A strain of Riemerella niger SHP-47 and its application

Abstract

The present invention relates to the field of microbial control, and in particular to a Kitasatosporia sp. SHP-47 and its application. The present invention provides a Kitasatosporia sp. SHP-47, and the deposit number of the Kitasatosporia sp. SHP-47 is CGMCC No. 33249. The strain has a significant inhibitory effect on the pathogenic bacteria of eucalyptus bacterial wilt, can effectively reduce the occurrence and spread of the disease, and provides a new way for the green control of eucalyptus; at the same time, it solves the problems of insufficient means for the control of eucalyptus bacterial wilt in the prior art and the negative impact of the use of chemical pesticides on the environment.

Description

Technical Field

[0001] The invention relates to the field of microbial control, in particular to a strain of Riemerella northernis SHP-47 and application thereof. Background Art

[0002] Eucalyptus (scientific name: Eucalyptus spp.) is a general term for plants of the genera Eucalyptus, Eucalyptus corymbosa and Eucalyptus cymbidium under the Myrtaceae family. Most of these plants are evergreen trees, with a total of 1,039 species (including subspecies and variants). The word eucalyptus was transliterated from French by modern person Wu Zonglian. "Eucalyptus" is the same as "case" in ancient Chinese, implying that there are materials that can be used to make utensils. In addition, eucalyptus has the function of curing diseases and preventing epidemics, so the name "Eucalyptus" also implies safety and no danger.

[0003] Eucalyptus is native to Australia, Papua New Guinea, Indonesia, the Philippines and other countries. It has a wide range of adaptability, strong resistance to adversity, and tolerance to drought and poor soil. Its growth environment covers a variety of terrain conditions from tropical to temperate, from coastal to inland, from plains to mountains. The main way of propagation of eucalyptus is through sowing, and it can also be achieved through self-pollination. It has strong adaptability, fast growth, good wood shape, and a wide range of uses. Eucalyptus can not only be used in industrial fields such as pulp and paper, wood-based panels and construction, but also as an important source of biomass energy. In addition, eucalyptus can also produce forest products such as eucalyptus oil and eucalyptus polyphenols.

[0004] Eucalyptus bacterial wilt is a systemic vascular bacterial disease caused by Ralstonia solanacearum. It is a common and serious bacterial disease in eucalyptus planting. The pathogen can invade the plant root system from the soil, causing the plant to wilt rapidly until the entire plant withers and dies. It can also invade the surrounding healthy plants and even harm the entire forest stand, seriously affecting the production of eucalyptus. At present, the prevention and control of eucalyptus bacterial wilt mainly relies on chemical pesticides, but chemical control methods have problems such as drug residues, environmental pollution and drug resistance. Therefore, the development of green, environmentally friendly, efficient and safe biological control methods has become a research focus. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Riemerella spp. SHP-47 and its application to solve the problems existing in the above-mentioned prior art. The present invention provides a strain of Riemerella spp. SHP-47 for preventing and controlling eucalyptus bacterial wilt, and studies its application in the prevention and control of eucalyptus bacterial wilt. Riemerella spp. SHP-47 provided by the present invention has the effect of significantly inhibiting the pathogenic bacteria of eucalyptus bacterial wilt, can effectively reduce the occurrence and spread of the disease, and provides a new way for the green prevention and control of eucalyptus; at the same time, it solves the problems of insufficient means for preventing and controlling eucalyptus bacterial wilt in the prior art, and the negative impact of the use of chemical pesticides on the environment.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a Kitasatosporia sp. SHP-47. The deposit number of the Kitasatosporia sp. SHP-47 is CGMCC No.33249.

[0008] The northern Riemerella SHP-47 of the present invention has morphological characteristics of ivory to light yellow, regular circle, and wrinkled surface; the Gram reaction is positive, and the bacteria are rod-shaped. It can grow well on LB medium, NA medium, and TSA medium. The strain is a facultative anaerobic bacterium with a growth temperature of 20°C-60°C; it can grow well in LB medium with a NaCl concentration of 2%-5%; it has the ability to produce hydrogen sulfide and can hydrolyze starch. The identification results of other physiological and biochemical characteristics are shown in Table 1.

