Streptomyces sp. and application thereof in preventing and treating tomato leaf mold

Abstract

This invention discloses a strain of *Streptomyces luteoverticillatus* and its application in controlling tomato leaf mold. *Streptomyces luteoverticillatus* RZ0102, which exhibits strong inhibitory effects against tomato leaf mold, was screened from river mud at the confluence of the Futong River and the sea in Rizhao. The strain's preservation number is CGMCC NO.15219. Experiments show that this strain possesses genetic stability, and its fermentation supernatant exhibits excellent thermal stability, pH stability, and UV irradiation stability. Both strain RZ0102 and its metabolites can effectively inhibit the growth of tomato leaf mold pathogens.

Description

Technical Field

[0001] This invention relates to a strain of *Streptomyces glaucus* and its application in the control of tomato leaf mold, belonging to the field of microbial technology. Background Technology

[0002] *Streptomyces luteoverticillatus* is an actinomycete in the genus *Streptomyces* with high development potential. Currently, research on *Streptomyces luteoverticillatus* is limited. CN108192829A reports that strain HY61 of *Streptomyces luteoverticillatus* can be used to control plant pathogens such as *Colletotrichum gloeosporioides*, *Botrytis cinerea*, *Fusarium oxysporum*, *Fusarium graminearum*, and *Curvularia zearalensis*. CN112899181A reports that strain CCTCC No: M2020262 of *Streptomyces luteoverticillatus* has inhibitory effects on *Sclerotium solani*, *Sclerotium radicans*, and *Sclerotium radicans*. However, there are currently no reports of *Streptomyces luteoverticillatus* being used to control tomato leaf mold.

[0003] Tomato leaf mold is an extremely serious foliar disease of tomatoes caused by Cladosporium fulvum. Once it occurs, leaf mold spreads rapidly and is highly contagious, causing yield reduction in mild cases and complete crop failure in severe cases. Currently, chemical control is the mainstream approach, but the environmental pollution and agricultural product safety issues caused by chemical pesticides are becoming increasingly serious. At present, biological control has received widespread attention due to its safe, healthy, and environmentally friendly principles; however, biological control of tomato leaf mold mainly focuses on Bacillus species, with less exploration of actinomycete resources. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a strain of *Streptomyces glaucus* and its application in the control of tomato leaf mold. This strain exhibits genetic stability, and its fermentation supernatant demonstrates excellent thermal stability, acid-base stability, and ultraviolet radiation stability. It can effectively antagonize *Brachysporum flavum*, the pathogen of tomato leaf mold, and can be developed into a microbial agent targeting tomato leaf mold, achieving green and efficient control of the disease.

[0005] This invention identifies *Streptomyces luteoverticillatus* RZ0102, a fungus with strong inhibitory effects on tomato leaf mold, from river mud at the confluence of the Futong River and the sea in Rizhao. This strain was deposited on January 15, 2018, at the China General Microbiological Culture Collection Center (CGMCC) (address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC NO. 15219. Experiments show that this strain exhibits genetic stability, and its fermentation supernatant demonstrates excellent thermal, acid-base, and UV stability. Both strain RZ0102 and its metabolites effectively inhibit the growth of tomato leaf mold, showing broad application prospects in the control of tomato leaf mold.

[0006] The present invention also provides the fermentation broth of the above-mentioned Streptomyces luteoverticillatus RZ0102.

[0007] The method for preparing the above-mentioned fermentation broth is characterized in that, after the strain is activated, it is cultured in Gao's No. 1 medium at a constant temperature of 28-32℃ with shaking for 10-15 hours to prepare a seed broth; the seed broth is inoculated into a large amount of fermentation medium at an inoculation rate of 1-10% and fermented at 28-32℃ for 16-20 hours to obtain the fermentation broth.

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

[0009] This invention also provides the application of Streptomyces gambogeyrsis RZ0102 or its fermentation liquid in the prevention and control of tomato leaf mold.

