Streptomyces hygroscopicus sh-37 strain for preventing and treating citrus canker and application of streptomyces hygroscopicus sh-37 strain
By isolating and identifying the strain of Streptomyces sh-37 and its fermentation broth, the pollution problem of chemical pesticides in the prevention and treatment of citrus canker disease in the prior art was solved, effectively inhibiting and controlling citrus canker bacteria was achieved, and green environmental protection was provided agricultural prevention and control methods.
Patent Information
- Application Number
- CN202411384502.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2024-12-20
AI Technical Summary
The prior art relies on chemical pesticides in the prevention and treatment of citrus canker diseases, resulting in frequent use of medicines, high costs and serious pollution, and lack of effective and green prevention and treatment methods.
Provide a strain of Streptocytica shi-37, which prevents and treats citrus canker disease, and its fermentation broth and biological agent, and isolates and chews and cultures in fermentation liquid culture medium at pH 7.3 to obtain chains. Mold fermentation broth is used to inhibit citrus canker bacteria.
The sh-37 strain has a significant inhibitory effect on citrus canker bacteria, and the fermentation broth has significant effect on the prevention and control of bacteria, providing a green and environmentally friendly comprehensive prevention and control method, meeting the needs of sustainable agricultural development.
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Figure CN119162040A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant disease prevention and control, and in particular to a Streptomyces hygroscopicus SH-37 strain for preventing and controlling citrus canker and an application thereof. Background Art
[0002] Citrus is an important fruit variety in my country, with the largest planting area, the highest yield and the largest consumption. my country is an important origin of citrus and the world's largest citrus producer, with a citrus planting area of more than 40 million mu and an annual output of more than 80 million tons, ranking first in the world in both planting area and output.
[0003] Citrus bacterial canker disease (CBCD) is caused by Xanthomonas campestris pv. citri, which can harm dozens of Rutaceae plants, especially important commercial cultivars in the genus Citrus. It is extremely harmful to production and is one of the three major diseases of citrus. It is a worldwide plant quarantine disease. Citrus bacterial canker disease originated in tropical southern Asia and is now mainly distributed in more than 30 countries including Asia, Pacific and Indian Ocean islands, and the Americas. The loss rate of orchards affected is generally 10-20%, and can reach more than 30% in severe cases. Under conditions of 25-30℃, rainfall is positively correlated with the incidence. There is a peak of disease every time new shoots grow, especially in summer and autumn.
[0004] Citrus canker damages citrus leaves, branches, and fruits, mainly causing significant scar-like damage. When the disease spreads seriously, it also causes leaf fall, withered tips, and fruit drop. Lightly affected fruits have scars, which reduces the commercial value of the fruit and makes it unstorable. In severe cases, the fruit falls and rots, reducing the yield. During the period of citrus shoots and young fruits, accompanied by hot and humid weather, canker is prone to occur. Citrus canker is a typical local leaf disease. Under suitable temperature conditions, rainfall is positively correlated with the occurrence of the disease. Hot and rainy seasons are conducive to the reproduction and spread of pathogens. The disease is serious. Generally, spraying is required for prevention about 20 days after the spring, summer, and autumn shoots are drawn out, that is, the first spray is done when the new shoots grow 1 cm long, and then once a week. Chemical pesticides are mainly used for prevention and control, which are frequently used, expensive, and cause serious pollution. Therefore, it is imperative to seek effective and green ecological prevention and control methods. Biological control methods are used to antagonize microorganisms to effectively reduce the density, vitality and ability of pathogens to infect crops, thereby achieving the effect of preventing and controlling plant diseases.
