Phenazine-1-carboxylic acid and streptomyces fradiae compound and application thereof in prevention and treatment of soil-borne diseases

Abstract

The invention discloses a phenazino-1-carboxylic acid and streptomyces fradiae compound agent and application thereof to prevention and treatment of soil-borne diseases. The compound agent is formed by compounding phenazino-1-carboxylic acid and streptomyces fradiae. The phenazino-1-carboxylic acid and the streptomyces fradiae are compounded, so that the antibacterial effect is enhanced, the acting time of the pesticide effect can be prolonged, and a better disease control effect is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological pesticides, and specifically relates to a compound preparation of a biological pesticide and an agricultural microbial agent and an application thereof. Background Art

[0002] With the advancement and implementation of green plant protection strategies, people's concern for their own health and the strengthening of environmental protection concepts, the research and development of pollution-free biological pesticides has become a hot spot in my country's biological industry, agricultural scientific research and application, and has been listed in the country's medium- and long-term scientific and technological development plan. As a naturally occurring substance or product, biological pesticides are safe and harmless to the environment other than the target, will not produce residues, and will not cause enrichment.

[0003] Shenqinmycin is a broad-spectrum, low-toxic, highly effective and environmentally compatible fungicide secreted by Pseudomonas aeruginosa M18 through biological culture. It is included in the list of pesticides allowed for use in both AA-level and A-level green foods in my country. It can be used to prevent and treat a variety of soil-borne diseases such as root rot, stem base rot, wilt, verticillium wilt, vine blight, grape white rot, and peanut white rot. Shenqinmycin mainly utilizes the redox ability of phenazine-1-carboxylic acid to accumulate active oxygen in fungal cells, inhibit the oxidative phosphorylation of the respiratory transport chain in mitochondria, thereby inhibiting the normal growth of hyphae and causing the mycelium of pathogenic fungi to break, swell, deform and lyse.

[0004] Actinomycetales are a type of filamentous prokaryotes that are between bacteria and filamentous fungi but close to bacteria. They are named because their aerial hyphae are radial. They mainly include Strepto-mycetes, Micromonospora, Nocardia, Actinomadura, and Actinoplanes, among which Streptomyces is the main one. Actinomycetes can produce rich and diverse secondary metabolites such as avermectin, streptomycin, ningnanmycin, polymycin and other antibiotic substances, which have good prevention and control effects on a variety of pathogenic bacteria and fungi, and have broad application prospects in agricultural production. As a type of Streptomyces, Streptomyces freundii can produce tylosin, neomycin, etc., and has good growth-promoting and disease-resistant effects.

[0005] As a broad-spectrum fungicide, shenqinmycin has a control effect on a variety of soil-borne diseases, but shenqinmycin is expensive, degrades quickly in the soil, and has a lower control effect on a single disease than chemical agents. Whether the biocontrol strain can colonize in the root system is a key factor in whether it can exert its biocontrol effect. Studies have shown that when Streptomyces S-101 is applied to the soil at 1×108cfu / g for 28 to 35 days, the number of viable bacteria tends to stabilize at 1×106cfu / g, and the number decreases fastest in the initial 0 to 14 days after application to the soil, and the decrease in the number of viable bacteria will lead to a decrease in the control effect of the bacterial agent. At the same time, it takes a certain amount of time for Streptomyces freundii to colonize the rhizosphere and exert its biocontrol effect. Therefore, shenqinmycin and Streptomyces freundii are compounded in a reasonable way and combined with appropriate methods and periods for the control of crop diseases. While reducing costs and increasing efficiency, it can improve the colonization effect of Streptomyces freundii in the soil and extend the protection period of crops. Therefore, it is of great practical significance to provide a compound pesticide of shenqinmycin and Streptomyces freundii. Summary of the invention

[0006] The object of the present invention is to provide a compound of sinocycline and Streptomyces freundii, which can enhance the antibacterial effect and prolong the duration of drug efficacy, thereby achieving a better disease prevention and control effect, and its use in preventing and controlling soil-borne diseases.

