Streptomyces rehmannia, fungicide and application of streptomyces rehmannia in preventing and treating ring spot of rehmannia

Abstract

The invention belongs to the technical field of microbial pesticides, and relates to a new strain of streptomyces, in particular to a strain of streptomyces rehmannia, a fungicide and application of the fungicide in prevention and treatment of rehmannia ring spot. The streptomyces rehmanniae is streptomyces rehmanniae Rer75, the streptomyces rehmanniae is preserved in the China Center for Type Culture Collection on September 20, 2023, and the preservation number of the streptomyces rehmanniae is CCTCC M 20231749. The Rer75 disclosed by the invention has good bacteriostatic activity on phomopsis graminicola, a biological agent prepared from the Rer75 has a relatively good prevention and treatment effect on the rehmannia ring spot, and both the protection effect and the treatment effect obviously reduce the attack degree of the rehmannia, so that the Rer75 has the potential of preventing and treating the rehmannia ring spot caused by the phomopsis graminicola.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial pesticides, and relates to a Streptomyces sp., in particular to a Rehmannia Streptomyces, a microbial agent and application of the Rehmannia Streptomyces in preventing and treating Rehmannia annularis. BACKGROUND

[0002] In recent years, with the continuous cropping of Rehmannia glutinosa, excessive fertilization and adjustment of the planting industry structure, the occurrence of Rehmannia diseases has also intensified year by year, especially the occurrence of annularis, which has seriously restricted the development of the Rehmannia industry. Rehmannia annularis is an important leaf disease, and when it occurs, the leaf appears a lesion, the lesion is round or long oval, has obvious concentric rings, the lesion is large, and perforation is caused in the later stage, and when the lesion is connected to the whole plant, death is caused; the disease is serious when the field humidity is large, resulting in a large reduction in yield, and even absolute yield. According to identification, the pathogen that can cause Rehmannia annularis-like symptoms includes various different pathogenic microorganisms, for example, Phyllosticta digitalis, Ascohyta molleriana Winter, Septoria digitalis and Phoma herbarum. Among them, Phoma herbarum is an important pathogenic bacterium of Rehmannia annularis.

[0003] At present, the prevention and treatment of Rehmannia annularis mainly relies on chemical pesticides, however, the prevention and treatment of Rehmannia annularis by chemical pesticides not only has poor effect, but also causes pesticide residue problems. With the improvement of people's food safety consciousness, the pesticide residue problem of traditional Chinese medicinal materials has attracted widespread attention. Therefore, it is of great significance to develop efficient, low-toxic and eco-friendly biological pesticides to realize the green prevention and control of Rehmannia and the development of traditional Chinese medicine industry. Actinomycetes are widely distributed in various environments and have strong resistance to harmful factors. In addition, actinomycetes can produce various secondary metabolites to inhibit harmful organisms, and are a kind of beneficial microorganisms. At present, many kinds of actinomycetes are developed as anti-insect and fungicide, and applied to the biological control of crops. As a member of actinomycetes, the biocontrol potential of Streptomyces has been further explored. In recent years, it has been found that Streptomyces not only has certain growth-promoting effect on plants, but also has good prevention and treatment effect on pine wood nematode, as well as on rice blast, Aspergillus niger, tobacco brown spot and powdery mildew and other fungal diseases, and can induce plants to produce disease resistance.

[0004] At present, there is no report on the prevention and treatment of Rehmannia annularis by Streptomyces, and further exploration of Streptomyces for efficiently preventing and treating Rehmannia annularis is needed. SUMMARY

[0005] The technical problem to be solved by the present invention is to provide a new strain of Streptomyces that is highly effective in controlling Rehmannia glutinosa ring spot disease, a fungal agent, and its application in controlling Rehmannia glutinosa ring spot disease.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, a Streptomyces rehmannia strain is provided, wherein Streptomyces rehmannia Rer75 was deposited at the China Center for Type Culture Collection on September 20, 2023, with accession number CCTCC M 20231749.

[0008] In a second aspect, the present invention provides a culture of the Streptomyces reticulatae, wherein the culture is a fermentation broth.

[0009] In one embodiment of the present invention, the method for preparing the fermentation culture medium includes:

[0010] The Streptomyces rehmanniae was inoculated into an activation medium and activated to obtain a seed culture.

