Streptomyces luteus and application thereof in prevention and treatment of tomato leaf mold

Abstract

The invention discloses streptomyces luteus and application of the streptomyces luteus in prevention and treatment of tomato leaf mold. According to the invention, streptomyces luteoverti RZ0102 with a relatively strong inhibition effect on tomato leaf mold is screened from river mud at the junction of the River and the sea, and the preservation number of the streptomyces luteoverti RZ0102 is CGMCC (China General Microbiological Culture Collection Center) NO.15219. The streptomyces luteoverti RZ0102 has the advantages that the streptomyces luteoverti RZ0102 has a relatively strong inhibition effect on the tomato leaf mold; experiments show that the strain has genetic stability in function, and fermentation supernate of the strain has excellent thermal stability, acid-base stability and ultraviolet irradiation stability; the strain RZ0102 and metabolites thereof can efficiently inhibit the growth of the tomato leaf mold bacteria.

Description

Technical Field

[0001] The invention relates to a Streptomyces luteus strain and application thereof in preventing and treating tomato leaf mold, belonging to the technical field of microorganisms. Background Art

[0002] Streptomyces luteoverticillatus is an actinomycete with high development value in the genus Streptomyces. At present, there are few studies on Streptomyces luteoverticillatus. CN108192829A reported that Streptomyces luteoverticillatus strain HY61 can be used to prevent and treat plant pathogens such as Colletotrichum gloeosporioides, Botrytis cinerea, Fusarium oxysporum, Gibberella tritici, and Curvularia zeae. CN112899181A reported that Streptomyces luteoverticillatus CCTCCNo: M2020262 has an inhibitory effect on peanut white rot, peanut root rot and peanut crown rot. However, there is no report on the use of Streptomyces luteoverticillatus for the prevention and treatment of tomato leaf mold.

[0003] Tomato leaf mold is an extremely serious leaf disease of tomatoes caused by Cladosporium fulvum. Once leaf mold occurs, it will spread rapidly and be highly epidemic. In mild cases, it will reduce production and yield, and in severe cases, it will cause total crop failure. At present, relying on chemical agents to control the disease is the mainstream, but the environmental pollution and agricultural product safety problems caused by chemical drugs are becoming increasingly serious. At present, biological control has received widespread attention for its safety, health and green concepts, but the biological control of tomato leaf mold mainly focuses on Bacillus, and less research has been done on actinomycete resources. Summary of the invention

[0004] In view of the above problems, the present invention provides a strain of Streptomyces luteus and its use in preventing and controlling tomato leaf mold. The strain is genetically stable in function, and its fermentation supernatant has excellent thermal stability, acid-base stability and ultraviolet irradiation stability; it can effectively antagonize the pathogen of tomato leaf mold, the chrysosporium, and can be developed into a microbial agent for tomato leaf mold, thereby achieving green and efficient prevention and control of tomato leaf mold.

[0005] The present invention screened out Streptomyces luteoverticillatus RZ0102, which has a strong inhibitory effect on tomato leaf mold, from the river mud at the junction of Futong River in Rizhao and the sea. The strain was deposited in the General Microbiological Center of China Microbiological Culture Collection Committee (deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​January 15, 2018, and the deposit number is CGMCC NO.15219. Experiments show that the strain has genetic stability in function, and its fermentation supernatant has excellent thermal stability, acid-base stability and ultraviolet irradiation stability; strain RZ0102 and its metabolites can effectively inhibit the growth of tomato leaf mold pathogens, and have broad application prospects in the prevention and control of tomato leaf mold.

[0006] The present invention also provides the fermentation bacterial liquid of the Streptomyces luteoverticillatus RZ0102.

[0007] The method for preparing the fermentation liquid is characterized in that after the bacteria are activated, Gao's No. 1 medium is used for constant temperature shaking culture at 28-32°C for 10-15 hours to prepare the seed liquid; the seed liquid is inoculated with a large amount of fermentation medium at an inoculation rate of 1-10% and fermented for 16-20 hours at 28-32°C to obtain the fermentation liquid.