[0007] The technical solution of the present invention is:

[0008] A compound of shinazomicin and Streptomyces freundii is characterized in that it is composed of shinazomicin and Streptomyces freundii.

[0009] The Streptomyces freundii is a Streptomyces freundii agent, which is prepared by the following method:

[0010] The Streptomyces freundii was prepared into a fermentation liquid, the Streptomyces freundii was inoculated on Gao's No. Ⅰ medium, cultured at 26-30°C for 3-5 days, then transferred to a seed medium, cultured at 26-30°C and 180-200 r / min for 24 hours, the seed liquid was transferred to a fermentation medium, cultured at 26-30°C and 180-200 r / min for 72-96 hours, and the addition amount was 4%;

[0011] Then, diatomaceous earth and sodium alginate are added to the fermentation liquid to prepare a Streptomyces freundii inoculum;

[0012] The mass ratio of Streptomyces freundii, diatomaceous earth and sodium alginate in the Streptomyces freundii inoculum is 20-5:10-2:5-1;

[0013] The compounded Streptomyces freundii inoculum is dried for later use.

[0014] The drying temperature of Streptomyces freundii inoculum is 30-60℃.

[0015] The formula of Gao's medium No. 1 is: soluble starch 20g, potassium nitrate 1g, sodium chloride 0.5g, dipotassium hydrogen phosphate 0.5g, ferrous sulfate 0.01g, agar 13g, water 1L, pH 7.2-7.4. The formula of seed and fermentation medium is: soluble starch 20-25g, corn flour 17-20g, ammonium sulfate 5-10g, calcium carbonate 0.5-1g, disodium hydrogen phosphate 0.5-1.5g, ferrous sulfate heptahydrate 0.05-0.1g, water 1L, pH 7.2-7.6.

[0016] The mass ratio of sinocycline to Streptomyces flexneri is 1:100.

[0017] An application of the compound of shinazomib and Streptomyces freundii in the preparation of a preparation for preventing and controlling soil-borne diseases.

[0018] The biological preparation obtained by compounding shinazomib and Streptomyces freundii was diluted with sterile water into preparations of different concentrations, mixed into PDA culture medium and made into plates. The pathogens were inoculated in the center of the plates. At the same time, single-dose treatments of shinazomib and Streptomyces freundii were set up. Sterile water was used as the control. The plates were cultured in an incubator at 28°C for an appropriate number of days for statistical analysis.

[0019] The pathogenic bacteria include: Rhizoctonia solani, Fusarium oxysporum, Alternaria alternata, Colletotrichum grisea, Pyricularia cinerea, Fusarium graminearum and Botrytis cinerea.

[0020] The said shinazomib improves the colonization effect of Streptomyces flexneri in the soil by sterilizing the soil sample at high temperature and high pressure (121°C, 20 minutes), then mixing the compound biological preparation of shinazomib and Streptomyces flexneri into the sterilized soil, stirring evenly and then filling into sterilized nutrient pots, sampling at 0, 5, 15 and 30 days respectively, and determining the colony count of Streptomyces flexneri in the soil by the dilution coating method.

[0021] The method of using the biological agent for preventing and controlling various soil-borne diseases includes soil treatment, root irrigation, leaf spraying, root dipping, etc.

[0022] Shenqinmycin is secreted by Pseudomonas aeruginosa M18 through biological culture, and is a broad-spectrum fungicide belonging to microbial antibiotics, which has a good inhibitory effect on the growth of pathogenic fungi of various soil-borne diseases. Streptomyces freundii can produce effective active ingredients such as tylosin and neomycin, and the antibacterial active substances produced by it have a good antibacterial effect on both bacteria and pathogenic fungi, and have a broad-spectrum antibacterial spectrum and strong antibacterial activity. The present invention can prolong the drug effect time while enhancing the antibacterial effect by compounding Shenqinmycin and Streptomyces freundii, thereby achieving a better disease prevention and control effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0024] Figure 1 The results of plate counting at 0d for Streptomyces freundii inoculum (a) and 1:100 compound inoculum (b);

[0025] Figure 2 The results of plate counting for Streptomyces freundii (a) and 1:100 compound bacterial agent (b) at 15 days.

