Heat-resistant sarcina sporogenes

Abstract

The invention discloses a strain of thermotolerant sporosarcina thermotolanan GS2, and the preservation number of the thermotolerant sporosarcina thermotolanan GS2 is CGMCC (China General Microbiological Culture Collection Center) NO.23984. The invention further discloses a method for preparing the thermotolerant sporosarcina thermotolanan. The thermotolerant sporosarcina GS2 biological agent disclosed by the invention has important significance on green prevention and control of plant diseases of apples, grapes, strawberries, rice, tobaccos and the like. The thermostable sporosarcina GS2 biological agent disclosed by the invention is simple in culture condition, high in breeding speed and easy to store and transport. The strain has strong stress resistance and is easy to colonize on soil and plants. The thermotolerant sporosarcina GS2 is obtained by being separated and purified from rhizosphere soil of an orchard, and is safe, non-toxic and environment-friendly. After the fertilizer is applied, the soil microenvironment can be improved, the chemical pesticide dosage is reduced, the ecological environment safety is protected, the fruit quality is improved, and good social, economic and ecological benefits are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a thermotolerant sporosarcina. Background Art

[0002] Plant fungal diseases have a great impact on plants, hindering their growth and development, leading to reduced yields, poor quality, and even death. The biological control method of plant fungal diseases is to use the growth-promoting and antibacterial properties of beneficial microorganisms to prevent and control diseases while promoting plant growth. In recent years, biological control has gradually become a hot topic in disease control due to its green and sustainable characteristics. Therefore, screening out biological resources that have antagonistic effects on diseases is a task of great significance.

[0003] At present, biocontrol bacteria have problems in plant disease control, such as poor colonization ability under natural conditions, weak drug resistance and unstable control effect. Therefore, further screening of microbial strains with excellent performance and good stability for plant disease control is the current research focus in this field.

[0004] Sporosarcina thermophila is a bacterium that can grow in high temperature environments and belongs to the genus Sporosarcina. This bacterium has the ability to form spores, which allows them to survive extreme conditions, such as high temperature, drought or nutrient-deficient environments. Spores are a highly resistant dormant state that protects bacteria from adverse environmental conditions. Bacteria of the genus Sporosarcina are usually classified as Gram-positive bacteria. When observed under a microscope, their cells are spherical or oval in shape and are about 1-2μm × 2-3μm in size. These bacteria are usually arranged in pairs or quadruplets, and sometimes in square piles. They are chemoheterotrophic bacteria and strictly aerobic, which means that they need oxygen for metabolic activities. On nutrient agar, cells of the genus Sporosarcina can form creamy to orange colonies. The excellent stress resistance of Sporosarcina thermophila has great development prospects in the development of biocontrol bacteria. Summary of the invention

[0005] The object of the present invention is to provide a thermotolerant sporosarcina strain.

[0006] The present invention provides a strain of Sporosarcina thermotoleran GS2, which is classified and named Sporosarcina thermotoleran. The strain was deposited in the General Microbiology Center of China National Microbiological Culture Collection on November 26, 2021, with the deposit number CGMCC NO.23984. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0007] The application of Sporosarcina thermotoleran GS2 in preventing and controlling plant fungal diseases.

[0008] Preferably, Sporosarcina thermotoleran GS2 is added at a concentration of 1.0×10 7 ~1.5×10 9 cfu / mL fermented bacterial solution spray or root irrigation treatment.

[0009] The application of Sporosarcina thermotoleran GS2 in inhibiting or killing plant disease pathogens.

[0010] Preferably, the plant disease pathogens include apple tree rot fungus (Valsa mali), apple anthracnose fungus (Colletotrichum acutatum), rice blast fungus (Magnaporthe oryzae), wheat fusarium (Fusarium graminearum), potato late blight fungus (Phytophthora infestans), tobacco black shank fungus (Phytophthora parasitica), grape gray mold fungus (Botrytis cinerea), apple ring rot fungus (Botryosphaeria dothidea), and strawberry powdery mildew fungus (Podosphaera aphanis).

[0011] A biological preparation comprises the Sporosarcina thermotoleran GS2 or its metabolite.

[0012] Preferably, the dosage form of the biological agent is an aqueous suspension, a powder, a wettable powder, an aqueous solution, a microemulsion, a granule or a water-dispersible granule.