[0008] The above-mentioned large-scale fermentation medium is, by weight ratio: soluble starch 2%, KNO3 0.1%, K2HPO4 0.05%, glucose 0.5%, urea 0.2%, peptone 0.2%, MgSO4·7H2O 0.05%, and the rest is water, with a pH of 7.2-7.5.

[0009] The present invention also provides the use of Streptomyces luteus RZ0102 or its fermented bacterial liquid in the prevention and treatment of tomato leaf mold.

[0010] The beneficial effects of the present invention are:

[0011] 1. High antibacterial efficiency

[0012] This strain is highly effective in antagonizing the pathogen of tomato leaf mold, F. fulva. The plate inhibition rate is as high as 90.83%; its fermented bacterial liquid can effectively inhibit the occurrence and spread of tomato leaf mold: the in vitro test of tomato leaves showed that its control effect reached 74.94%; the field effect test showed that its control effect reached 86.85%, showing its potential in controlling tomato leaf mold.

[0013] 2. Antibacterial stability

[0014] The strain is functionally genetically stable, and its fermentation supernatant has excellent thermal stability, acid-base stability and ultraviolet irradiation stability.

[0015] The present invention screened out an actinomycete, Streptomyces luteus, which has a good control effect on tomato leaf mold, which is beneficial to further explore actinomycete resources and enrich the resource library of tomato leaf mold biocontrol bacteria strains, and is of great significance for carrying out green comprehensive prevention and control of tomato leaf mold. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The antibacterial activity of the fermentation supernatant of four bacterial strains;

[0017] Figure 2 The effect of different temperatures on the antibacterial stability of the fermentation supernatants of four bacterial strains;

[0018] Figure 3 Effect of different pH values ​​on the antibacterial stability of the fermentation supernatants of four bacterial strains

[0019] Figure 4 The effect of ultraviolet irradiation at different times on the antibacterial stability of the fermentation supernatant of four bacterial strains;

[0020] Figure 5 This is the colony morphology of Streptomyces luteus strain RZ0102;

[0021] Figure 6 The effect of passage number on the antibacterial effect of Streptomyces luteus RZ0102;

[0022] Figure 7 The control effect of Streptomyces luteus RZ0102 on tomato leaf mold. DETAILED DESCRIPTION

[0023] The following will combine the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1: Isolation and purification of Streptomyces luteoverticillatus strain RZ0102

[0025] The Streptomyces luteoverticillatus RZ0102 of the present invention is isolated from river mud at the junction of Futong River and the sea in Rizhao by using the dilution plate method and the plate streak method. The specific isolation method is:

[0026] (1) Preparation of river mud suspension: Weigh 1 g of river mud sample and add it to a conical flask containing 99 mL of sterile physiological saline near a flame. The conical flask contains glass beads; place it in a shaker at 28 to 30°C and shake it evenly at 120 to 140 rpm for 10 to 20 min to evenly disperse the bacteria, spores or spores in the river mud, thereby preparing a river mud suspension;

[0027] (2) Dilution coating: Use the 10-fold dilution method to dilute the river mud suspension into 10 -1 ~10 -6 Dilution of bacterial samples; respectively from 10 -4 , 10 -5 and 10 -6 100 μL of the diluted bacterial sample was spread on a plate containing Gao's medium No. 1, and three parallel plates were spread for each gradient;

[0028] (3) Cultivation: Seal the coated plate and place it in an incubator at 28-30°C for 3-5 days, and observe the growth of bacterial colonies on the plate regularly;

[0029] (4) Single-bacterial purification: Preliminarily select strains with colony morphology similar to that of actinomycetes, streak single-bacterial plates, and perform purification culture at a temperature of 28-30°C for 3-5 days. If the strain is impure, repeat the operation. The obtained strains are numbered.

[0030] Using the above method, a total of 34 bacterial strains were isolated from four samples, and were numbered RZ0101~RZ0111, RZ0201~RZ0207, RZ0301~RZ0307, ​​and RZ0401~RZ0409 respectively.

[0031] Example 2: Screening of the highly effective antagonistic strain RZ0102 against tomato leaf mold

[0032] (1) Initial screening

[0033] The plate confrontation method was used to screen the antagonistic strains with high efficiency against tomato leaf mold pathogen.