[0026] Figure 3 This is the antibacterial effect diagram of the mixture of 1:20 of shenqinmycin and Streptomyces freundii on Botrytis cinerea in a dish;

[0027] Figure 4 This is the antibacterial effect diagram of a mixture of 1:80 of sinocycline and Streptomyces freundii on Rhizoctonia solani in a dish.

[0028] Figure 3 , Figure 4 Middle: a: blank control, b: sinocycline, c: Streptomyces freundii, d: sinocycline and Streptomyces freundii mixed. DETAILED DESCRIPTION

[0029] Example 1: Preparation of Streptomyces freundii inoculum

[0030] The preparation of the Streptomyces freundii inoculum comprises the following steps:

[0031] (1) Preparation of seeds and fermentation medium: 25 g soluble starch, 17 g corn flour, 8 g ammonium sulfate, 1 g calcium carbonate, 1.5 g disodium hydrogen phosphate, 0.1 g ferrous sulfate heptahydrate, 1 L water, adjust pH to 7.4, divide into Erlenmeyer flasks 50 / 250 mL, prepare fermentation medium, and sterilize at 121°C under high temperature and high pressure for 20 min.

[0032] (2) Preparation of seed solution: Streptomyces freundii activated on Gao's solid medium No. 1 was inoculated into the seed culture medium and cultured at 28°C and 200 r / min for 24 h to prepare the seed solution.

[0033] (3) Preparation of fermentation broth: The seed liquid in step (2) was inoculated into the fermentation medium at an inoculum rate of 4%, and cultured at 28° C. and 200 r / min for 96 h to prepare a fermentation broth.

[0034] (4) Add diatomaceous earth and sodium alginate to the fermentation liquid collected in step (3), dry at 45°C, and place at room temperature for later use, with the ratio of fermentation liquid: diatomaceous earth: sodium alginate being 15:10:4. The viable bacterial count was measured by dilution coating to be 1×109 cfu / g.

[0035] Example 2: Preparation of a 1:100 compound bacterial agent of shinazomicin and Streptomyces freundii

[0036] The Streptomyces freundii agent obtained in step (4) of Example 1 and the original drug of sinocycline were mixed in a mass ratio of 1:100.

[0037] Example 3: Preparation of a 1:20 composite bacterial agent of shinazomicin and Streptomyces freundii

[0038] The Streptomyces freundii agent obtained in step (4) of Example 1 and the original drug of sinocycline were mixed in a mass ratio of 1:20.

[0039] Example 4: In vitro antibacterial effect of a 1:20 mixture of shinazomicin and Streptomyces freundii on Botrytis cinerea

[0040] (1) Activate Botrytis cinerea on PDA medium until it fills the plate

[0041] (2) Dissolve shinazomib with DMF and dilute with 0.1% Tween 80 water to different concentration gradients. Dissolve the Streptomyces flexneri agent in water in step (4) of Example 1 and dilute to different concentration gradients. Inoculate 5uL of Streptomyces flexneri agent at 3cm from the center of the PDA plate; After dissolving shinazomib with different concentrations, mix them into the PDA culture medium, and then pour them into 6 culture dishes with a diameter of 9cm in equal amounts to prepare drug-containing plates of corresponding concentrations, of which 3 are treated with shinazomib alone and the other 3 are treated with mixed use. Inoculate 5uL of Streptomyces flexneri agent at 3cm from the center of the plate. A blank control without drug treatment is set up for the experiment, and each treatment is repeated 3 times.

[0042] (3) Cut the activated Botrytis cinerea on the PDA plate from the edge of the colony using a sterile borer with a diameter of 5 mm under sterile conditions, inoculate the cake with an inoculation needle into the center of the drug-containing plate with the mycelium facing upward, cover with a cover dish, and place in an incubator at a suitable temperature for culture.

[0043] (4) Investigate the growth of pathogenic fungi based on the growth of bacteria in the blank control culture dish. Use a caliper to measure the diameter of the colony in millimeters (mm). Use the cross method to measure the diameter of each colony vertically once and take the average value.