[0013] Beneficial effects of the present invention: The thermotolerant sporosarcina GS2 of the present invention is of great significance for the green prevention and control of plant diseases such as apples, grapes, strawberries, rice and tobacco. The thermotolerant sporosarcina GS2 biological preparation of the present invention has simple culture conditions, fast reproduction speed, and is easy to store and transport. It has strong stress resistance and is easy to colonize in soil and plants. The thermotolerant sporosarcina GS2 of the present invention is isolated and purified from the rhizosphere soil of the orchard, and is safe, non-toxic and environmentally friendly. After application, it can improve the soil microenvironment, reduce the amount of chemical pesticides used, protect the ecological environment, improve the quality of fruits, and has good social, economic and ecological benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Colony morphology of Thermosporosarcina GS2.

[0015] Figure 2 Plate inhibition effect of strain GS2. DETAILED DESCRIPTION

[0016] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0017] Example 1 Isolation and identification of strains

[0018] Rhizosphere soil from healthy apple trees was collected and separated and purified from the water dilution of soil samples by dilution plating method. Weigh 1g of rhizosphere soil, add it to 10ml of sterile water, culture at 180rpm for 30min, then dilute it with water, the dilution gradient is 10×, 100× and 1000×, take 100μl of each gradient dilution and spread it on LB medium, culture at 28℃ for 1d, pick a single colony and purify it by streaking on LB medium plate. The purified single colony was transferred to new LB medium for storage.

[0019] The plate confrontation antagonism test was used to screen highly active antagonistic strains, and it was found that GS2 showed high antagonistic activity against fungi. Based on the results of colony and bacterial morphology observation, physiological and biochemical indicators, 16S and gyrA gene sequence determination, GS2 was identified as Sporosarcina thermophila ( Figure 1 ).

[0020] Example 2 Determination of the antibacterial activity of Thermosporosarcina GS2 against plant pathogenic fungi

[0021] The plate confrontation method was used to determine the inhibitory activity of GS2 strain against plant pathogenic fungi. The target plant pathogenic strains included apple tree rot pathogen (Valsa mali), apple anthracnose pathogen (Colletotrichum gloeosporioides), rice blast pathogen (Magnaporthe oryzae), wheat fusarium (Fusarium graminearum), potato late blight pathogen (Phytophthora infestans), tobacco black shank pathogen (Phytophthora parasitica), grape gray mold pathogen (Botrytis cinerea), and apple ring rot pathogen (Botryosphaeria dothidea). The specific operation is as follows: the target plant pathogens are cultured at 28°C for 5 days, the hyphae at the edge of the colony are scraped, and the hyphae are rolled into a 2mm hyphae ball, which is inoculated in the center of the PDA medium plate, and then the cultured bacterial culture solution is adjusted to an OD600 of about 1, and 1 μl is dripped around the hyphae ball about 2.5 cm away, and the plate inoculated with only the target plant pathogens is used as the control, and cultured at 28°C for 4 days, and the inhibitory effect of bacteria on the growth of the target strain is observed, and the diameters of the control and treated anthracnose fungi are measured, and the inhibitory rate of the GS2 strain on the plant pathogenic fungi is calculated. Inhibition rate (%) = (control group colony diameter - treatment group colony diameter) / control group colony diameter × 100%.

[0022] The results of the antibacterial spectrum test showed that the GS2 strain had good antagonistic activity against the tested pathogenic fungi, with an inhibition rate of 51.5-71.4% (Table 1, Figure 2 GS2 strain had a strong inhibitory effect on the mycelial growth of the above 8 pathogens. The mycelial growth at the edge of the colony of the 8 pathogens cultured against GS2 strain was sparse or stopped.

[0023] Table 1 Inhibitory effect of Thermosporosarcina GS2 on various pathogenic fungi

[0024]

[0025]

[0026] Example 3 In vitro activity of Thermosporosarcina GS2 against apple anthracnose

[0027] Apple anthracnose was inoculated on a PDA plate and cultured at 28°C for 9 days. Mycelium and spores on the surface of the culture medium were scraped and shaken with sterile water for 1 min. The spore solution was filtered with lens paper and the concentration was adjusted to 10 5 Thermotolerant sporogenous Sarcina GS2 was inoculated into LB liquid medium and cultured in a shaking incubator at 200 rpm and 30°C for 6 days until the fermentation liquid concentration reached 109 cfu / mL. Cut the unfolded leaves of the Golden Crown apple tree, dilute the fermentation liquid 500 times and spray it, use clean water as the control, and treat 10 leaves for each treatment. After 8 hours, spray and inoculate the suspension of apple anthracnose spores, keep the moisture incubated for 5 days and observe the disease condition of the leaves. The grading method and calculation formula are as follows:

[0028] The grading method is based on the percentage of the lesion area on each leaf to the entire leaf area;

[0029] Level 0: no disease;

[0030] Level 1: The lesion area accounts for less than 5% of the entire leaf area;

[0031] Level 3: The lesion area accounts for 6% to 15% of the entire leaf area;

[0032] Level 5: The lesion area accounts for 16% to 25% of the entire leaf area;

[0033] Level 7: The lesion area accounts for 26% to 50% of the entire leaf area;

[0034] Level 9: The lesion area accounts for more than 51% of the entire leaf area.