[0009] The present invention provides the use of the above-mentioned Riemerella sp. SHP-47 in the preparation of a biological microbial agent for preventing and treating eucalyptus bacterial wilt.

[0010] The invention provides a biological microbial agent for preventing and treating eucalyptus bacterial wilt, wherein the biological microbial agent comprises the above-mentioned Riemerella northern Riemerella SHP-47.

[0011] Preferably, the biological bacterial agent also includes auxiliary materials.

[0012] The present invention provides the use of the above-mentioned Riemerella sp. SHP-47 or the above-mentioned biological bacterial agent in preventing and controlling eucalyptus bacterial wilt.

[0013] The invention provides a method for preventing and treating eucalyptus bacterial wilt, comprising the step of treating eucalyptus trees infected with eucalyptus bacterial wilt by using the above-mentioned Riemerella SHP-47 or the above-mentioned biological microbial agent.

[0014] The present invention discloses the following technical effects:

[0015] The present invention provides a Kitasatosporia sp. SHP-47, the deposit number of which is CGMCC No. 33249. The strain has a significant effect of inhibiting the pathogenic bacteria of eucalyptus bacterial wilt, can effectively reduce the occurrence and spread of the disease, and provides a new way for the green prevention and control of eucalyptus; at the same time, it solves the problems of insufficient means for the prevention and control of eucalyptus bacterial wilt in the prior art, and the negative impact of the use of chemical pesticides on the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 The graphs are the inhibitory effect of strain SHP-47 on the pathogen of eucalyptus bacterial wilt; wherein A: blank control, i.e., the inhibitory effect graph of the LB liquid culture medium inoculated; B: the inhibitory effect graph of the inoculated strain SHP-47;

[0018] Figure 2 The results of the fermentation culture broth experiment of the strain SHP-47 are shown in FIG. A: blank control, i.e., a plate of LB solid culture medium without bacteria; B: a plate of fermentation centrifugation supernatant of the strain SHP-47;

[0019] Figure 3 The colony macromorphology and individual morphology of strain SHP-47; A: colony macromorphology of strain SHP-47; B: individual morphology of strain SHP-47;

[0020] Figure 4 is the Gram staining result of strain SHP-47;

[0021] Figure 5 is the phylogenetic tree diagram of strain SHP-47;

[0022] Figure 6 This is the diameter of the inhibition zone of Kitasatosporia sp. SHP-47 treated with different temperatures;

[0023] Figure 7 The graphs show the inhibitory effect of Kitasatosporia sp. SHP-47 on the pathogen of eucalyptus bacterial wilt after being treated at different temperatures; A: blank control, i.e., the inhibitory effect graph without temperature treatment; B: the inhibitory effect graph under 30℃ treatment; C: the inhibitory effect graph under 40℃ treatment; D: the inhibitory effect graph under 50℃ treatment; E: the inhibitory effect graph under 60℃ treatment; F: the inhibitory effect graph under 70℃ treatment;

[0024] Figure 8 This is the diameter of the inhibition zone of Kitasatosporia sp. SHP-47 treated with ultraviolet light at different times;

[0025] Fig. 9The graphs show the inhibitory effect of Kitasatosporia sp. SHP-47 on the pathogen of eucalyptus bacterial wilt after being treated with ultraviolet light for different time periods; A: blank control, i.e., the inhibitory effect graph without ultraviolet light treatment; B: the inhibitory effect graph after being treated with ultraviolet light for 30 minutes; C: the inhibitory effect graph after being treated with ultraviolet light for 60 minutes; D: the inhibitory effect graph after being treated with ultraviolet light for 90 minutes; E: the inhibitory effect graph after being treated with ultraviolet light for 120 minutes;

[0026] Fig.10 This is the diameter of the inhibition zone of Kitasatosporia sp. SHP-47 treated with different organic reagents;

[0027] Fig.11 The graph shows the inhibitory effect of Kitasatosporia sp. SHP-47 on the pathogen of eucalyptus bacterial wilt after being treated with different organic reagents; A: blank control, that is, the inhibitory effect graph after being treated with sterile water; B: the inhibitory effect graph after being treated with methanol; C: the inhibitory effect graph after being treated with anhydrous ethanol; D: the inhibitory effect graph after being treated with isopropanol; E: the inhibitory effect graph after being treated with chloroform. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Example 1 Isolation, screening and identification of antagonistic strains.