[0011] The seed culture was inoculated into a fermentation medium for fermentation culture to obtain a fermentation culture broth.

[0012] In one embodiment of the present invention, the activation culture medium consists of: 2% oats, 0.02% MgSO4, 0.02% potassium nitrate, 0.05% K2HPO4, 2% agar, and water to 1L.

[0013] In one embodiment of the present invention, the fermentation culture medium is composed of: 0.4% yeast extract, 0.4% glucose, 1% malt extract, 0.2% CaCO3, water added to 1L, pH 7.2-7.4.

[0014] In one embodiment of the present invention, the inoculation amount of the seed liquid is 5%-7% of the mass of the fermentation medium.

[0015] In one embodiment of the present invention, the activation culture conditions are a temperature of 27-29°C, a rotation speed of 200-240 r / min, and a shaking culture for 72 h. The preferred culture conditions are 28°C and 220 r / min.

[0016] In one embodiment of the present invention, the fermentation culture conditions are the same as those for activation culture, and the fermentation culture time is 6-8 days, preferably 7 days.

[0017] A third aspect of the present invention provides a biological agent with the Streptomyces reticulatae or the culture as the active ingredient.

[0018] In one embodiment of the present invention, the biological agent is a liquid preparation.

[0019] In one embodiment of the present invention, the biological agent is directly the fermentation culture medium, i.e., the spore suspension.

[0020] In another embodiment of the present invention, the biological agent is obtained by centrifuging the fermentation culture at 12,000 rpm for 20 min, taking the supernatant, and filtering it with a 0.22 μm sterile filter membrane to remove bacteria, resulting in a sterile fermentation filtrate.

[0021] A fourth aspect of the present invention provides the application of the aforementioned biological agent in the prevention and control of rehmannia ring rot. The pathogen causing rehmannia ring rot is *Phoma herbarum*.

[0022] Optionally, during the growth period of Rehmannia glutinosa, the biological agent can be sprayed on the leaves of Rehmannia glutinosa to prevent the occurrence of Rehmannia glutinosa ring spot disease.

[0023] Optionally, during the growth period of Rehmannia glutinosa, the aforementioned biological agent can be sprayed on leaves of Rehmannia glutinosa that have been infected with sclerotium rotundum to reduce the disease index of Rehmannia glutinosa.

[0024] The present invention has the following beneficial effects:

[0025] The *Streptomyces rehmannia* Rer75 provided by this invention exhibits good antibacterial activity against *Streptomyces rehmannia*, and its antibacterial activity increases with increasing concentration of the fermentation broth. At a mixing ratio of 55%, the inhibition rate against *Streptomyces rehmannia* mycelial growth is 100%. Potted plant efficacy tests also showed good control effects; both protective and curative effects significantly reduced the severity of disease in *Rehmannia glutinosa*, indicating that Rer75 has the potential to control *Rehmannia glutinosa* ring spot disease caused by *Streptomyces rehmannia*.

[0026] The biological agent with Rer75 as the active ingredient of the present invention is pollution-free and does not cause environmental pollution. It can reduce or even eliminate the need for other corresponding chemical pesticides and has good prospects for development and application. Attached Figure Description

[0027] Figure 1 The results show the plate confrontation between Streptomyces reticulatae Rer7 and Pteryces cerevisiae. Detailed Implementation

[0028] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0029] Example 1: Obtaining Streptomyces rehmannia

[0030] (1) The Streptomyces rehmanniae was isolated using conventional actinomycete isolation methods;

[0031] (2) Separation matrix: It was isolated from the rhizosphere soil of Rehmannia glutinosa plants in Wenxian County, Henan Province;

[0032] (3) Separation time: May 2022;

[0033] (4) Identification: Morphological identification and 16S rRNA molecular identification were performed respectively. The identification result was that it was a new species of Streptomyces and named Streptomyces rehmannia Rer75.

[0034] The Streptomyces rehmannia Rer75 was deposited on September 20, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC M 20231749.