[0044] (5) The results are shown in Table 1

[0045] Table 1 The antibacterial effect of the mixed bacterial agent of 1:20 of shinazomicin and Streptomyces freundii on Botrytis cinerea in the dish

[0046]

[0047] Example 5: In vitro antibacterial effect of a mixture of 1:80 of shinazomicin and Streptomyces freundii on Rhizoctonia solani

[0048] (1) activating Rhizoctonia solani on PDA medium until it fills the plate;

[0049] (2) Dissolve shinazomib with DMF and dilute with 0.1% Tween 80 water to different concentration gradients. Dissolve the Streptomyces flexneri agent in water in step (4) of Example 1 and dilute to different concentration gradients. Inoculate 5uL of Streptomyces flexneri agent at 3cm from the center of the PDA plate; After dissolving shinazomib with different concentrations, mix them into the PDA culture medium, and then pour them into 6 culture dishes with a diameter of 9cm in equal amounts to prepare drug-containing plates of corresponding concentrations, of which 3 are treated with shinazomib alone and the other 3 are treated with mixed use. Inoculate 5uL of Streptomyces flexneri agent at 3cm from the center of the plate. A blank control without drug treatment is set up for the experiment, and each treatment is repeated 3 times.

[0050] (3) Using a sterile borer with a diameter of 5 mm, cut the activated Rhizoctonia solani on the PDA plate into a cake from the edge of the colony under sterile conditions. Use an inoculation needle to inoculate the cake into the center of the drug-containing plate with the mycelium side facing up. Cover the plate with a cover dish and place it in an incubator at a suitable temperature for culture.

[0051] (4) Investigate the growth of pathogenic fungi based on the growth of bacteria in the blank control culture dish. Use a caliper to measure the diameter of the colony in millimeters (mm). Use the cross method to measure the diameter of each colony vertically once and take the average value.

[0052] (5) The results are shown in Table 2

[0053] Table 2 The antibacterial effect of the mixed bacterial agent of 1:80 of shinazomicin and Streptomyces freundii on Rhizoctonia solani in the dish

[0054]

[0055] Example 6: Colonization effect of Streptomyces freundii in soil in a 1:100 mixed bacterial agent of shinazomicin and Streptomyces freundii

[0056] (1) Soil sterilization: The collected soil was placed in a sterilization bag and sterilized at 121°C for 20 min. The soil was cooled and then used for later use.

[0057] (2) Weigh 10 g of the Streptomyces freundii agent prepared in step (4) of Example 1 and 10.1 g of a 1:100 composite agent of sinocycline and Streptomyces freundii, respectively, and mix them evenly into the sterilized soil until the total weight is 1 kg. Transfer the mixture into a sterilized flowerpot, seal it with plastic wrap, and repeat the experiment three times.

[0058] (3) Samples were collected at 0, 5, 15, and 30 days, and the number of viable Streptomyces freundii in each treatment was determined by the dilution coating method.

[0059] (4) The results are shown in Table 3

[0060] Table 3 Plate count results

[0061] deal with 0d(cfu / g) 5d(cfu / g) 10d(cfu / g) 15d(cfu / g) 30d(cfu / g) Streptomyces freundii <![CDATA[0.99×10 7 ]]> <![CDATA[0.62×10 6 ]]> <![CDATA[0.56×10 5 ]]> <![CDATA[0.42×10 5 ]]> <![CDATA[0.40×10 5 ]]> 1:100 compound bacterial agent <![CDATA[1.0×10 7 ]]> <![CDATA[1.31×10 6 ]]> <![CDATA[1.77×10 5 ]]> <![CDATA[0.88×10 5 ]]> <![CDATA[0.85×10 5 ]]>

[0062] Example 7: Control effect of a 1:20 mixed bacterial agent of shinazomicin and Streptomyces freundii on cucumber gray mold