[0034] 1. Culture medium

[0035] Beef extract peptone medium (Nutrient Agar, NA): peptone 10.0 g / L, sodium chloride 5.0 g / L, beef extract powder 3.0 g / L, agar powder 15.0 g / L.

[0036] LB nutrient agar medium (Luria-Bertani Agar, LB): peptone 10.0 g / L, sodium chloride 5.0 g / L, yeast extract 5.0 g / L, agar powder 15.0 g / L.

[0037] Potato Dextrose Agar (PDA): peeled potatoes 200.0 g / L, glucose 20.0 g / L, agar powder 20.0 g / L.

[0038] V8 Juice Agar (V8): V8 juice 100.0 mL / L, agar powder 20.0 g / L, calcium carbonate 3.0 g / L.

[0039] Corn meal medium (CMA): corn meal 10.0 g / L, agar powder 15.0 g / L.

[0040] After the above culture media were prepared, they were dissolved in distilled water to a volume of 400 mL and sterilized at 121°C for 100 min.

[0041] 2. Experimental steps

[0042] (1) Sample collection

[0043] Healthy forest land in a forest with severe eucalyptus bacterial wilt in Qingyuan City, Guangdong Province was selected. The surface vegetation and other impurities were removed with a shovel. The root soil sample of 30 cm underground at about 50 cm (within 1 m) of the eucalyptus was taken and placed in a 50 mL centrifuge tube. The tube was promptly placed in an ice box (dry ice + foam box) for low-temperature transportation and transported back to the laboratory for processing.

[0044] (2) Isolation and purification of antagonistic bacteria

[0045] Place the rooted soil sample in a conical flask, add an appropriate amount of sterile water and small steel balls, and place it in a shaker at 160r / min for 20 min at room temperature. Add 9 mL of sterile water to 4 small test tubes, draw 1 mL of the shaken soil suspension into the first test tube, draw 1 mL of the diluted soil suspension from the first test tube into the second test tube, and so on. After gradient dilution, the concentration is 10 -1 , 10 -2 , 10 -3 , 10 -4 80 µL of each gradient dilution was spread on NA, LB, PDA, V8, and CMA solid culture media, respectively. After inverted culture at 28°C for 1-2 days, single colonies were picked and isolated and purified on the corresponding solid culture media using the plate partitioning method. The purified strains were then stored at -20°C for a long term using 50% (volume fraction) glycerol as a protective agent.

[0046] (3) Activation of pathogens

[0047] The pathogen of eucalyptus bacterial wilt was provided by the Institute of Forest Ecology and Protection, Chinese Academy of Forestry. Wipe the bacterial tube with cotton wool soaked in 75% alcohol, heat the upper end of the bacterial tube over the flame of an alcohol lamp, drop sterile water onto the heated upper end of the bacterial tube to crack the glass, and tap the cracked upper end of the bacterial tube with the bottom of tweezers. Use a pipette to absorb 0.4 mL of sterile water to dissolve the bacterial powder, absorb 0.1 mL into LB solid culture medium, spread evenly, and culture at 28°C for 1-2 days.

[0048] (4) Initial screening and rescreening of antagonistic strains

[0049] The inhibition zone method was used to screen strains that have inhibitory effects on eucalyptus bacterial wilt. In a sterile clean bench, an appropriate amount of LB liquid culture medium was taken into a centrifuge tube, and several sterile filter paper pieces with a diameter of 0.5 cm were placed at the same time. Single colonies of purified strains were picked and inoculated into centrifuge tubes respectively, and cultured in a shaker at 28°C and 180 r / min for 1~2 days until turbidity. Single colonies of pathogens were picked and inoculated into LB liquid culture medium, and cultured in a shaker at 28°C and 180 r / min for 1 day. LB solid culture medium was heated to about 50°C, and pathogenic bacteria liquid was added to LB solid culture medium at a ratio of pathogenic bacteria liquid: culture medium = 1:10 (volume ratio) to prepare bacteria-containing plates. Filter paper pieces in each centrifuge tube were picked and placed on the bacteria-containing plate, and four filter paper pieces were placed symmetrically on each plate. The filter paper pieces inoculated with only LB liquid culture medium were used as blank controls. They were cultured at 28°C for 2~3 days to observe whether there was an inhibition zone. From the initial screening results, select strains with better antibacterial effects for rescreening. Place a filter paper in the center of each plate and repeat 3 groups. When the size of the inhibition zone no longer changes, measure the diameter of the inhibition zone and screen out the antagonistic strain with the strongest antibacterial effect. The results are as follows: Figure 1 The experimental results showed that the strain SHP-47 had a high inhibitory effect on the pathogen of eucalyptus bacterial wilt, and the diameter of the inhibition zone reached 25.17 mm.