[0005] Actinomycetes are an important branch of the bacterial domain and have great potential to produce resistant metabolites. More than 10,000 antibiotics have been discovered so far, and more than half of them are produced by actinomycetes. In agricultural production, Streptomyces-related preparations are also often used to prevent and control plant diseases. Streptomyces hygroscopicus can produce a variety of important metabolically active substances, which plays an important role in agriculture and medicine, and has great potential for development and utilization. For example, patent CN117660260 A discloses two strains of Streptomyces hygroscopicus Sh1 and Sh2, and their use in preventing and treating melon wilt, plant anthracnose, plant leaf spot, plant gray mold, plant sclerotinia, melon vine blight and / or pitaya canker; patent CN117965370A discloses a strain of Streptomyces hygroscopicus JN3-4, and its use in preventing and treating pathogenic bacteria such as Aggregatibacterium sphaerocephala, red bean anthracnose, red pine blight, corn leaf spot, tobacco target spot, rice seedling pathogen, melon leaf spot pathogen, pepper anthracnose, pear fruit rot, cucumber sclerotinia, blueberry canker, larch dieback, etc. However, there is no relevant report on Streptomyces hygroscopicus used to prevent and treat citrus canker. Summary of the invention
[0006] The object of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and to provide a Streptomyces hygroscopicus sh-37 strain for preventing and treating citrus canker.
[0007] The second object of the present invention is to provide a Streptomyces fermentation liquid and biological preparation for preventing and treating citrus canker.
[0008] The third object of the present invention is to provide a method for preventing and treating citrus canker by using Streptomyces, Streptomyces fermentation broth or Streptomyces biological preparation.
[0009] The present invention provides a Streptomyces hygroscopicus sh-37 strain for preventing and treating citrus canker. The strain was deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on April 26, 2024, with a deposit number of GDMCC No: 64572.
[0010] The present invention isolates a strain sh-37 from banana orchard soil collected from Sihui, Zhaoqing on December 30, 2021 by conventional dilution plate method. The strain was observed after culturing on Gao's medium No. 1 for 5 days, and its single colony was round, purplish gray, with neat edges and no wrinkles, and the aerial hyphae were white and underdeveloped. The 16S rRNA gene sequence of the strain is SEQ ID No. 1. Through molecular biological identification combined with its morphological characteristics, the sh-37 strain was identified as Streptomyces hygroscopicus of the genus Streptomyces. The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on April 26, 2024, with a strain collection number of GDMCC No: 64572, and the classification name is Streptomyces sp.sh-37. The storage address is No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province.
[0011] The invention also provides a streptomyces fermentation liquid containing the hygroscopic streptomyces sh-37 strain for preventing and treating citrus canker.
[0012] Specifically, the Streptomyces hygroscopicus sh-37 strain is shaken and cultured in a fermentation liquid culture medium with a pH of 7.3 to obtain a Streptomyces fermentation liquid.
[0013] The present invention also provides a streptomyces biological preparation for preventing and treating citrus canker containing the streptomyces hygroscopicus sh-37 strain or the streptomyces fermentation liquid or its fermentation supernatant.
[0014] The present invention also provides a method for preparing the streptomyces biological agent, which comprises inoculating and culturing the above-mentioned streptomyces sh-37 strain to obtain seed liquid, then inoculating the seed liquid into a fermentation liquid culture medium to obtain fermentation liquid, or further centrifuging to obtain a fermentation supernatant.
[0015] Preferably, the above-mentioned Streptomyces hygroscopicus sh-37 strain is inoculated into PD culture medium and cultured by shaking for 3 days to obtain seed liquid; the seed liquid is then inoculated into fermentation liquid culture medium and cultured by shaking for 10 days to obtain fermentation liquid, or the fermentation supernatant is obtained by centrifugation, which is the biological preparation.
[0016] The present invention determines that the sh-37 strain has a significant inhibitory effect on citrus canker bacteria, and the diameter of the inhibition zone in the dish is more than 1.5 cm. At the same time, the results of further in vitro leaf protection efficiency determination show that the fermentation liquid of the sh-37 strain has a significant preventive and therapeutic effect on citrus canker bacteria.
[0017] Therefore, the present invention also provides the use of the above-mentioned Streptomyces hygroscopicus sh-37 strain, the above-mentioned Streptomyces fermentation liquid or the above-mentioned Streptomyces biological preparation in preventing and treating citrus canker or in preparing a preparation for preventing and treating citrus canker.
