH-AZ.

[0009] Based on a general technical concept, the present invention also provides a method for preparing the biocontrol agent, which comprises the following steps:

[0010] S1. Inoculate a single colony of R. salsa RH-AZ into a serum bottle, perform seed culture in R. salsa liquid culture medium, and obtain a bacterial solution in the logarithmic growth phase;

[0011] S2. inoculating the bacterial liquid into a production culture medium, and carrying out production culture until the logarithmic growth phase to obtain a biocontrol agent of the Rhizoctonia solani strain.

[0012] In the above preparation method, further, the temperature of the seed culture in S1 is 28°C to 32°C, and the light condition is 2500 Lx to 3500 Lx.

[0013] In the above preparation method, further, the bacterial liquid is inoculated in S2 according to 2% to 5% of the total volume of the production culture medium of the Rhizopus spp.

[0014] In the above preparation method, further, the temperature of the production culture in S2 is 28°C to 32°C, and the lighting conditions are 2500 Lx to 3500 Lx.

[0015] In the above preparation method, further, the components of the sea red bacteria liquid culture medium include: 1.5 g / L yeast extract powder, 1 g / L NaAc, 1.5 g / L (NH4)2SO4, 0.5 g / L MgSO4·7H2O, 1 g / L KH2PO4, 0.1 g / LCaCl2, 3g / L NaCl, 2g / L K2HPO4, 0.5g / L H3BO3, 0.03g / L NaMoO4·2H2O, and the pH value is 7.0.

[0016] In the above preparation method, further, the components of the production medium include: 1.5 g / L yeast extract powder, 1 g / L NaAc, 1.5 g / L (NH4)2SO4, 0.5 g / L MgSO4·7H2O, 1 g / L KH2PO4, 0.1 g / L CaCl2, 3g / LNaCl, 2g / L K2HPO4, 0.5g / L H3BO3, 0.03g / L NaMoO4·2H2O, and the pH value is 7.0.

[0017] Based on a general technical concept, the present invention also provides an application of a biocontrol agent in improving the growth and development of rice seedlings under salt stress.

[0018] Based on a general technical concept, the present invention also provides a fermentation broth, which is obtained by fermenting and culturing the biocontrol agent and centrifuging the supernatant.

[0019] Based on a general technical concept, the present invention also provides a method for preparing the fermentation broth, the preparation method comprising the following steps:

[0020] S3. Fermenting and culturing the biocontrol agent according to claim 3 at a temperature of 30° C. and a light condition of 3000 Lx for 7 to 10 days;

[0021] S4. Centrifuge the fermented solution at 6000 rpm for 15 min, collect the supernatant, and obtain the fermentation broth.

[0022] Based on a general technical concept, the present invention also provides an application of the fermentation liquid in improving the growth and development of rice seedlings under salt stress.

[0023] Compared with the prior art, the advantages of the present invention are:

[0024] (1) The present invention provides a Rhizopus virgatus strain RH-AZ of the present invention, which has a simple culture process, short culture time and low cost, and can be used to improve the growth and development of rice seedlings under salt stress.

[0025] (2) The present invention provides a biocontrol agent of the Rhizopus spp. strain of the present invention, which is environmentally friendly, non-toxic to humans and animals, harmless to crops, and easy and convenient to apply. Its preparation method has the advantages of being simple, fast, and low-cost; it can significantly improve the salt tolerance of rice and promote its growth.

[0026] (3) The present invention provides a fermentation liquid of the sea red bacteria strain of the present invention, which is environmentally friendly, non-toxic to humans and animals, harmless to crops, and easy and convenient to apply. Its preparation method has the advantages of being simple, fast, and low-cost; it can significantly improve the salt tolerance of rice and promote its growth.

[0027] A Rhodobium marinum sp. RH-AZ strain, whose preservation unit is the China Center for Type Culture Collection (CCTCC), located at Wuhan University, China, with the preservation number being CCTCC NO: M 20221252 and the preservation date being August 8, 2022.

[0028] In addition to the above-described purposes, features and advantages, the present invention has other purposes, features and advantages. The present invention will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings constituting a part of this application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0030] Figure 1 Schematic diagram of the strain of the sea red bacteria strain of Example 1 of the present invention.

[0031] Figure 2 It is a schematic diagram of the biocontrol agent of the sea red bacteria strain of Example 2 of the present invention.

[0032] Figure 3 It is a schematic diagram of the fermentation broth of the Rhizopus spp. strain of Example 3 of the present invention.