[0063] Potted experiment for the prevention and control of cucumber gray mold: cucumber seedlings were transplanted with two leaves and one heart after two leaves were raised. One week later, the bacterial agent was sprayed on the leaves according to the treatment. 0.05g of shenqinmycin, 1g of Streptomyces freundii, and 1.05g of the compound bacterial agent were accurately weighed and dissolved in 1L of water, and sprayed on the leaves at 20mL / plant. The control group was sprayed with an equal amount of water, 20mL of the 1:20 compound bacterial agent, and 24 hours later, the cucumber gray mold pathogen was inoculated on the leaves, and the inoculated leaves were covered with a fresh-keeping bag to keep them moist. After 4 days, the diameter of the lesions in each treatment group was counted, and the prevention effect was calculated.

[0064]

[0065] Table 4 The control effect of the mixed bacterial agent of 1:20 of shenjinmycin and Streptomyces freundii on cucumber gray mold

[0066] deal with <![CDATA[Lesion area / cm 2 > Prevention effect / % Clear water control 5.65a / Shinqin 1.85c 67.3 Streptomyces freundii 3.24b 42.7 Compound Bacterial Agent 0.96d 83.0

[0067] As can be seen from Examples 4 and 5, the experimental results of the inhibitory effects of single agents of shinazomicin and Streptomyces freundii and the mixture of the two on Botrytis cinerea and Rhizoctonia solani show that their co-toxicity coefficients are 102 and 108, respectively, showing an additive effect. As can be seen from Example 6, under the condition of the same number of viable bacteria, after the Streptomyces freundii was mixed with soil for treatment, the survival rate of the Streptomyces freundii treated with shinazomicin was improved. As can be seen from Example 7, the control effect of foliar spraying of the composite bacterial agent on cucumber gray mold was increased from 67.3% of a single agent of shinazomicin and 42.7% of the Streptomyces freundii agent to 83%, which can better prevent and control cucumber gray mold.

Claims

1. A compound of shinazomicin and Streptomyces freundii, characterized in that: It is composed of a combination of sinocycline and Streptomyces freundii.

2. The compound of shinazomib and Streptomyces freundii according to claim 1, characterized in that: The Streptomyces freundii is a Streptomyces freundii agent, which is prepared by the following method: Prepare Streptomyces freundii into fermentation liquid, inoculate Streptomyces freundii on Gao's medium No. Ⅰ, culture at 26-30℃ for 3-5 days, then transfer to seed medium, culture at 26-30℃ and 180-200r / min for 24 hours, transfer seed liquid to fermentation medium, culture at 26-30℃ and 180-200r / min for 72-96 hours, and add 4%; Then, diatomaceous earth and sodium alginate are added to the fermentation liquid to prepare a Streptomyces freundii inoculum; The mass ratio of Streptomyces freundii, diatomaceous earth and sodium alginate in the Streptomyces freundii inoculum is 20~5:10~2:5~1; The compounded Streptomyces freundii inoculum is dried for later use.

3. The compound of shinazomib and Streptomyces freundii according to claim 2, characterized in that: The drying temperature of Streptomyces freundii inoculant is 30~60℃.

4. The compound of shinazomib and Streptomyces freundii according to claim 2, characterized in that: The formula of Gao's medium No. 1 is: soluble starch 20g, potassium nitrate 1g, sodium chloride 0.5g, dipotassium hydrogen phosphate 0.5g, ferrous sulfate 0.01g, agar 13g, water 1L, pH 7.2~7.

4. The formula of seed and fermentation medium is: soluble starch 20~25g, corn flour 17~20g, ammonium sulfate 5~10g, calcium carbonate 0.5~1g, disodium hydrogen phosphate 0.5~1.5g, ferrous sulfate heptahydrate 0.05~0.1g, water 1L, pH 7.2~7.

6.

5. The compound of shinazomib and Streptomyces freundii according to claim 1, 2, 3 or 4, characterized in that: The mass ratio of sinocycline to Streptomyces flexneri is 1:

100.

6. Use of the combination of shinazomib and Streptomyces freundii as claimed in claim 1 in the preparation of a preparation for preventing and controlling soil-borne diseases.

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