[0035] Example 2: Inhibitory effect of Rer75 on the mycelial growth of *Pseudomonas stolonifera*

[0036] The effect of Rer75 on the mycelial growth of *Pteris vittata* was determined using the plate confrontation method. First, a 5 mm diameter mycelial cake of activated *Pteris vittata* (isolated from *Rehmannia glutinosa* leaves) was inoculated in the center of a PDA plate. Then, Rer75 was inoculated 2 cm from the center of the PDA plate using the streak method. Plates without Rer75 inoculation served as controls. Each treatment was repeated in triplicate. The plates were incubated at 28°C in the dark for 3 days. The mycelial length of each group was measured, and the inhibition rate of the treatment groups was calculated using formula (1). The results are shown in Table 1.

[0037]

[0038] Table 1. Inhibitory effect of Rer75 on the mycelial growth of *Pyrophyllus gracilis*

[0039] Treatment Hyphal growth diameter (mm) Inhibition rate (%) Control 74.0±0.00 / Rer75 23.3±0.057 73.50

[0040] Result image Figure 1 As shown in Table 1, Rer75 has a strong inhibitory effect on the mycelial growth of *Pseudomonas stolonifera*, with an inhibition rate as high as 73.50%. The mycelial growth of the control group was vigorous, while there was an obvious inhibition zone around Rer75, and the mycelial growth of *Pseudomonas stolonifera* was sparse.

[0041] Example 3: Inhibitory effect of fermentation supernatant of different mixed concentrations on mycelial growth of *Pterocarya stylosa*

[0042] Preparation of sterile fermentation filtrate: Rer75 was transferred to ISP 3 (formulation: 2% oats, 0.02% MgSO4, 0.02% potassium nitrate, 0.05% K2HPO4, 2% agar) for activation, and cultured with shaking at 28℃ and 220 r / min. The resulting seed culture, after 72 h of culture, was transferred at a 6% inoculum to GM medium (formulation: 0.4% yeast extract, 0.4% glucose, 1% malt extract, 0.2% CaCO3, pH 7.2-7.4) for fermentation for 7 days. The fermentation broth was centrifuged at 12,000 rpm for 20 min, and the supernatant was collected and filtered through a 0.22 μm sterile membrane to obtain sterile fermentation filtrate.

[0043] Assay for the antibacterial activity of sterile filtrate plates: Sterile filtrate was mixed with sterile PDA medium at ratios of 10%, 20%, 30%, 40%, 50%, and 55%, and poured into 9 mm diameter petri dishes to prepare antibacterial media. 5 mm diameter mycelial cakes were obtained by punching holes at the edge of the activated *Pseudomonas aeruginosa* hyphae and inoculated into the center of the antibacterial plate. The control medium was prepared by mixing GM liquid medium with PDA at the same ratio, with three replicates for each treatment. When the control pathogen hyphae covered approximately two-thirds of the plate, the diameter was measured, and the inhibition rate was calculated according to formula (1).

[0044] Table 2. Inhibitory effect of different mixed concentrations of Rer75 fermentation supernatant on mycelial growth of *Pyrophyllus gracilis*.

[0045]

[0046] Note: Data in the table are mean ± standard error. Different letters in the same column indicate significant differences at the p-level (P < 0.05) according to the least significant difference test.

[0047] As shown in Table 2, after 7 days of fermentation, the inhibition rate of Rer75 against *Pterygophyton spp.* increased with the increase of the mixing ratio of the fermentation filtrate. When the mixing concentration reached 30%, it had a significant inhibitory effect; when the mixing concentration was 55%, the inhibition rate was 100%, indicating that Rer75 has a strong inhibitory effect on *Pterygophyton spp.*

[0048] Example 4: The potted control effect of Rer75 on Rehmannia glutinosa ring spot disease

[0049] The Rehmannia glutinosa pot experiment used Huai Di Huang Jin Jiu. The same size and weight of Rehmannia glutinosa tubers were selected, disinfected with 75% alcohol, rinsed multiple times with sterile water, and planted in pots. Subsequent experiments were conducted when the Rehmannia glutinosa plants grew to the seedling stage.

[0050] Preparation of spore suspension: Rer75 cells were transferred to ISP3 for activation. Well-growing single colonies were selected for streak subculturing. After 7 days of growth at 28°C, a spore suspension was obtained and adjusted to 10⁻⁶ g / L with sterile water. 6 CFU / mL.