[0033] Figure 4 These are the results of the effects of the biocontrol agent and biocontrol fermentation liquid treatment of the Rhizoctonia solani strain in Example 4 of the present invention on the growth of rice seedlings under salt stress.

[0034] Figure 5 These are the results of the effects of the biocontrol agent and biocontrol fermentation liquid treatment of the Rhizoctonia solani strain in Example 4 of the present invention on the fresh weight of the stems of rice seedlings under salt stress.

[0035] Figure 6 These are the results of the effects of the biocontrol agent and biocontrol fermentation liquid treatment of the Rhizoctonia solani strain in Example 4 of the present invention on the stem weight of rice seedlings under salt stress.

[0036] Figure 7 These are the results of the effects of the biocontrol agent and biocontrol fermentation liquid treatment of the Rhizoctonia solani strain in Example 4 of the present invention on the stem length of rice seedlings under salt stress. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with the accompanying drawings and specific preferred embodiments, but the protection scope of the present invention is not limited thereby. The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0038] The materials and instruments used in the following examples are all commercially available.

[0039] Example 1

[0040] A strain of the present invention is the strain of the present invention, namely, RH-AZ ( Rhodobium marinum sp. RH-AZ), which was isolated and purified from Qingdao seawater and deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M20221252. The depository address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hunan Province, China.

[0041] The gene sequence of the RH-AZ strain of Rhizoctonia solani is shown in SEQ ID NO.1, specifically:

[0042]

[0043] The Rhizoctonia solani RH-AZ of this embodiment was obtained by screening using the following method:

[0044] (1) Separation: 5.0 mL of seawater sample collected from Qingdao was added to 120 mL of R. marinum enrichment liquid culture medium and cultured for 5 to 7 days to obtain a dark red mixed bacterial liquid. The formula of R. marinum liquid culture medium is: 1.5 g / L yeast extract powder, 1 g / L NaAc, 1.5 g / L (NH4)2SO4, 0.5 g / L MgSO4·7H2O, 1 g / L KH2PO4, 0.1 g / L CaCl2, 3 g / L NaCl, 2 g / L K2HPO4, 0.5 g / L H3BO3, 0.03 g / L NaMoO4·2H2O, pH value is 7.0.

[0045] (2) Use an inoculation loop to pick up an appropriate amount of the dark red mixed bacterial liquid and streak it on the double-layer plate medium enriched with R. marinum. After culturing at 30℃±2℃ and 3000 Lx light for 7 to 10 days, a large number of dark red single colonies were obtained. The solid culture medium of R. marinum culture medium has the following formula: 1.5 g / L yeast extract powder, 1 g / L NaAc, 1.5 g / L (NH4)2SO4, 0.5 g / LMgSO4·7H2O, 1 g / L KH2PO4, 0.1 g / L CaCl2, 3g / L NaCl, 2g / L K2HPO4, 0.5g / L H3BO3, 0.03g / L NaMoO4·2H2O, pH value is 7.0. (Agar powder of the upper layer of the double-layer plate medium is 18 g / L, and agar powder of the lower layer is 13 g / L).

[0046] The colony is round, with smooth edges and a colony diameter of 0.20 mm to 0.60 mm. Use a sterilized toothpick to pick a single colony with a regular, smooth and large shape and enrich it in the liquid culture medium of the red bacteria for 7 to 10 days to obtain a rose-red bacterial solution. Take the rose-red bacterial solution and streak it, pick a single cell to enrich the bacterial solution, and repeat 3 times to obtain a pure culture. The biomass of the strain in the pure culture finally obtained by enrichment was determined by the gradient dilution method and the dilution plate culture method. The concentration of the red bacteria agent used in the subsequent experiments was about 1×10 6 cfu / mL~1×10 8 cfu / mL.

[0047] Figure 1 Schematic diagram of the strain of the sea red bacteria strain of the preferred embodiment of the present invention. The sea red bacteria RH-AZ has the following main characteristics: -The individual morphology is rod-shaped, and it can grow in 1% to 9% NaCl. The optimal growth temperature is 28℃ to 32℃, and the optimal growth pH is 6.8 to 7.2. It grows well under anaerobic and microaerobic conditions. The characteristic absorption peaks of the bacteria are 300 nm, 380 nm, 520 nm, 880 nm and 890 nm, and the measured 16S rDNA sequence length is 1335 bp.

[0048] Example 2

[0049] A biocontrol agent of the present invention is obtained by separating and purifying the Rhizoctonia solani strain of Example 1, carrying out seed culture and production culture.