[0018] The present invention also provides the use of the above-mentioned Streptomyces hygroscopicus SH-37 strain, the above-mentioned Streptomyces fermentation liquid or the above-mentioned Streptomyces biological preparation in inhibiting citrus canker pathogens or in preparing a preparation for inhibiting citrus canker pathogens. Specifically, the above-mentioned Streptomyces hygroscopicus SH-37 strain, the above-mentioned Streptomyces fermentation liquid or the above-mentioned Streptomyces biological preparation is directly applied on the diseased plants after being prepared.
[0019] The present invention provides a method for inhibiting citrus canker pathogen in vitro, which comprises directly inoculating the hygroscopic streptomyces sh-37 strain, the streptomyces fermentation liquid or the streptomyces biological preparation onto the surface of the citrus canker pathogen.
[0020] The invention provides a method for preventing and treating citrus canker in agricultural production, wherein the hygroscopic streptomyces sh-37 strain, the streptomyces fermentation liquid or the streptomyces biological preparation are used to treat plants suffering from citrus canker.
[0021] Furthermore, the diseased plant is citrus.
[0022] Furthermore, the citrus canker is citrus canker caused by Xanthomonas citrus pathotype.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] The present invention provides a Streptomyces hygroscopicus sh-37 strain for preventing and treating citrus canker, and the sh-37 strain was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on April 26, 2024, with a deposit number of GDMCC No: 64572. The sh-37 strain has a strong antibacterial effect on citrus canker pathogens, has a significant preventive and therapeutic effect on citrus canker, can provide new effective prevention and control means and technical support for the comprehensive prevention and control of citrus canker, and meets the needs of green environmental protection and sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is the colony morphology of actinomycete sh-37 strain on Gao's medium No. 1.
[0026] Figure 2 This is the phylogenetic tree of actinomycete sh-37 strain.
[0027] Figure 3 This is the inhibition zone produced when the hygroscopic Streptomyces sh-37 strain was spot-inoculated on a plate containing citrus canker bacteria.
[0028] Figure 4 To determine the protective effect of fermentation broth of Streptomyces hygroscopicus sh-37 strain on detached citrus leaves. DETAILED DESCRIPTION
[0029] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.
[0030] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.
[0031] Example 1 Collection method and morphological identification of actinomycete sh-37 strain
[0032] A strain was isolated from the soil of a banana orchard collected from Sihui, Zhaoqing on December 30, 2021 by the conventional dilution plate method, and its morphology was observed after being cultured on Gao's No. 1 medium for 5 days.
[0033] The conventional dilution plate separation method is as follows: the collected soil samples are naturally air-dried, sieved, 10 g are placed in a conical bottle, 90 mL of sterile water is added, and the mixture is shaken at 180 rpm for 20 min; the soil suspension is then diluted with sterile water to 10 4 Times, measure 0.1mL and drop it on the surface of Gao's medium No. 1, and spread it evenly with a triangular spreading stick; then culture it in a constant temperature incubator at 28℃, observe the growth of the colonies at any time, pick a single colony from the plate and streak it to another Gao's medium No. 1 for culture, after 3 times of streaking, separate and purify, obtain a single colony, inoculate the slant and store it at 4℃.
[0034] The formula of Gao's medium No. 1 is: soluble starch 20g, NaCl 0.5g, KNO 3 1g, K 2 HPO 4 0.5 g, MgSO 4 7H 2 O 0.5g, FeSO 4 7H 2 O 0.01g, agar 20g, dilute to 1000mL, adjust pH to 7.3.
[0035] The results are as follows Figure 1 As shown, after the strain was cultured on Gao's medium No. 1 for 5 days, the single colony was round, with neat edges and no wrinkles, limited growth, white aerial hyphae and underdeveloped, and spores were pale purple-gray and round. According to the "Streptomyces Identification Manual", the sh-37 strain was preliminarily identified as Streptomyces.