[0034] The isolated strains were streaked on Gao's solid medium No. 1 with a sterile inoculation loop for 48 hours to activate the strains; a bacterial cake with a diameter of 5 mm was punched on the edge of the activated Cladosporium fulvum, the pathogen of tomato leaf mold, with a cork hole puncher, and transplanted to the center of the PDA plate; the activated strains to be tested were spotted on both sides 2.0 cm away from the center; the control group was not treated at 2.0 cm away from the center on the other two sides of the plate; this was repeated 3 times; after the treatment was completed, the plates were cultured at a constant temperature of 28°C, and the inhibitory effect of the selected strains on the pathogens was observed daily. After the pathogens in the control group filled the plate, the radius of the lesions in the experimental group was measured, and the inhibition rate was calculated.

[0035] Inhibition rate = [(control colony radius - bacterial cake radius) - (treated colony radius - bacterial cake radius)] / (control colony radius - bacterial cake radius) × 100%.

[0036] The results showed that among the 34 strains tested, RZ0101, RZ0102, RZ0104, RZ0107, RZ0108, RZ0109, RZ0111, RZ0203, RZ0205, RZ0301, RZ0305, RZ0306, RZ0307, ​​RZ0403, RZ0408 and RZ0409 had inhibitory effects on the growth of Flavour Mold (see Table 1); among them, the inhibition rates of RZ0102, RZ0111, RZ0403 and RZ0409 were above 85%, which were significantly higher than those of other strains (see Table 1).

[0037] Table 1 Antibacterial rate of different strains

[0038]

[0039]

[0040] In order to further identify strains with high efficiency in antagonizing Flavour Mold, we tested the antagonistic activity and stability of the fermentation supernatants of RZ0102, RZ0111, RZ0403 and RZ0409.

[0041] (2) Rescreening

[0042] RZ0102, RZ0111, RZ0403 and RZ0409 were rescreened by testing the antagonistic activity and stability of the fermentation supernatant. The specific method is as follows:

[0043] ① Preparation of fermentation broth: Use a sterile inoculating spatula to scoop up a piece of the strain to be tested that has been activated on the Gao's No. 1 solid plate, inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of Gao's No. 1 liquid culture medium, and culture it at 30°C and 180 r / min for 48 h to obtain the fermentation broth.

[0044] ② Preparation of sterile fermentation supernatant: The obtained fermentation broth was placed in a centrifuge tube and centrifuged at 4000 r / min for 15 min. The supernatant was filtered through a 0.22 μm filter to obtain the fermentation supernatant.

[0045] ③ Determination of antibacterial activity by toxic medium method: the fermentation supernatant was fully mixed with PDA medium at a ratio of 1:19 to prepare a toxic plate; the experimental group was inoculated with a 5-mm-diameter Cladosporium fulvum cake in the center of the toxic plate; the control group was added with sterile water instead of the fermentation supernatant at the same ratio, and a 5-mm-diameter Cladosporium fulvum cake was inoculated in the center; after the treatment, the plates were cultured at 30°C for 4 days, the lesion radius of the experimental and control groups was measured, and the inhibition rate was calculated; each treatment was repeated 3 times.

[0046] The results showed that the antibacterial activities of the fermentation supernatants of RZ0102, RZ0111, RZ0403 and RZ0409 were 80%, 79.17%, 80.13% and 79.17%, respectively, with no significant difference (refer to Figure 1 ).

[0047] Then we used the toxic medium method to test the antibacterial stability of the fermentation supernatants of RZ0102, RZ0111, RZ0403 and RZ0409. The strains with better resistance were screened through temperature resistance, acid and alkali resistance, and UV resistance tests.

[0048] Temperature resistance test: 20 mL of the treated supernatant was taken in batches and put into 50 mL centrifuge tubes, and placed in a 4 ℃ refrigerator, a 20 ℃ incubator, a 30, 40, 50, 60, 70, 80, 90, 100, 121 ℃ water bath and a sterilizer for treatment for 60 min. After the supernatant of each treatment returned to room temperature, the toxic medium method was used to determine the inhibitory effect of the supernatant of different strains after heat treatment on Flavosporium. Untreated sterile water was used as the control. Each treatment was repeated 3 times.

