Streptomyces for preventing and treating sugarcane pokkah boeng rot and application thereof

Abstract

The invention discloses a streptomyces strain for preventing and treating sugarcane pokkah boeng rot, which is streptomyces noursei NN-Q01-01 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.28993, and application of fermentation liquor of the streptomyces strain in the aspect of preventing and treating the sugarcane pokkah boeng rot. The streptomyces for preventing and treating the sugarcane pokkah boeng rot is separated from healthy passion fruit rhizosphere soil, has an antagonistic effect on the sugarcane pokkah boeng rot and can effectively prevent and treat the sugarcane pokkah boeng rot.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Streptomyces strain for preventing and treating sugarcane tip rot and application thereof. Background Art

[0002] Sugarcane pokkah boeng disease (PBD) is a global fungal disease caused by a complex infection of multiple Fusarium species. Currently, the dominant pathogen of sugarcane pokkah boeng disease in my country is Fusarium sacchari. This infection causes a dramatic drop in sugarcane yields, resulting in significant economic losses for the sugarcane industry and becoming a major obstacle to its development.

[0003] Currently, the prevention and control of sugarcane tip rot primarily relies on disease-resistant varieties and the application of chemical pesticides. However, the predominant sugarcane varieties cultivated in my country are generally susceptible to tip rot, and the breeding of traditional disease-resistant varieties has significant limitations, such as the long time required. Furthermore, the excessive use of chemical pesticides leads to environmental pollution, soil residues, and the development of pesticide resistance, which poses a potential threat to human health through accumulation in the food chain. Therefore, the development of environmentally friendly biological control technologies has become an urgent challenge in the prevention and control of sugarcane tip rot. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a strain of Streptomyces knowlesi NN-Q01-01 isolated from the rhizosphere soil of healthy passion fruit, which has an antagonistic effect on sugarcane tip rot. Its antagonistic effect is utilized to carry out safe, effective and environmentally friendly biological control of sugarcane tip rot.

[0005] To achieve the above objectives, the present invention provides the following technical solutions:

[0006] A strain of Streptomyces for controlling sugarcane tip rot is Streptomyces noursei NN-Q01-01, with a preservation number of CGMCC No. 28993. It was deposited in the General Microbiology Center of the China Culture Collection Administration on November 14, 2023, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] As described above, the application of Streptomyces for preventing and controlling sugarcane tip rot is described.

[0008] The application of the fermentation liquid of Streptomyces for preventing and controlling sugarcane tip rot as described above in preventing and controlling sugarcane tip rot.

[0009] Preferably, the fermentation broth is a fermentation broth prepared by adding sterile fermentation filtrate and sterile water to a volume concentration of 2%.

[0010] Preferably, the sterile fermentation filtrate is obtained by inoculating the seed liquid of Streptomyces noursei NN-Q01-01 into the fermentation medium at an inoculum volume ratio of 1%, culturing at 28° C. and 200 r / min for 7 days, centrifuging and absorbing the supernatant, and filtering with a 0.22 μm bacterial filter to obtain the sterile fermentation filtrate.

[0011] Preferably, the seed liquid is obtained by activating the sugarcane tip rot-control Streptomyces, i.e., Streptomyces noursei NN-Q01-01, on a PDA plate. That is, under sterile operating conditions, a bacterial block of Streptomyces noursei NN-Q01-01 is transferred to a PDA plate and cultured at 28° C. for 7 days. Eight bacterial cakes are taken from the edge of the colony on the PDA plate and inoculated into LB medium, and cultured at 28° C. and 200 r / min for 2 days to obtain the seed liquid of Streptomyces noursei NN-Q01-01.

[0012] Preferably, the fermentation medium comprises 30.0 g of soluble starch, 20.0 g of sucrose, 2.0 g of peptone, 8.0 g of soybean powder, 2.0 g of NaCl, 0.5 g of MgSO4·7H2O, 3.0 g of CaCO3, 0.5 g of K2HPO4·7H2O, and deionized water to 1.0 L, with a pH of 8.0; and sterilized at 115°C for 20 min.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The sugarcane tip rot preventing and controlling Streptomyces (Streptomyces noursei NN-Q01-01) of the present invention is isolated and obtained from the rhizosphere soil of healthy passion fruit, has an antagonistic effect on the sugarcane tip rot, and can effectively prevent and control the sugarcane tip rot.

[0015] Preservation Information

[0016] Streptomyces noursei NN-Q01-01 was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC) on November 14, 2023, with the deposit number CGMCC No. 28993. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The present invention shows the inhibitory effect of the Streptomyces for preventing and controlling sugarcane tip rot on the mycelial growth of Fusarium spp., wherein A is CK and B is Streptomyces noursei NN-Q01-01.