[0050] The preparation method of the biocontrol agent comprises the following steps:

[0051] (1) The single colony of R. salsa RH-AZ in Example 1 was inoculated into a serum bottle, and seed culture was performed in a R. salsa liquid culture medium. The temperature of the seed culture was 28°C to 32°C, the light conditions were 2500 Lx to 3500 Lx, and the pH value of the liquid culture medium was 7.0 to 7.2, and a bacterial solution in the logarithmic growth phase was obtained. The components of the R. salsa liquid culture medium were: 1.5 g / L yeast extract powder, 1 g / L NaAc, 1.5 g / L (NH4)2SO4, 0.5 g / L MgSO4·7H2O, 1 g / L KH2PO4, 0.1 g / LCaCl2, 3 g / L NaCl, 2 g / L K2HPO4, 0.5 g / L H3BO3, 0.03 g / L NaMoO4·2H2O, and the pH value was 7.0.

[0052] (2) Production culture: The above-mentioned bacterial liquid is inoculated at an inoculation rate of 2% to 5% of the total volume of the production culture medium of the sea red bacteria strain for production culture. The temperature is 28°C to 32°C, the light conditions are 2500 Lx to 3500 Lx, the production culture medium formula is consistent with the liquid culture medium, the pH value is 7.0 to 7.2, and the biocontrol agent of the sea red bacteria strain is obtained by culturing to the logarithmic growth phase. The bacterial concentration of the biocontrol agent is 1×10 6 cfu / mL~1×10 8 cfu / mL.

[0053] Figure 2 Schematic diagram of the appearance of the biocontrol agent of the sea red bacteria strain of the present invention. The biocontrol agent of the sea red bacteria strain of the present invention has the characteristics of being environmentally friendly, harmless to humans and animals, no phytotoxicity to crops, simple and convenient application, and is not easy to cause pests and diseases to develop drug resistance. The biocontrol agent of the sea red bacteria strain of the present invention and its fermentation liquid can improve the growth and development of rice under salt stress.

[0054] Example 3

[0055] A fermentation broth of the present invention is obtained by fermenting and culturing the Hydatidella strain of Example 2.

[0056] The biocontrol agent of the RH-AZ strain of the sea red bacteria obtained in Example 2 was fermented for 7 to 10 days, the fermentation temperature was 30°C, the light condition was 3000 Lx, and the fermentation medium was consistent with the formula of the S1 sea red bacteria liquid culture medium. After fermentation, centrifugation was performed at 6000 rpm for 15 min, and the supernatant was collected to obtain the fermentation liquid of the sea red bacteria RH-AZ bacteria, which was stored at 4°C for later use.

[0057] Figure 3 Schematic diagram of the appearance of the fermentation liquid of the Rhizopus spp. strain of Example 3 of the present invention. The fermentation liquid of this example has the characteristics of being environmentally friendly, non-toxic to humans and animals, harmless to crops, and simple and convenient to apply.

[0058] Embodiment 4:

[0059] An application of the biocontrol agent of Example 2 and the fermentation liquid of Example 3 in improving the growth and development of rice seedlings under salt stress.

[0060] (1) Rice cultivation: Rice seedlings were cultivated in a hydroponic box under greenhouse conditions at 26°C, 16 h light, 8 h dark. Hoagland hydroponic nutrient solution was added to the hydroponic box.

[0061] The formula of Hoagland hydroponic nutrient solution is: 945 mg / L Ca(NO3)2, 607 mg / L KNO3, 115 mg / L NH4NO3, 136 mg / L NH3H2PO3, 493 mg / L MgSO4, 2.5 mL iron salt solution (iron salt solution contains: 2.78g ferrous sulfate heptahydrate, 3.73 g EDTA-2Na, 500 mL distilled water, pH5.5), 5 mL trace elements (trace elements are: 0.83 mg / L KI, 6.2 mg / L H3BO3, 22.3 mg / L MnSO4, 8.6 mg / L ZnSO4, 0.25 mg / L NaMoO3, 0.025 mg / L CuSO4, 0.025 mg / L CuCl2), pH: 6.0. When the rice seedlings grow to the stage of two leaves and one heart, the seedlings are treated.

[0062] (2) Group A: blank hydroponic nutrient solution.

[0063] Group B: NaCl was added to the hydroponic nutrient solution, and the final concentration of NaCl was 8‰.

[0064] Group C: 25 mL of the Red Bacteria liquid culture medium and NaCl were added to 1 L of the hydroponic nutrient solution, and the final concentration of NaCl was 8‰.

[0065] Group D: 25 mL of the biocontrol agent prepared in Example 2 and NaCl were added to 1 L of hydroponic nutrient solution, and the final concentration of NaCl was 8‰.