[0049] Acid and alkali resistance test: Take 20 mL of the supernatant stored at low temperature and use 1 mol·L -1 Concentrated HCl and 1 mol·L -1 The pH values ​​of the supernatant were adjusted to 3, 4, 5, 6, 7, 8, 9 and 10 with NaOH, respectively. After standing in a 4°C refrigerator for 24 h, the pH of each treatment was adjusted back to pH 7. Sterile water was used as the control, and each treatment was repeated 3 times. The toxic medium method was used to detect the inhibitory effect of the fermentation broth of different strains after pH treatment on Flavosporium.

[0050] UV resistance test: Take a certain amount of fermentation supernatant in a sterilized culture dish and place it under a UV lamp at 70uW / cm 2 The samples were irradiated for 12 h, 24 h, 36 h and 48 h respectively and then taken out. Sterile water was used as the control. Each treatment was repeated 3 times. The toxic medium method was used to detect the inhibitory effect of the fermentation broth of different strains treated with ultraviolet light on Flavosporium.

[0051] The results of the temperature test showed that: within the range of 20-40℃, the antibacterial activity of the fermentation supernatants of the four strains was high and stable, and the antibacterial rate was stable at more than 70%; both too high and too low temperatures could significantly reduce the antibacterial activity of the fermentation supernatants of RZ0111, RZ0403 and RZ0409; too low temperature had no significant effect on the antibacterial activity of the fermentation supernatant of RZ0102, and the temperature exceeding 60℃ had a reduced effect on the antibacterial activity of the fermentation supernatant of RZ0102, but it still had antibacterial activity after treatment at 121℃ (refer to Figure 2 ). The above results show that the fermentation supernatant of RZ0102 has good stability in the range of 4-60℃.

[0052] The results of pH test showed that the fermentation supernatants of RZ0102, RZ0111, RZ0403 and RZ0409 were suitable for functioning under neutral conditions. Excessive acidity and alkali could affect the antibacterial activity of the fermentation supernatants of the four strains, but the antibacterial activity of the fermentation supernatant of RZ0102 was above 70% in the pH range of 3-10 (refer to Figure 3 ).

[0053] The results of the ultraviolet test showed that the antibacterial activity of the fermentation supernatants of RZ0102, RZ0111, RZ0403 and RZ0409 did not change significantly after being irradiated with ultraviolet rays of the same intensity but different durations (refer to Figure 4 ).

[0054] In summary, the fermentation supernatant of RZ0102 has excellent thermal stability, acid-base stability and UV irradiation stability, and can be developed and utilized as a functional strain for the prevention and control of tomato leaf mold.

[0055] Example 3: Identification of strain RZ0102

[0056] 1. Morphological characteristics

[0057] The activated RZ0102 was streaked on Gao's medium No. 1, and the morphology of single colonies was observed daily. After 4 days of culture, the colony morphology was irregularly round, the surface of the colony was dry and wrinkled, the edge was irregular and powdery, almost grayish white, and there was a distinct muddy smell (refer to Figure 5 ).

[0058] 2. Physiological and biochemical characteristics

[0059] According to the Manual of Identification of Common Bacteria Systems, the strain RZ0102 was subjected to physiological and biochemical identification. The results are shown in Table 2:

[0060] Table 2 Physiological and biochemical characteristics of strain RZ0102

[0061]

[0062] Positive: +; Negative: -

[0063] 3. Molecular biological characteristics

[0064] Through relevant molecular biological methods, DNA extraction, amplification and sequencing, the full-length 16S rDNA base sequence of the strain (SEQ-1) was obtained, and the results are as follows:

[0065]

[0066] Based on the comprehensive morphological, physiological and biochemical and molecular biological characteristics, strain RZ0102 was finally identified as Streptomyces luteoverticillatus; the strain has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit date of January 15, 2018 and the deposit number of CGMCC NO.15219.