[0018] Figure 21. The effect of the Streptomyces sp. for controlling sugarcane tip rot of the present invention on the hyphae of Fusarium spp.; wherein, row A is CK, the leftmost frame is a colony of Fusarium spp. on a PDA culture medium, the middle frame is a 2000-fold magnified image of the mycelium at the edge of the colony of Fusarium spp. on a PDA culture medium, and the rightmost frame is a 5000-fold magnified image of the mycelium at the edge of the colony of Fusarium spp. on a PDA culture medium;

[0019] Behavior B shows mycelia of Fusarium spp. treated with fermentation liquid of Streptomyces for controlling sugarcane tip rot. The leftmost frame shows colonies of Fusarium spp. on PDA medium containing 10% sterile fermentation filtrate, the middle frame shows mycelia at the edge of colonies of Fusarium spp. on PDA medium containing 10% sterile fermentation filtrate at 2000 times magnification, and the rightmost frame shows mycelia at the edge of colonies of Fusarium spp. on PDA medium containing 10% sterile fermentation filtrate at 5000 times magnification.

[0020] Figure 3 This is a statistical graph showing the effect of the fermentation liquid of Streptomyces for preventing and controlling sugarcane tip rot on the germination of sugarcane Fusarium spores.

[0021] Figure 4 These are magnified images of whole plants and parts of each treatment in a potted plant experiment using Streptomyces fermentation liquid of different concentrations for controlling sugarcane tip rot according to the present invention; A is the whole plant, and B is the part corresponding to the whole plant. DETAILED DESCRIPTION

[0022] The following is a detailed description of the specific embodiments in conjunction with the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Unless otherwise specified, the raw materials and reagents used in the examples are all commercially available.

[0023] Fusarium sacchari was isolated and purified from diseased sugarcane plants collected from a sugarcane base in Quli Town, Fusui County, Chongzuo City, Guangxi Province. Koch's postulates confirmed it as the pathogen causing sugarcane tip rot. The strain was identified as Fusarium sacchari and stored in a -80°C freezer at the State Key Laboratory for Conservation and Utilization of Subtropical Agricultural Bioresources for future use. This strain of Fusarium sacchari was used in subsequent experiments.

[0024] In the following examples, Streptomyces noursei NN-Q01-01 is a Streptomyces species for controlling sugarcane tip rot.

[0025] The culture medium used in the examples is as follows:

[0026] Potato dextrose agar (PDA): 6.0 g potato flour, 20.0 g glucose, 20.0 g agar, and dilute to 1.0 L with deionized water.

[0027] Potato dextrose water (PDW): 6.0 g potato flour, 20.0 g glucose, and dilute to 1.0 L with deionized water.

[0028] LB medium: tryptone 10.0 g, yeast extract 5.0 g, NaCl 10.0 g, and deionized water to 1.0 L.

[0029] Fermentation medium: soluble starch 30.0 g, sucrose 20.0 g, peptone 2.0 g, soybean powder 8.0 g, NaCl 2.0 g, MgSO4·7H2O 0.5 g, CaCO3 3.0 g, K2HPO4·7H2O 0.5 g, dilute to 1.0 L with deionized water, pH 8.0.

[0030] All the above culture media were sterilized at 115℃ for 20min.

[0031] Example 1

[0032] Antagonistic Effect of Streptomyces noursei NN-Q01-01 on Fusarium spp. in Sugarcane

[0033] The Streptomyces noursei NN-Q01-01, which is deposited in the General Microbiology Center of the China Culture Collection Administration Committee with the accession number CGMCC No. 28993, was activated: under sterile operation conditions, the bacterial block of Streptomyces noursei NN-Q01-01 was transferred to the center of the PDA plate, placed in a constant temperature incubator at 28°C and cultured for 7 days, and then a sterile pipette tip with a diameter of 0.6 cm was used to hit the bacterial cake at the edge of the colony for later use.

[0034] Using the plate standoff method, Fusarium saccharum was used as the target bacteria. A 0.6 cm diameter sterile pipette tip was used to poke a bacterial cake from the edge of a Fusarium saccharum colony. This cake was then inoculated onto the center of a PDA plate. The previously mentioned Streptomyces noursei NN-Q01-01 bacterial cake (0.6 cm diameter) was then inoculated 2.5 cm to either side of the plate. A PDA plate inoculated with Fusarium saccharum alone served as the control (CK). The plates were incubated in a 28°C incubator for observation. Four biological replicates were performed for each strain. After 5 days, the colony diameters of the test bacteria were measured to calculate the inhibitory effect.