[0010] The beneficial effects of this invention are:

[0011] 1. High antibacterial efficacy

[0012] This strain is highly effective against *Pseudomonas aeruginosa*, the pathogen causing tomato leaf mold. The plate inhibition rate reached 90.83%; its fermented bacterial solution effectively inhibited the occurrence and spread of tomato leaf mold: in vitro experiments on tomato leaves showed a control effect of 74.94%; field trials showed a control effect of 86.85%, demonstrating its potential in controlling tomato leaf mold.

[0013] 2. Antibacterial stability

[0014] This strain exhibits genetic stability in function, and its fermentation supernatant demonstrates excellent thermal stability, acid-base stability, and UV irradiation stability.

[0015] This invention has screened a strain of actinomycete *Streptomyces glaucus* that has a good control effect on tomato leaf mold, which is conducive to further exploring actinomycete resources, enriching the resource bank of biocontrol bacteria strains for tomato leaf mold, and is of great significance for carrying out green integrated control of tomato leaf mold. Attached image description:

[0016] Figure 1 The antibacterial activity of the fermentation supernatant of four strains of bacteria was evaluated.

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

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

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

[0020] Figure 5 Colony morphology of Streptomyces gamboge strain RZ0102;

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

[0022] Figure 7 The efficacy of *Garcinia cambogia* RZ0102 in controlling leaf mold disease in tomato leaves. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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 this invention was isolated from river mud at the junction of the Futong River and the sea in Rizhao using the dilution plate method and the streak plate method. The specific isolation method is as follows:

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

[0027] (2) Dilution and Coating: The river mud suspension was diluted sequentially to 10 times using a 10-fold dilution method. -1 ~10 -6 Bacterial samples at dilutions; from 10 -4 10 -5 and 10 -6 Take 100 μL of the diluted bacterial sample and spread it on a plate containing Gao Shi No. 1 medium, with three replicates for each gradient.

[0028] (3) Cultivation: Seal the coated plates and incubate them in an incubator at 28-30℃ for 3-5 days, and observe the growth of colonies on the plates regularly.

[0029] (4) Single bacterial purification: Select strains with colony morphology similar to that of actinomycetes, streak single bacteria on plates, and carry out purification culture at a temperature of 28-30℃ for 3-5 days; if the strain is not pure, repeat the operation; number the obtained strains.

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

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

[0032] (1) Initial screening

[0033] The plate confrontation method was used to screen antagonistic strains that effectively antagonize the pathogen of tomato leaf mold.

[0034] The isolated strains were streaked onto Gao's No. 1 solid medium for 48 hours using a sterile inoculation loop to activate the strains. A 5mm diameter mycelial cake was punched from the edge of the activated *Cladosporium fulvum*, the pathogen causing tomato leaf mold, and transplanted into the center of a PDA plate. Activated strains were sampled at 2.0cm from the center on both sides. The control group received no treatment at 2.0cm from the center on the other two sides of the plate. This process was repeated three times. After treatment, the plates were incubated at 28℃, and the inhibitory effect of the selected strains on the pathogen was observed daily. Once the pathogen in the control group had completely covered 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 - Mycelium cake radius) - (Treatment colony radius - Mycelium cake radius)] / (Control colony radius - Mycelium cake radius) × 100%.

[0036] The results showed that among the 34 selected 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 *Pseudomonas aeruginosa* (see Table 1). Among them, the inhibition rates of RZ0102, RZ0111, RZ0403, and RZ0409 were above 85%, which was significantly higher than that of other strains (see Table 1).

[0037] Table 1 Antibacterial rate of different strains

[0038]

[0039]

[0040] To further identify strains that effectively antagonize *Pseudomonas aeruginosa*, we tested the antagonistic activity and stability of the fermentation supernatants of RZ0102, RZ0111, RZ0403, and RZ0409.

[0041] (2) Second screening

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

[0043] ① Preparation of fermentation broth: Take a piece of the activated test strain on Gao's No. 1 solid plate with a sterile inoculation spatula, inoculate it into an Erlenmeyer flask containing 100mL of Gao's No. 1 liquid culture medium (250mL), and incubate at 30℃ and 180r / min for 48h to obtain its fermentation broth.