[0066] Group E: 25 mL of the fermentation broth prepared in Example 3 and NaCl were added to 1 L of hydroponic nutrient solution, and the final concentration of NaCl was 8‰.

[0067] The hydroponic nutrient solutions of groups A, B, C, D, and E were applied to rice seedlings, and the stem fresh weights of rice seedlings in the five treatment groups A, B, C, D, and E were measured on 0d, 3d, 6d, 9d, 12d, and 15d, respectively (see Figure 5 ), stem weight (see Figure 6 ), stem length (see Figure 7 ) and other indicators. Each indicator was measured three times, and the results were as follows: Figure 5 ~ Figure 7 shown.

[0068] Figure 4 It is a schematic diagram of the appearance of the effect of biocontrol agents and fermentation liquid treatment of the Rhizoctonia solani strain on the growth of rice seedlings under salt stress according to a preferred embodiment of the present invention.

[0069] Figure 5 The effect of biocontrol agents and fermentation broth of Rhizoctonia solani strain on the fresh stem weight of rice seedlings under salt stress.

[0070] Figure 6 The effect of biocontrol agents and fermentation broth of Rhizoctonia solani strain on the stem weight of rice seedlings under salt stress.

[0071] Figure 7 The effect of biocontrol agents and fermentation broth of Rhizoctonia solani strain on the stem length of rice seedlings under salt stress.

[0072] from Figures 4 to 7 The results show that when the sea red bacteria biocontrol agent and fermentation liquid in this embodiment are applied to rice seedlings, they can significantly increase the stem length, stem fresh weight and stem dry weight of rice seedlings under salt stress.

[0073] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as above in the preferred embodiment, it is not used to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A strain of Rhizopus spp., characterized in that: The sea red bacteria strain is sea red bacteria RH-AZ ( Rhodobium marinum RH-AZ), the deposit number is CCTCC NO: M 20221252, and the deposit date is August 8, 2022.

2. A biocontrol agent, characterized in that: The biocontrol agent is obtained by seed culture and production culture of the Rhizoctonia solani RH-AZ described in claim 1.

3. A method for preparing the biocontrol agent according to claim 2, characterized in that: The preparation method comprises the following steps: S1. Inoculate a single colony of R. salsa RH-AZ into a serum bottle, and perform seed culture with R. salsa liquid culture medium to obtain a bacterial solution in the logarithmic growth phase; the components of the R. salsa liquid culture medium include: 1.5 g / L yeast extract powder, 1 g / L NaAc, 1.5 g / L (NH4)2SO4, 0.5 g / L MgSO4·7H2O, 1 g / L KH2PO4, 0.1 g / L CaCl2, 3g / L NaCl, 2g / LK2HPO4, 0.5g / L H3BO3, 0.03g / L NaMoO4·2H2O, and a pH value of 7.0; S2. Inoculate the bacterial liquid into a production culture medium, and carry out production culture to the logarithmic growth phase to obtain a biocontrol agent of the sea red bacteria strain, wherein the components of the production culture medium include: 1.5 g / L yeast extract powder, 1 g / L NaAc, 1.5 g / L (NH4)2SO4, 0.5 g / L MgSO4·7H2O, 1 g / L KH2PO4, 0.1 g / L CaCl2, 3g / L NaCl, 2g / L K2HPO4, 0.5g / LH3BO3, 0.03g / L NaMoO4·2H2O, and a pH value of 7.

0.

4. The preparation method according to claim 3, characterized in that The temperature of the seed culture in S1 is 28°C to 32°C, and the light condition is 2500 Lx to 3500 Lx; And / or, in said S2, the bacterial solution is inoculated according to 2% to 5% of the total volume of the production culture medium of the Rhizopus marinum strain; And / or, the temperature of the production culture in S2 is 28°C to 32°C, and the lighting conditions are 2500 Lx to 3500 Lx.

5. Use of the biocontrol agent according to claim 2 in improving the growth and development of rice seedlings under salt stress.

6. A fermentation broth, characterized in that: The fermentation liquid is obtained by fermenting and culturing the biocontrol agent according to claim 2 and centrifuging the supernatant.

7. A method for preparing the fermentation liquid according to claim 6, characterized in that: The preparation method comprises the following steps: Fermenting the biocontrol agent according to claim 2 at a temperature of 30° C. and a light condition of 3000 Lx for 7 to 10 days; The fermented solution was centrifuged at 6000 rpm for 15 min, and the supernatant was collected to obtain the fermentation broth.

8. Use of the fermentation liquid according to claim 6 in improving the growth and development of rice seedlings under salt stress.

Images