[0035] Calculation of the inhibition rate: Inhibition rate (%) = (colony diameter of the control group - colony diameter of the treated group) / (colony diameter of the control group) × 100

[0036] Test results ( Figure 1 ) showed that Streptomyces noursei NN-Q01-01 had significant inhibitory activity against Fusarium saccharum, with an inhibition rate of 64.62%.

[0037] Example 2

[0038] Effects of the Fermentation Broth of Streptomyces noursei NN-Q01-01 on the Mycelia of Fusarium spp. in Sugarcane

[0039] The Streptomyces noursei NN-Q01-01, which is deposited in the General Microbiology Center of the China Culture Collection Administration Committee with the accession number CGMCC No. 28993, was activated: under sterile operation conditions, the Streptomyces noursei NN-Q01-01 bacterial block was transferred to a PDA plate and cultured in a constant temperature incubator at 28°C for 7 days. Eight bacterial cakes were taken from the edge of the colony on the PDA plate and inoculated into 100 mL of LB medium. The culture was carried out at 28°C and 200 rpm for 2 days to prepare the seed solution of Streptomyces noursei NN-Q01-01 for later use.

[0040] A 250 mL Erlenmeyer flask was inoculated with 100 mL of fermentation medium using the previously prepared Streptomyces noursei NN-Q01-01 seed solution at a volume ratio of 1% and cultured at 28°C and 200 rpm for 7 days. The fermentation broth was centrifuged, the supernatant removed, and filtered through a 0.22 μm bacterial filter to obtain a sterile fermentation filtrate. A PDA plate containing 10% sterile fermentation filtrate was prepared (i.e., 10% sterile fermentation filtrate was added to the PDA plate) and inoculated with a 0.6 cm diameter Fusarium saccharum cake in the center. A PDA plate inoculated with Fusarium saccharum cake without sterile fermentation filtrate served as a control (CK). After 5 days of culture at 28°C, 2-3 cakes were collected from the edge of the colony, sampled with electron microscopy fixative, and sent to Chengdu Lilai Biotechnology Co., Ltd. for electron microscopy observation.

[0041] The electron microscopy results are as follows Figure 2 As shown, the mycelia of Fusarium saccharum cultured without sterile fermentation filtrate were relatively smooth, while the mycelia of Fusarium saccharum cultured with sterile fermentation filtrate showed obvious shrinkage and curling deformities, indicating that the fermentation broth of Streptomyces knowlesi NN-Q01-01 can inhibit the growth of Fusarium saccharum.

[0042] Example 3

[0043] Effects of the fermentation broth of Streptomyces knowlesi NN-Q01-01 on the germination of spores of Fusarium spp. in sugarcane

[0044] Add 20 mL of sterile water to a culture dish of Fusarium saccharum colony cultured on a PDA plate for 5 days, scrape the hyphae with a sterile coating stick, and filter the spore solution of Fusarium saccharum with 4 layers of sterile lens paper into a triangular flask to obtain the spore solution of Fusarium saccharum. Pipette 1.5 mL and add it to 150 mL of PDW medium. Adjust the spore concentration to 1 × 10 6 / mL, and then added with 10% by volume of the fermentation broth of Streptomyces noursei NN-Q01-01 (the fermentation broth refers to the sterile fermentation filtrate prepared in Example 2) for treatment, and the fermentation broth without Streptomyces noursei NN-Q01-01 was used as the control (CK). The mixture was then incubated in a shaking incubator at 28°C and 200 rpm, and observed every 2 hours. The proportion of germinated spores in each time period was observed and counted under a microscope.

[0045] The results showed (Table 1 and Figure 3 ), the spore germination rate of Fusarium spp. in the CK group was higher than that in the fermentation broth treatment from 2h to 12h. The spore germination rate of the CK group reached 88.30% at 8h and then did not change much, entering a plateau period of germination. However, the spore germination rate of the spores treated with the fermentation broth of Streptomyces noursei NN-Q01-01 was only 5.45% at 8h. The experimental data showed that the fermentation broth of Streptomyces noursei NN-Q01-01 had a significant inhibitory effect on the spore germination of Fusarium spp. within 12 hours.