[0050] (5) Fermentation culture broth experiment of antagonistic strains

[0051] Activate strain SHP-47 and pathogens, pick single colonies and inoculate them into 2 mL centrifuge tubes containing LB liquid culture medium, and culture them in a shaker at 28°C and 180 r / min for 1~2 days until they become turbid, and obtain the fermentation culture broth of strain SHP-47 and the fermentation culture broth of pathogens. Centrifuge the fermentation culture broth of strain SHP-47 at 13000 r / min for 10 min to obtain the fermentation centrifugation supernatant of strain SHP-47. Heat the LB solid culture medium to about 50°C, and prepare bacterial plates at a ratio of 1 mL of fermentation centrifugation supernatant and 9 mL of culture medium. Pick a filter paper piece of the fermentation culture broth containing pathogens on the bacterial plate, repeat 3 groups, use a non-bacterial LB solid culture medium plate as a blank control, culture at 28°C for 2~3 days, and observe whether there is an inhibition zone. The results are as follows: Figure 2 The results showed that an inhibition zone was generated in the plate containing the centrifugal supernatant of the fermentation of the antagonistic strain, indicating that the metabolites of the antagonistic strain SHP-47 can also antagonize eucalyptus bacterial wilt.

[0052] (6) Identification of strain SHP-47

[0053] The strain was classified and identified according to the Manual of Common Bacteria Identification. Figure 3As shown, the colonies of strain SHP-47 on the LB plate are ivory to light yellow, regular circles, and have wrinkles on the surface. The Gram reaction of strain SHP-47 is positive, see Figure 4 The bacteria are rod-shaped and can grow well on LB medium, NA medium and TSA medium. The physiological and biochemical characteristics of strain SHP-47 are shown in Table 1.

[0054] Table 1 Physiological and biochemical characteristics of strain SHP-47

[0055]

[0056] The bidirectional sequences obtained by sequencing were spliced ​​using SEQMAN software, aligned and cut using MEGA 6.0 software, and the phylogenetic tree was constructed using FigTree software. The results are shown in Figure 5 As shown. After comprehensive identification of molecular biology, morphology and physiological and biochemical characteristics, the results showed that strain SHP-47 was a new species of Kitasatosporia sp., and its classification was named Kitasatosporia sp. The present invention named strain SHP-47 Kitasatosporia sp. SHP-47, and it was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Strain Collection Committee on December 30, 2024, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33249.

[0057] Example 2 Performance measurement of Kitasatosporia sp. SHP-47.

[0058] (1) Antagonistic stability test of antagonistic bacterial solution against pathogens after treatment at different temperatures

[0059] Activate Kitasatosporia sp. SHP-47, pick a single colony and culture it at 28℃ and 180 r / min for 1~2 days until it becomes turbid. Activate the pathogen of eucalyptus bacterial wilt and prepare bacterial plates. Take Kitasatosporia sp. SHP-47 bacterial solution and treat it in a water bath for 30 minutes, and set up a control group (Kitasatosporia sp. SHP-47 bacterial solution without temperature treatment, room temperature is about 23℃). Place a sterile filter paper with a diameter of 0.5 cm on the bacterial plate, and take 2 µL of Kitasatosporia sp. SHP-47 bacterial solution under different water bath treatments (30, 40, 50, 60, 70℃) onto the filter paper, and set up 3 replicates for each treatment. Culture at 28℃ for 2~5 days, observe the inhibition zone and measure the diameter of the inhibition zone. The results are as follows Figure 6 The antagonistic stability test of Kitasatosporia sp. SHP-47 culture liquid against pathogenic bacteria after being treated at different temperatures is shown in the figure. Figure 7The results showed that the antibacterial activity was enhanced at 30℃, slightly decreased at 40℃, significantly decreased at 50℃, and almost disappeared at 60℃ and 70℃. It can be seen that the thermal stability of Kitasatosporia sp. SHP-47 can meet the application temperature environment of biological agents or biological fertilizers.