[0036] Example 2 Molecular Biological Identification of Actinomycetes sh-37 Strain
[0037] The sh-37 strain isolated in Example 1 was streaked and inoculated into Gao's medium No. 1, cultured in an incubator at 28°C for 7 days, and the bacteria were collected. The genomic DNA of the sh-37 strain was extracted using a bacterial genome extraction kit, and the 16S rRNA gene was PCR amplified and sequenced using bacterial universal primers F: 5'-AGAGTTTGATCCTGGCTCAG-3' and R: 5'-GGTTACCTTGTTACGACTT-3'. The sequencing results were compared with the 16S rRNA gene sequences of prokaryotes in the GenBank gene library, and the 16S rRNA gene sequences of strains with higher sequence homology were retrieved.
[0038] The formula of Gao's medium No. 1 is: soluble starch 20g, NaCl 0.5g, KNO 3 1g, K 2 HPO 4 0.5 g, MgSO 4 7H 2 O 0.5g, FeSO 4 7H 2 O 0.01g, agar 20g, dilute to 1000mL, adjust pH to 7.3.
[0039] The measured 16S rRNA gene sequence is 1440 bases in total. The 16S rRNA gene sequence of the strain is shown in SEQ ID No. 1. The top 100 sequences with the highest similarity in the GenBank gene library are all Streptomyces strains. Combined with morphological and molecular biological characteristics, the sh-37 strain was identified as a Streptomyces sp. strain. The 16S rRNA gene sequences of 10 typical strains were selected to construct a phylogenetic tree using MEGA5.0 software. The results are as follows: Figure 2 As shown, the sh-37 strain and Streptomyces hygroscopicus belong to the same branch, with a similarity of 99%. Combined with the morphological and cultural characteristics, the sh-37 strain was identified as Streptomyces hygroscopicus. The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on April 26, 2024, with the strain collection number GDMCC No: 64572, and the classification name is Streptomyces sp.sh-37. The storage address is No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.
[0040] Example 3 Preparation of biological preparation containing Streptomyces hygroscopicus sh-37 strain
[0041] The isolated and identified Streptomyces hygroscopicus sh-37 strain was inoculated into PD liquid culture medium, and cultured in a shaking incubator at 28°C and 160 r / min for 3 days to prepare a seed solution; 10 mL of the seed solution was inoculated into a fermentation liquid culture medium with an inoculation amount of 10%, i.e., the bottling amount was 100 mL / 250 mL bottle, and then cultured at 28°C and 160 r / min for 10 days to obtain a preparation.
[0042] The formula of PD culture medium is: 20 g glucose, 200 g potato juice, and the volume is fixed to 1000 mL.
[0043] The fermentation liquid medium formula is (g / L): glucose 10g, soluble starch 20g, yeast powder 5g, NaCl 0.5g, KNO 3 1g, K 2 HPO 4 0.5 g, MgSO 4 7H 2 O 0.5g and FeSO 4 7H 2 O 0.01g.
[0044] Example 4 In vitro antibacterial activity of Streptomyces hygroscopicus sh-37 against citrus canker pathogen
[0045] Inoculate citrus canker bacteria into beef extract peptone liquid culture medium and culture in a constant temperature shaking incubator at 28°C and 160r / min for 2 days. Take 1mL of citrus canker bacteria fermentation liquid and add it to 100mL of beef extract peptone culture medium cooled to 45-50°C, shake it evenly and pour it into a plate. After the plate solidifies, inoculate the strain sh-37 at an equilateral triangle position 1.5cm away from the edge of the plate. After inoculation, measure the diameter of the inhibition zone at the inoculation point and culture it for 3 days. Use the bacterial plate without sh-37 as a control.
[0046] The formula of beef extract peptone medium is: 3g beef extract, 10g peptone, 5g NaCl, 20g agar, 1000mL water, and adjust the pH to 7.4.
[0047] The formula of beef extract peptone liquid culture medium is: 3g beef extract, 10g peptone, 5g NaCl, 1000mL water, and adjust the pH to 7.4.
[0048] The results are as follows Figure 3 As shown, the fermentation liquid of sh-37 strain was determined to have a significant inhibitory effect on citrus canker bacteria, with the inhibition diameter reaching more than 1.5 cm.