[0044] ② Preparation of sterile fermentation supernatant: Place the obtained fermentation broth in a centrifuge tube, centrifuge at 4000 r / min for 15 min, and filter the supernatant using a 0.22 μm filter to obtain the fermentation supernatant.

[0045] ③ Determination of antibacterial activity using the toxic medium method: The fermentation supernatant was thoroughly mixed with PDA medium at a ratio of 1:19 to prepare toxic plates. For the experimental group, a 5 mm diameter Cladosporium fulvum mycelial cake was inoculated in the center of the toxic plate. For the control group, sterile water was added in the same proportion to replace the fermentation supernatant, and a 5 mm diameter Cladosporium fulvum mycelial cake was inoculated in the center. After treatment, the plates were incubated at 30℃ 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 differences (refer to...). Figure 1 ).

[0047] We then used the toxic medium method to test the antibacterial stability of the fermentation supernatants of RZ0102, RZ0111, RZ0403, and RZ0409. This was mainly achieved through temperature resistance, acid and alkali resistance, and UV resistance tests, to screen for strains with better resistance.

[0048] Temperature resistance test: 20 mL of the treated supernatant was taken in portions and placed into 50 mL centrifuge tubes. The tubes were then placed in a 4℃ refrigerator, a 20℃ incubator, a water bath at 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, 100℃, and a sterilizer at 121℃ for 60 min. After the supernatant of each treatment returned to room temperature, the inhibitory effect of the supernatant of different strains on *Pseudomonas aeruginosa* was determined using the toxic medium method. Untreated sterile water was used as a 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 acid and alkali resistance test. -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. After standing in a refrigerator at 4°C for 24 hours, the pH of each treatment was adjusted back to pH 7. Sterile water was used as a control. Each treatment was repeated 3 times. The inhibitory effect of different strains of fermentation broth on *Pseudomonas aeruginosa* after pH treatment was detected using the toxic medium method.

[0050] UV resistance test: Take a certain amount of fermentation supernatant into a sterile petri dish and place it under a 70uW / cm UV lamp. 2 After irradiation for 12h, 24h, 36h and 48h respectively, the samples were taken out, with sterile water as a control. Each treatment was repeated 3 times. The inhibitory effect of different strains of fermentation broth treated with ultraviolet light on *Pseudomonas aeruginosa* was detected by the toxic medium method.

[0051] Temperature test results showed that within the temperature range of 20–40℃, the fermentation supernatant of the four strains exhibited high and stable antibacterial activity, with an inhibition rate consistently above 70%. Both excessively high and low temperatures significantly reduced the antibacterial activity of the fermentation supernatant of RZ0111, RZ0403, and RZ0409. Low temperatures had no significant effect on the antibacterial activity of the fermentation supernatant of RZ0102, while temperatures exceeding 60℃ reduced its antibacterial activity. However, treatment at 121℃ still maintained antibacterial activity (see reference). Figure 2 The above results indicate that the fermentation supernatant of RZ0102 has good stability in the range of 4-60℃.

[0052] pH test results showed that the fermentation supernatants of RZ0102, RZ0111, RZ0403, and RZ0409 were all suitable for functioning under neutral conditions. Excessive acidity or alkalinity affected the antibacterial activity of the fermentation supernatants of all four strains. However, the antibacterial activity of the fermentation supernatant of RZ0102 remained above 70% within the pH range of 3–10 (refer to pH 3). Figure 3 ).

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

[0054] In summary, the fermentation supernatant of RZ0102 exhibits excellent thermal stability, acid-base stability, and UV irradiation stability, making it suitable for development and utilization as a functional strain for controlling tomato leaf mold.

[0055] Example 3: Identification of strain RZ0102

[0056] 1. Morphological characteristics

[0057] The activated RZ0102 was streaked onto Gao's No. 1 medium, and the morphology of single colonies was observed daily. After 4 days of incubation, the colonies were irregularly round, with a dry and wrinkled surface, irregularly powdery edges, and a nearly grayish-white color, exhibiting a distinct muddy odor (see reference). Figure 5 ).