[0035] Disease index = 100 × ∑ (number of diseased leaves at each level × relative level value) / (total number of leaves surveyed × 9).

[0036] In the in vitro inoculation test, the control had severe disease, with large and continuous lesions, while the leaves treated with 500-fold dilution of the fermentation liquid of strain GS2 had mild disease, with smaller lesions. The 500-fold dilution of the fermentation liquid of strain GS2 had a protective effect of 68.5% against apple anthracnose, which had a significant protective effect.

[0037] Example 4 Control effect of thermotolerant sporogenes GS2 on strawberry powdery mildew

[0038] Experimental method: Spray inoculation with spore suspension was used. Neat and consistent potted strawberry seedlings were selected, and the GS2 bacterial solution that had been fermented for 6 days was diluted 250, 500, and 1000 times for spray treatment. Each treatment was repeated 4 times, with 4 pots in each repeat. The treatment of spraying with clean water was used as a control. After 24 hours, take the strawberry leaves covered with powdery mildew, gently wash the fresh spores on the back of the leaves with sterile water, filter with double-layer lens paper, make a spore suspension, and spray inoculate. The inoculated strawberry plants were moved to a 23°C greenhouse, and the disease was graded and investigated until the control without spraying GS2 bacterial solution was fully diseased. The disease index was used to calculate the prevention effect. The grading method and calculation formula are as follows:

[0039] The classification method is as in Example 3;

[0040] Disease index = 100 × ∑ (number of diseased leaves at each level × relative level value) / (total number of leaves investigated × 9);

[0041] Control effect (%) = 100 × (control disease index - treatment disease index) / control disease index.

[0042] In the strawberry powdery mildew pot test, different dilution multiples of the fermentation liquid of Thermosporosarcina GS2 can inhibit the occurrence of strawberry powdery mildew, among which the prevention effect of 250 times liquid was as high as 88.92%, 500 times liquid was slightly lower than 250 times liquid, and 1000 times liquid was slightly worse. It is recommended to use the fermentation liquid of Thermosporosarcina GS2 at a multiple of 250-500 times.

[0043] Table 2 The control effect of thermotolerant sporogenes GS2 on powdery mildew of potted strawberry

[0044]

[0045]

[0046] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A strain of Sporosarcina thermotoleran GS2, characterized in that: Its deposit number is CGMCC NO.23984.

2. Use of the Sporosarcina thermotoleran GS2 according to claim 1 in preventing and controlling plant fungal diseases.

3. The use of Sporosarcina thermotoleran GS2 according to claim 2 in preventing and controlling plant fungal diseases, characterized in that: Sporosarcina thermophila was added to the culture medium at a concentration of 1.0×10 7 ~1.5×10 9 cfu / mL fermented bacterial solution spray or root irrigation treatment.

4. Use of the Sporosarcina thermotoleran GS2 according to claim 1 in inhibiting or killing plant disease pathogens.

5. The use of Sporosarcina thermotoleran GS2 according to claim 1 in inhibiting or killing plant disease pathogens, characterized in that: The plant disease pathogens include apple tree rot fungus (Valsa mali), apple anthracnose fungus (Colletotrichum acutatum), rice blast fungus (Magnaportheoryzae), wheat fusarium (Fusarium graminearum), potato late blight fungus (Phytophthora infestans), tobacco black shank fungus (Phytophthora parasitica), grape gray mold fungus (Botrytiscinerea), apple ring rot fungus (Botryosphaeria dothidea), and strawberry powdery mildew fungus (Podosphaeraaphanis).

6. A biological agent, characterized in that: It comprises the Sporosarcinathermotoleran GS2 or its metabolites as described in claim 1.

7. The biological agent according to claim 6, characterized in that: The dosage form of the biological preparation is water suspension, powder, wettable powder, aqueous solution, microemulsion, granule or water dispersible granule.

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