[0049] Example 5 Determination of the protective effect of Streptomyces hygroscopicus sh-37 on detached leaves
[0050] The actinomycete sh-37 strain cultured on Gao's medium No. 1 plate was inoculated into PD culture medium and cultured at 28°C and 160r / min for 3 days to prepare seed culture medium. 10mL of sh-37 seed culture medium was inoculated into fermentation liquid medium and cultured at 28°C and 160r / min for 10 days. The fermentation liquid was collected and centrifuged at 8000rpm / min for 10min to obtain the supernatant. Citrus leaves were pricked with needles and inoculated with citrus canker pathogens (the bacterial liquid concentration was 10 4 After 24 hours of moisturizing, the sh-37 fermentation supernatant (1 mL / leaf) was applied for protection efficacy determination, and the control was to apply water after inoculation of pathogens. The disease condition of the leaves was observed after 5 days.
[0051] PD medium: 20 g glucose, 200 g potato juice, dilute to 1000 mL.
[0052] The formula of the fermentation liquid medium is (g / L): glucose 10g, soluble starch 20g, yeast powder 5g, NaCl 0.5g, KNO 3 1g, K 2 HPO 4 0.5 g, MgSO 4 7H 2 O 0.5g and FeSO 4 7H 2 O 0.01g.
[0053] The results are as follows Figure 4 As shown, it was determined that the fermentation liquid of the sh-37 strain had a significant preventive and therapeutic effect on citrus canker bacteria. The three diseased leaves in the upper part of the figure were inoculated with pathogens and then coated with clean water as a control, and the three leaves in the lower part were treated with the sh-37 fermentation liquid.
Claims
1. A Streptomyces sp. sh-37 strain for preventing and treating citrus canker, characterized in that: The strain was deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on April 26, 2024, with the deposit number GDMCCNo: 64572.
2. A Streptomyces fermentation liquid for preventing and treating citrus canker, characterized in that: The Streptomyces fermentation broth contains the hygroscopic Streptomyces sh-37 strain according to claim 1.
3. A Streptomyces biological preparation for preventing and treating citrus canker, characterized in that: The Streptomyces biological preparation contains the hygroscopic Streptomyces SH-37 strain according to claim 1, the Streptomyces fermentation broth according to claim 2, or the supernatant of the hygroscopic Streptomyces SH-37 strain fermentation broth according to claim 2.
4. The method for preparing the Streptomyces biological preparation for preventing and treating citrus canker according to claim 3, characterized in that: The hygroscopic Streptomyces sh-37 strain of claim 1 is cultured to obtain seed liquid, and the seed liquid is then inoculated into a fermentation liquid culture medium to obtain a fermentation liquid, or is then centrifuged to obtain a fermentation supernatant.
5. Use of the strain of Streptomyces hygroscopicus sh-37 according to claim 1, the Streptomyces fermentation broth according to claim 2, or the Streptomyces biological preparation according to claim 3 in preventing and treating citrus canker or in preparing a preparation for preventing and treating citrus canker.
6. Use of the hygroscopic Streptomyces sh-37 strain according to claim 1, the Streptomyces fermentation broth according to claim 2, or the Streptomyces biological preparation according to claim 3 in inhibiting citrus canker bacteria or in preparing a preparation for inhibiting citrus canker bacteria.
7. The use according to claim 5, characterized in that: The hygroscopic Streptomyces sh-37 strain is prepared into a bacterial liquid or a biological preparation and then directly applied on plants suffering from citrus canker.
8. A method for inhibiting citrus canker bacteria in vitro, characterized in that: The hygroscopic Streptomyces sh-37 strain according to claim 1, the Streptomyces fermentation broth according to claim 2, or the Streptomyces biological preparation according to claim 3 is inoculated onto the surface of citrus canker bacteria.
9. A method for preventing and controlling citrus canker in agricultural production, characterized in that: The hygroscopic Streptomyces sh-37 strain according to claim 1, the Streptomyces fermentation liquid according to claim 2 or the Streptomyces biological preparation according to claim 3 is used to treat plants suffering from citrus canker.
10. The use according to claim 7 or the method according to claim 9, characterized in that: The plant is citrus.