[0067] Example 4: Genetic stability detection of Streptomyces luteus RZ0102

[0068] In order to further clarify whether Streptomyces luteus RZ0102 has development value, we used the streak method to test its genetic stability. The streak was cultured 100 times on Gao's medium No. 1, and the difference in the effect of Streptomyces luteus RZ0102 on inhibiting the growth of F. chrysosporium after 1 passage and 100 passages was compared by plate confrontation culture (refer to the initial screening step of Example 2).

[0069] The results showed that there was no significant difference in the inhibitory effect of Streptomyces luteus RZ0102 on the growth of F. chrysosporium between the 1st and 100th generations (refer to Figure 6 , Table 2). The results show that after multiple passages, the antibacterial effect of Streptomyces luteus RZ0102 has no significant degradation and is genetically stable in function, which also lays the foundation for subsequent industrial production.

[0070] Table 2 Diameters of inhibition zones of Streptomyces luteus RZ0102 at different passage times

[0071]

[0072] Example 5: Control of tomato leaf mold by Streptomyces luteus RZ0102

[0073] In order to explore the control effect of Streptomyces luteus RZ0102 on tomato leaf mold, we used 'extra large' tomato detached leaves as experimental materials and explored the control effect of Streptomyces luteus RZ0102 on tomato leaf mold.

[0074] 1. Test methods

[0075] Take tomato leaves that are 4-5 weeks old, have the same growth and leaf position after transplanting and place them in a 90 mm culture dish. Place 3 layers of moist absorbent paper in the culture dish to keep it moist. Place 4 tomato leaves in each culture dish and repeat 5 culture dishes.

[0076] Two treatments were set for each leaf: ①CK: select an area equidistant from the center of the plate and make a cake of 5 mm yellow brown spore mold cake (subsequent treatments are the same); 100 μL of sterile water was evenly applied to the center of the left tomato leaf, and after air drying, the yellow brown spore mold cake was inoculated for infection for 6 hours. ②CT: 100 μL of diluted bacterial solution of Streptomyces luteus RZ0102 (concentration of 5.0×10 8 CFU / mL), and then inoculated with the fungus cake of Flavour Mold for infection for 6 h after air drying.

[0077] Each treatment culture dish was placed in an incubator at 28°C for incubation. The leaf disease status was observed 24h and 48h after incubation, the lesion diameter of each treatment was quantified, the disease index of each treatment was calculated, and the control effect of Streptomyces luteus RZ0102 was calculated.

[0078] 2. Results Analysis

[0079] The experimental results showed that the diluted bacterial solution of Streptomyces luteus RZ0102 could effectively inhibit the expansion of lesions (refer to Figure 7 ), significantly reducing the disease index of tomato leaf mold (see Table 3); the 48h control effect of Streptomyces luteus RZ0102 was calculated to be 74.94% (see Table 3). This shows that Streptomyces luteus RZ0102 has a good control effect on tomato leaf mold, and spraying it before the onset of tomato leaf mold can effectively prevent the occurrence and spread of the disease.

[0080] Table 3 Statistics of disease index and control effect of different treatments at 48h

[0081]

[0082] Example 6: Preparation of fermentation broth of Streptomyces luteus RZ0102

[0083] The preparation process of the fermentation liquid of Streptomyces luteus RZ0102 is as follows:

[0084] (1) Seed solution acquisition: Streptomyces luteus RZ0102 was streaked on Gao's medium No. 1 under sterile conditions and activated at 30°C, while observing whether there was any contamination; after activation, a loop of bacterial moss was taken with an inoculation loop and inoculated into Gao's medium No. 1, and cultured at 30°C and 180 r / min for 12 h to prepare the seed solution of Streptomyces luteus RZ0102;

[0085] (2) Large-scale fermentation culture: 60-70% of the tank volume is filled with water, and the raw materials are added according to the formula of the large-scale fermentation medium. After the raw materials are completely dissolved, the pH is adjusted to 7.2-7.3, and 0.1% soybean oil defoamer is added. Sterilize at 121° C. and 0.1-0.12 MPa for 30 min. After sterilization, the seed liquid is inoculated into the fermentation tank (500 L) at a 5% inoculation rate, and fermented and cultured at 30° C. for 16-20 h. The bacterial count can reach about 5 billion / ml, which is the fermentation liquid of Streptomyces luteus RZ0102.