[0058] 2. Physiological and biochemical characteristics

[0059] According to the "Handbook of Systematic Identification of Common Bacteria", 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] Using relevant molecular biology techniques, the full-length 16S rDNA sequence of this strain (SEQ-1) was obtained through DNA extraction, amplification, and sequencing. The results are as follows:

[0065]

[0066] Based on comprehensive morphological, physiological and biochemical, and molecular biological characteristics, strain RZ0102 was finally identified as Streptomyces luteoverticillatus. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 15, 2018, with accession number CGMCC NO.15219.

[0067] Example 4: Genetic stability test of *Streptomyces gambogey* RZ0102

[0068] To further clarify whether *Streptomyces chamaejasminoides* RZ0102 has development value, we tested its genetic stability using the streak plating method. The culture was continuously streaked 100 times on Gao's No. 1 medium, and the difference in the inhibitory effect of *Streptomyces chamaejasminoides* RZ0102 on the growth of *Pseudomonas aeruginosa* between one and 100 subcultures was compared using plate confrontation culture (refer to the initial screening steps in Example 2).

[0069] The results showed that there was no significant difference in the inhibitory effect of *Streptomyces glaucus* RZ0102, which was passaged once and 100 times, on the growth of *Pseudomonas aeruginosa* (refer to...). Figure 6 (Table 2). The results show that after multiple generations, the antibacterial effect of *Streptomyces gambogey* RZ0102 did not significantly degrade, and it has genetic stability in function, which lays the foundation for subsequent industrial production.

[0070] Table 2. Diameter of inhibition zones for *Streptomyces garcinosus* RZ0102 at different passage numbers.

[0071]

[0072] Example 5: Control of leaf mold disease on detached tomato leaves by *Garcinia cambogia* RZ0102

[0073] To investigate the control effect of *Streptomyces garcinella* RZ0102 on tomato leaf mold, we used detached leaves of 'extra-large' tomatoes as experimental materials to explore the control effect of *Streptomyces garcinella* RZ0102 on tomato leaf mold.

[0074] 1. Test Methods

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

[0076] Two treatments were set up for each leaf: ①CK: A 5mm *Bacillus flavus* mycelium cake was evenly spread in a region equidistant from the center of the plate (subsequent treatments were the same); 100μL of sterile water was evenly applied to the center of the left tomato leaf, and after air drying, it was inoculated with *Bacillus flavus* mycelium cake for 6 hours. ②CT: 100μL of diluted *Streptomyces glaucus* RZ0102 bacterial solution (concentration 5.0×10⁻⁶) was evenly applied to the center of the right tomato leaf. 8 After air-drying, inoculate with *Pseudomonas aeruginosa* mycelium cakes for 6 hours.

[0077] Each treatment petri dish was placed in an incubator and incubated at 28℃. Leaf disease status was observed at 24h and 48h after incubation, the diameter of lesions in each treatment was quantified, the disease index of each treatment was calculated, and the control effect of Streptomyces garcinella RZ0102 was calculated.

[0078] 2. Results Analysis

[0079] Experimental results showed that diluted bacterial suspensions of *Streptomyces glaucus* RZ0102 effectively inhibited the spread of lesions (refer to...). Figure 7 The application of *Streptomyces glaucus* RZ0102 significantly reduced the disease index of tomato leaf mold (see Table 3). Calculations showed that the 48-hour control efficacy of *Streptomyces glaucus* RZ0102 was 74.94% (see Table 3). This indicates that *Streptomyces glaucus* RZ0102 has a good control effect on tomato leaf mold, and spraying it before the onset of the disease can effectively prevent its occurrence and spread.

[0080] Table 3. Statistics on disease index and prevention and control effects of different treatments at 48 hours.

[0081]

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

[0083] The preparation process of fermentation broth of *Garcinia cambogia* RZ0102 is as follows:

[0084] (1) Seed liquid acquisition: Under aseptic conditions, Streptomyces garcinella RZ0102 was streaked on Gao's No. 1 medium and activated at 30℃. At the same time, the presence of contamination was observed. After activation, a loopful of fungal growth was inoculated into Gao's No. 1 medium and cultured at 30℃ and 180r / min for 12h to prepare the seed liquid of Streptomyces garcinella RZ0102.