[0051] Two treatment groups were set up: Rer75 spore suspension and water control (CK).

[0052] Protective effect determination: Rehmannia glutinosa seedlings of similar size were selected and sprayed with Rer75 spore suspension on the leaves. Water was used as a blank control. 5 mL was applied to each seedling. Each treatment was repeated three times, with 12 seedlings per replicate. Two days after spraying with the spore suspension, two 7 mm diameter fungal cakes were inoculated onto the leaves of each seedling to detect Rehmannia glutinosa ring rot. Disease incidence was assessed on day 7 post-treatment.

[0053] Therapeutic effect determination: *Rehmannia glutinosa* scab fungus was cultured for 7 days. A 7mm mycelial cake was collected from the inner edge of the hyphae and inoculated onto the leaves of *Rehmannia glutinosa*. Two days later, each plant was sprayed with 5mL of Rer75 spore suspension. Water was used as a blank control. Each treatment was repeated three times, with 12 *Rehmannia glutinosa* plants per replicate. Disease incidence was assessed on the *Rehmannia glutinosa* plants on day 7 after treatment.

[0054] The disease severity grading criteria for Rehmannia glutinosa were as follows: Grade 0: No symptoms; Grade 1: Lesions only at the inoculated site; Grade 3: Less than 1 / 3 of the plant showed systemic symptoms (such as stem shrinkage and wilting); Grade 5: Less than 2 / 3 of the plant showed systemic symptoms; Grade 7: More than 2 / 3 of the entire plant showed systemic symptoms; Grade 9: The plant completely withered and died. After recording the disease severity for each treatment, the disease index and control effect under different treatments were calculated according to formulas (2) and (3). The results are shown in Table 3.

[0055]

[0056]

[0057] Table 3. Disease incidence in the control group and the Rer75 treatment group.

[0058]

[0059] Table 4 shows the potted plant control efficacy of Rer75 against Rehmannia glutinosa ring spot.

[0060]

[0061] Table 4 shows that Streptomyces rehmannia Rer75 has a good preventive and therapeutic effect on Rehmannia glutinosa ring spot disease. The protective and therapeutic efficacy reached 39.56% and 40.15%, respectively. The efficacy of the two treatments was similar, and the protective efficacy was slightly higher than that of the therapeutic efficacy. Both significantly reduced the disease index of Rehmannia glutinosa, proving that Rer75 has great application potential in the prevention and treatment of Rehmannia glutinosa ring spot disease.

[0062] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0063] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A strain of Streptomyces retusus, characterized in that, The Streptomyces rehmannia mentioned is Streptomyces rehmannia Rer75, which was deposited at the China Center for Type Culture Collection on September 20, 2023, with accession number CCTCC M20231749.

2. The culture of *Streptomyces reticulata* according to claim 1, characterized in that, The culture is a fermentation broth.

3. The method for preparing the culture according to claim 2, characterized in that, include: The Streptomyces rehmanniae was inoculated into an activation medium and activated to obtain a seed culture. The seed culture was inoculated into a fermentation medium for fermentation culture to obtain a fermentation culture broth.

4. The preparation method according to claim 3, characterized in that, The activation medium consists of: 2% oats, 0.02% MgSO4, 0.02% potassium nitrate, 0.05% K2HPO4, 2% agar, and water to a final volume of 1L.

5. The preparation method according to claim 3, characterized in that, The fermentation medium consists of: 0.4% yeast extract, 0.4% glucose, 1% malt extract, 0.2% CaCO3, water added to 1L, pH 7.2-7.

4.

6. The preparation method according to claim 3, characterized in that, The inoculation amount of the seed liquid is 5%-7% of the mass of the fermentation medium.

7. The preparation method according to claim 3, characterized in that, The fermentation conditions are: temperature 27-29℃, rotation speed 200-240 r / min, and time 6-8 days.

8. A biological agent with Streptomyces rehmanniae as described in claim 1 or the culture as described in claim 2 as the active ingredient.

9. The application of the biological agent according to claim 8 in the prevention and control of Rehmannia glutinosa ring spot disease.

10. The application according to claim 9, characterized in that, The pathogen causing the ring spot disease of Rehmannia glutinosa is Phona herbarum.

Images