[0086] The seed liquid culture medium is Gao's medium No. 1.

[0087] Formula of bulk fermentation medium (mass ratio): soluble starch 2%, KNO3 0.1%, K2HPO4 0.05%, glucose 0.5%, urea 0.2%, peptone 0.2%, MgSO4·7H2O 0.05%, and the rest is water, pH 7.2-7.5.

[0088] Example 7: Field verification of the effect of fermentation liquid of Streptomyces luteus RZ0102 on tomato leaf mold

[0089] 1. Test methods

[0090] The test site was set up in Shouguang City, Shandong Province. In previous years, leaf mold disease occurred severely in the area, and the cultivation conditions of the test site were uniform. The control effect of tomato leaf mold disease was determined by foliar spraying. Each treatment group was randomly selected from 7 rows of tomatoes, with a total of 140 trees, and a protective row was set between each treatment group. From the beginning of flowering to 20-25 days after fruit planting, different preparations were evenly sprayed on the tomato plants of each treatment group at a dosage of 10mL / tree; 10 days later, spray again at the same dosage, and use it for 3 times; 10 days after the last spraying of the bacterial solution, check the incidence of leaf mold disease on the tomato leaves of each treatment group.

[0091] The experiment set up three treatments as follows:

[0092] (1) Clean water control group (CK): spray clean water on the leaves.

[0093] (2) Carbendazim treatment (T1): spray on leaves at a dilution of 500 times.

[0094] (3) Bacterial solution treatment (T2): Spray the leaves with a diluted solution of Streptomyces luteus RZ0102 fermentation solution (the concentration of the diluted solution was 5.0×10 8 CFU / mL).

[0095] Disease grading standards: Level 0: no lesions; Level 1: lesion area accounts for less than 1 / 4 of the leaf area; Level 2: lesion area accounts for 1 / 4-2 / 4 of the leaf area; Level 3: lesion area accounts for 2 / 4-3 / 4 of the leaf area; Level 4: lesion area accounts for more than 3 / 4 of the leaf area.

[0096] Disease index = [∑ (disease level value × number of diseased leaves at that level) / (total number of leaves surveyed × highest disease level value)] × 100%

[0097] Control efficiency = [1-(disease index of the treatment group / disease index of the control group)] × 100%

[0098] 2. Results Analysis

[0099] The results showed that after the fermentation liquid of Streptomyces luteus RZ0102 was sprayed on the leaves, the disease index of tomato leaf mold was reduced to 11.26, which was significantly lower than that of the water control and carbendazim treatment; further calculation of its control effect showed that the control effect of the fermentation liquid of Streptomyces luteus RZ0102 was 86.85% (see Table 4). The results show that the fermentation liquid of Streptomyces luteus RZ0102 can effectively prevent and control the occurrence of tomato leaf mold in the field, and can be promoted and applied as a biocontrol agent for tomato leaf mold.

[0100] Table 4 Control effect of dilution of fermentation liquid of Streptomyces luteus RZ0102

[0101]

Claims

1. A Streptomyces luteoverticillatus RZ0102, the deposit number of which is CGMCC NO.15219.

2. The fermentation broth of Streptomyces luteus RZ0102 according to claim 1.

3. The method for preparing the fermented bacterial liquid according to claim 2, characterized in that: After activating the Streptomyces luteus RZ0102 strain, Gao's No. 1 medium was used for constant temperature shaking culture at 28-32° C. for 10-15 hours to prepare seed liquid; the seed liquid was inoculated into a large amount of fermentation medium at an inoculation rate of 1-10%, and fermented and cultured at 28-32° C. for 16-20 hours to obtain a fermentation culture liquid.

4. The method for preparing a fermented bacterial liquid as claimed in claim 3, characterized in that: The bulk fermentation medium comprises, by weight: 2% soluble starch, 0.1% KNO3, 0.05% K2HPO4, 0.5% glucose, 0.2% urea, 0.2% peptone, 0.05% MgSO4·7H2O, and the rest water, with a pH of 7.2-7.

5.

5. Use of the Streptomyces luteus RZ0102 according to claim 1 or the fermentation liquid according to claim 2 in the prevention and treatment of tomato leaf mold.

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