[0060] (2) Antagonistic stability test of antagonistic bacteria solution against pathogens after being treated with ultraviolet light for different time periods

[0061] Activate Kitasatosporia sp. SHP-47, pick a single colony and culture it at 28℃ and 180 r / min for 1~2 days until it becomes turbid. Activate the pathogen of eucalyptus bacterial wilt and prepare bacterial plates. Take Kitasatosporia sp. SHP-47 bacterial solution and place it at a distance of 20 cm from a 30W ultraviolet lamp for different time periods, and set up a control group (Kitasatosporia sp. SHP-47 bacterial solution not treated with ultraviolet light). Place a sterile filter paper with a diameter of 0.5 cm on the bacterial plate, and take 2 µL of Kitasatosporia sp. SHP-47 bacterial solution under different ultraviolet irradiation times (30 min, 60 min, 90 min, 120 min) onto the filter paper, and set 3 replicates for each treatment. Incubate at 28°C for 2-5 days, observe the inhibition zone and measure the diameter of the inhibition zone. Figure 8 The results of the antagonistic stability test of Kitasatosporia sp. SHP-47 bacterial solution after ultraviolet treatment for different time periods are shown in Fig. 9 The results showed that compared with the control group, the antibacterial activity of the bacterial solution after ultraviolet treatment for 30 min, 60 min, 90 min, and 120 min against pathogenic bacteria was significantly reduced, and the longer the irradiation time, the lower the antibacterial activity. It can be seen that the antagonistic effect of Kitasatosporia sp. SHP-47 is limited by the ultraviolet irradiation time, but it still has a good antibacterial effect under long-term ultraviolet irradiation.

[0062] (3) Antagonistic stability test of antagonistic bacterial solution against pathogens after treatment with different organic reagents

[0063] Activate Kitasatosporia sp. SHP-47, pick a single colony and culture it at 28℃ and 180 r / min for 1~2 days until it becomes turbid. Activate the pathogen of eucalyptus bacterial wilt and prepare bacterial plates. Add organic solvents (methanol, anhydrous ethanol, isopropanol or chloroform) to the bacterial solution of Kitasatosporia sp. SHP-47 in a volume ratio of 1:9, and set up a control group (an equal volume of clean water was added to the bacterial solution of Kitasatosporia sp. SHP-47), and culture at 28℃ for 5h. Place a sterile filter paper with a diameter of 0.5 cm on the bacterial plate, take 2 µL of the antagonistic bacterial solution containing different organic solvents onto the filter paper, and set up 3 replicates for each treatment. Culture at 28℃ for 2~5 days, observe the inhibition zone and measure the diameter of the inhibition zone. The results are as follows Fig.10 As shown in Figure 2, the antagonistic stability test results of Kitasatosporia sp. SHP-47 bacterial solution after being treated with different organic reagents are as follows: Fig.11 The results showed that compared with the control group, the Kitasatosporia sp. SHP-47 solution treated with chloroform lost its antibacterial activity, the Kitasatosporia sp. SHP-47 solution treated with isopropanol had a significantly reduced antibacterial activity against pathogens, and the Kitasatosporia sp. SHP-47 solution treated with methanol and anhydrous ethanol had a relatively stable antibacterial activity against pathogens. This indicates that the antagonistic substance of Kitasatosporia sp. SHP-47 has good water solubility and alcohol solubility.

[0064] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of sporospora noritis ( Kitasatosporia sp.) SHP-47, characterized in that The deposit number of the sp. neri SHP-47 is CGMCC No.33249.

2. Use of the Riemerella sp. SHP-47 according to claim 1 in the preparation of a biological agent for preventing and treating eucalyptus bacterial wilt.

3. A biological agent for preventing and treating eucalyptus bacterial wilt, characterized in that: The biological bacterial agent includes the Riemerella vulgaris SHP-47 described in claim 1.

4. The biological agent according to claim 3, characterized in that: The biological bacterial agent also includes auxiliary materials.

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