[0046] Table 1 Statistics of spore germination of Fusarium spp. in sugarcane by fermentation broth of Streptomyces noursei NN-Q01-01

[0047]

[0048] Example 4

[0049] Potted experiment on the control of sugarcane tip rot by Streptomyces knowlesi NN-Q01-01

[0050] Take the sugarcane Fusarium spp. cultured on the PDA plate for 5 days, add 20 mL of sterile water, scrape the hyphae with a sterile coating stick, and filter the sugarcane Fusarium spp. spore solution with 4 layers of sterile lens paper. Use a hemocytometer to adjust the final concentration of the spore solution to 1×10 5 Spores / mL were used, and 500 μL of spore liquid was inoculated from the growing point of the sugarcane into the interior of healthy sugarcane seedlings with basically the same growth status using a syringe. Sterile water injection was used as a negative control (CK1). One day later, fermentation broth with volume concentration gradients of 4%, 2%, and 1% (the fermentation broths with different concentration gradients were prepared by adding different amounts of sterile water to the sterile fermentation filtrate prepared in Example 2) was evenly sprayed on the sugarcane seedlings, and spraying with clear water was used as a control (CK2). On the 14th day after sugarcane inoculation, the disease situation was investigated, and the disease situation of the sugarcane was recorded by taking photos, and the incidence rate, disease index, and control effect were calculated. The field grading standards for sugarcane tip rot are shown in Table 2. The calculation formulas for incidence rate, disease index, and control effect are as follows:

[0051]

[0052] Table 2 Grading standards for sugarcane tip rot

[0053]

[0054] The results showed (Table 3 and Figure 4 ) The incidence rate and disease index of sugarcane treated with fermented liquid were significantly lower than those of the control. Sugarcane treated with 2% fermented liquid after inoculation with Fusarium spores had the lowest incidence rate, only 57.00%, and the best control efficacy, reaching 72.46%. However, sugarcane treated with 4% fermented liquid had an incidence rate of 75.00% and a control efficacy of 47.88%, indicating that excessively high concentrations of fermented liquid have a certain adverse effect on sugarcane, resulting in a reduced control efficacy. Sugarcane treated with 1% fermented liquid had an incidence rate of 78.33% and a control efficacy of 56.36%. This indicates that spraying an appropriate concentration of fermented liquid can effectively control sugarcane tip rot.

[0055] Table 3 Statistics of NN-Q01-01 fermentation liquid for the prevention and treatment of sugarcane tip rot

[0056]

[0057]

[0058] Note: Different letters (a, b and c) in the same column indicate significant differences at P < 0.05 in one-way analysis of variance.

[0059] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A Streptomyces strain for controlling sugarcane tip rot, characterized in that: The strain is Streptomyces noursei NN-Q01-01, with a deposit number of CGMCC No. 28993.

2. Use of the Streptomyces for controlling sugarcane tip rot according to claim 1 in controlling sugarcane tip rot.

3. Use of the fermentation liquid of the Streptomyces for controlling sugarcane tip rot according to claim 1 in controlling sugarcane tip rot.

4. The use of the fermentation liquid of the Streptomyces for controlling sugarcane tip rot according to claim 3 in controlling sugarcane tip rot, characterized in that: The fermentation liquid is a fermentation liquid prepared by adding sterile fermentation filtrate and sterile water to form a volume concentration of 2%.

5. Use of the fermentation liquid of the Streptomyces for controlling sugarcane tip rot according to claim 4 in controlling sugarcane tip rot, characterized in that: The sterile fermentation filtrate is prepared by inoculating the seed liquid of Streptomyces for preventing and treating sugarcane tip rot into the fermentation medium at an inoculum volume ratio of 1%, culturing at 28°C and 200r / min for 7 days, centrifuging and absorbing the supernatant, and filtering to obtain the sterile fermentation filtrate.

6. Use of the fermentation liquid of the Streptomyces for controlling sugarcane tip rot according to claim 5 in controlling sugarcane tip rot, characterized in that: The seed liquid is obtained by activating Streptomyces noursei NN-Q01-01 on a PDA plate, that is, under sterile operating conditions, transferring the Streptomyces noursei NN-Q01-01 bacterial block to the PDA plate, culturing at 28°C for 7 days, taking 8 bacterial cakes from the edge of the colony on the PDA plate and inoculating them into LB medium, culturing at 28°C and 200 r / min for 2 days to obtain the seed liquid of Streptomyces noursei NN-Q01-01.

7. Use of the fermentation liquid of the Streptomyces for controlling sugarcane tip rot according to claim 5 in controlling sugarcane tip rot, characterized in that: The fermentation medium comprises 30.0 g of soluble starch, 20.0 g of sucrose, 2.0 g of peptone, 8.0 g of soybean powder, 2.0 g of NaCl, 0.5 g of MgSO4·7H2O, 3.0 g of CaCO3, 0.5 g of K2HPO4·7H2O, and is diluted to 1.0 L with deionized water, with a pH of 8.

0. The medium is sterilized at 115° C. for 20 min.

Images