[0085] (2) Mass fermentation culture: Fill the tank with water to 60-70% of its volume, add the raw materials according to the mass fermentation culture medium formula, and after the raw materials are completely dissolved, adjust the pH to 7.2-7.3, add 0.1% soybean oil defoaming agent, and sterilize at 121℃ and 0.1-0.12MPa for 30 minutes. After sterilization, inoculate the seed liquid into the fermentation tank (500L) at a 5% inoculation rate, and ferment at 30℃ for 16-20 hours. The bacterial count can reach about 5 billion / ml. At this time, it is the fermentation liquid of Streptomyces garcinella RZ0102.

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

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

[0088] Example 7: Field efficacy verification of fermentation broth of *Garcinia cambogia* RZ0102 against tomato leaf mold.

[0089] 1. Test Methods

[0090] The experimental site was located in Shouguang City, Shandong Province, where leaf mold outbreaks have been severe in previous years. The cultivation conditions at the experimental site were uniform. The efficacy of foliar spraying in controlling tomato leaf mold was determined. Each treatment group consisted of 140 tomato plants from 7 randomly selected rows, with protective rows between treatment groups. From the beginning of flowering until 20-25 days after fruit transplanting, different formulations were evenly sprayed onto the tomato plants in each treatment group at a dosage of 10 mL per plant. Ten days later, the same dosage was applied again, for a total of three applications. Ten days after the final spray, the leaf mold disease incidence in each treatment group was examined.

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

[0092] (1) Control group (CK): Foliar spray with water.

[0093] (2) Carbendazim treatment (T1): Foliar spray diluted 500 times.

[0094] (3) Bacterial solution treatment (T2): Foliar spray with diluted fermentation solution of *Garcinia cambogia* RZ0102 (diluted solution concentration: 5.0 × 10⁻⁶). 8 (CFU / mL)

[0095] Disease grading standards: Grade 0: No lesions; Grade 1: Lesions cover less than 1 / 4 of the leaf area; Grade 2: Lesions cover 1 / 4 to 2 / 4 of the leaf area; Grade 3: Lesions cover 2 / 4 to 3 / 4 of the leaf area; Grade 4: Lesions cover more than 3 / 4 of the leaf area.

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

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

[0098] 2. Results Analysis

[0099] The results showed that foliar spraying with diluted fermented Streptomyces garciniae RZ0102 reduced the tomato leaf mold disease index to 11.26, significantly lower than the water control and carbendazim treatment. Further calculations revealed that the control efficacy of diluted fermented Streptomyces garciniae RZ0102 was 86.85% (see Table 4). These results indicate that fermented Streptomyces garciniae RZ0102 can effectively control tomato leaf mold in the field and can be promoted and applied as a biocontrol agent for tomato leaf mold.

[0100] Table 4. Control efficacy of diluted fermentation broth of *Streptomyces garcinosus* RZ0102.

[0101]

Claims

1. A type of *Garcinia cambogia* ( Streptomyces luteoverticillatus The preservation number of *Garcinia cambogia* RZ0102 is CGMCC NO.15219.

2. The fermentation broth of *Streptomyces gambogey* RZ0102 as described in claim 1.

3. The method for preparing the fermentation broth according to claim 2, characterized in that, After activating the *Streptomyces gambogey* strain RZ0102, a seed culture was prepared by constant temperature shaking culture at 28–32℃ for 10–15 h using Gao's No. 1 medium. The seed culture was then inoculated into the fermentation medium at an inoculation rate of 1–10% and fermented at 28–32℃ for 16–20 h to obtain the fermentation culture.

4. The method for preparing fermentation broth as described in claim 3, characterized in that, The fermentation medium is composed of, by weight: 2% soluble starch, 0.1% KNO3, 0.05% K2HPO4, 0.5% glucose, 0.2% urea, 0.2% peptone, 0.05% MgSO4·7H2O, with the remainder being water, and a pH of 7.2–7.

5.

5. The application of the *Streptomyces glaucus* RZ0102 as described in claim 1 or the fermentation liquid as described in claim 2 in the prevention and control of tomato leaf mold.

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