CN108048380B - Streptomyces debaryensis QY-3 and application thereof - Google Patents
Streptomyces debaryensis QY-3 and application thereof Download PDFInfo
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Abstract
The invention discloses a Streptomyces debarkii QY-3 and application thereof, wherein the latin name of the Streptomyces debarkens is the strain number QY-3, the Streptomyces debarkii is preserved in China general microbiological culture collection center, and the preservation number is CGMCC No. 14467. The streptomyces debarkii QY-3 has broad-spectrum activity of resisting plant pathogenic fungi, has obvious inhibiting effect on plant pathogenic bacteria such as colletotrichum gloeosporioides, banana fusarium oxysporum, wheat gibberellic disease, cucumber fusarium oxysporum, dragon fruit fester bacteria, apple ring rot bacteria, mango stem rot bacteria or guava pseudoplectania solani, especially has good preventing and treating effect on colletotrichum gloeosporioides, and is a biocontrol actinomycete strain with great development potential.
Description
Technical Field
The invention relates to the field of biological control of plant diseases, in particular to streptomyces debarkensis QY-3 and application thereof.
Background
Rubber trees (Hevea brasiliensis) are tree species of trees with perennial heat producing zones and rainforest, are mainly distributed in areas of Hainan, Yunnan, Guangdong, Guangxi, Fujian and the like in China, and are main sources of natural rubber. Natural rubber is an important industrial raw material and strategic resource, is closely related to daily life, national defense and industrial construction of people, can not be completely replaced by synthetic rubber at present due to superior performance, and plays an important strategic position in national economy. Rubber anthracnose is an important leaf disease which damages rubber trees in China, is one of four main diseases in the current rubber production, can damage rubber seedlings and field young trees until the trees are grown, can cause repeated leaf fall and young tips of the rubber trees to wither when serious, delays the cutting time, and obviously reduces the yield of rubber. Rubber anthracnose has great harm to rubber in China, such as Hainan, Yunnan, Guangdong and other areas, and is one of important biological factors threatening the sustainable and healthy development of China's natural rubber industry.
Colletotrichum gloeosporioides (Penz.) Penz.And Sacc is an important pathogenic bacterium causing rubber tree anthracnose, agricultural measures and chemical control are mainly adopted for controlling the pathogenic bacterium at present, the control effect is direct when a bactericide is used, but the bactericide is easy to generate drug resistance, a large amount of manpower, material resources and time are required for breeding disease-resistant varieties, and the resistance of the varieties is easy to lose. Therefore, it is imperative to develop safe and effective control measures, with biological control being one of the most promising measures. Actinomycetes are a class of microorganisms having great practical value, and nearly 70% of the currently found bioactive substances of about 8000 kinds among microorganisms are produced by actinomycetes. Actinomycetes are important sources of agricultural antibiotics, and microbial pesticides prepared by using secondary metabolites generated by actinomycetes have the characteristics of no pollution, difficulty in causing pests to have drug resistance and the like, so that actinomycetes become the main body of pollution-free pesticides and the development direction of future pesticides.
The screening of anthrax biocontrol actinomycetes is also partially carried out at home and abroad, Choudhary and the like are separated from India local soil to obtain a biocontrol actinomycetes MT7 which is identified as Streptomyces violaceus (Streptomyces violoscens), and the biocontrol actinomycetes has broad-spectrum antibacterial effect on postharvest pathogenic fungi of various fruits, for example, the Minimum Inhibitory Concentration (MIC) on colletotrichum gloeosporioides is 0.0312 mg/mL; the fermentation liquor crude extract of S.malaysiensis MJM1968 discovered by Palaniyandi and the like has strong inhibition effect on the yam colletotrichum, and 50 mu g/mL of the fermentation liquor crude extract can completely inhibit conidium germination of colletotrichum gloeosporioides; duyixin and the like separate and screen an actinomycete S15 from a soil sample collected from Jinggang mountain area in the west of Jiang, the culture filtrate of the actinomycete has strong inhibition effect on various important plant pathogenic fungi such as soybean anthracnose pathogen, citrus anthracnose pathogen, cucumber anthracnose pathogen and the like, has a wide antagonistic spectrum and is identified as streptomyces bordetella (S.bottropensis); an actinomycete DTJ-24 is separated from strawberry rhizosphere soil by the plum-silk tree and the like, the inhibition rate of the actinomycete anthracnose of strawberry colletotrichum reaches 69.89%, and the actinomycete is identified as streptomyces lavendulae (S.lavendonulae). The currently screened biocontrol actinomycetes of the anthrax bacteria are mostly directed at the crops such as yam, strawberry, orange, grape, soybean and the like, but the research on the biocontrol actinomycetes screening of the rubber anthrax bacteria is rarely reported.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a streptomyces dirhamiana QY-3 strain and application thereof.
The technical scheme adopted by the invention is as follows:
the Streptomyces debarkii strain QY-3 has the latin name of Streptomyces debarkensis and the strain number of QY-3, is preserved in China general microbiological culture collection center with the preservation number of CGMCC No.14467 and the preservation address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
Preferably, the streptomyces debarkensis QY-3 is used for inhibiting plant pathogenic fungi.
Preferably, the plant pathogenic fungi include anthrax pathogenic bacteria, banana fusarium oxysporum, wheat gibberellic disease, cucumber fusarium oxysporum, dragon fruit fusarium, dragon fruit canker, apple ring rot, mango stem rot or guava pestalotiopsis.
More preferably, the anthrax pathogenic bacteria are colletotrichum gloeosporioides or banana gloeosporioides.
The invention also provides a fermentation liquid, which is prepared from the streptomyces dirhamiana QY-3.
Preferably, the fermentation broth is obtained by: fermenting and culturing streptomyces debarkii QY-3, and filtering to remove thalli to obtain fermentation liquor; the fermentation culture medium adopted by the fermentation culture is as follows: 20g of millet, 3g of peptone, 20g of glucose, CaCO32g, 2.5g of NaCl, 1000mL of water, pH7.2-7.4, and the culture conditions of 28 ℃ and 180rpm for shaking culture for 7 d.
The invention also provides a screening and separating method of the streptomyces debarkensis QY-35, which comprises the following steps:
taking a glacier sediment sample, grinding, adding the glacier sediment sample into a container filled with sterile water and glass beads, oscillating, taking supernatant, diluting to obtain a diluent, taking the diluent, uniformly coating the diluent on a Gao-shi culture medium I flat plate, culturing for 5-10d, selecting actinomycete colonies, and further separating and purifying by using a scribing method until obtaining an actinomycete pure culture; a strain QY-3 with high inhibitory activity to colletotrichum gloeosporioides is screened by taking colletotrichum gloeosporioides as a target strain and adopting a confronting culture method.
Preferably, the screening and separating method of streptomyces debarkensis QY-3 is obtained by the following steps:
taking 10g glacier sediment samples, fully grinding, adding the samples into a container filled with 100mL of sterile water and glass beads, oscillating the container on a shaking table at 100rpm for 20min, taking supernatant, carrying out gradient dilution by 10 times to obtain diluent, taking 0.1mL of the diluent, uniformly coating the diluent on a Gaoshi I culture medium flat plate, culturing the flat plate in a constant temperature incubator at 28 ℃ for 5-10d, selecting actinomycete colonies, and further separating and purifying the actinomycete colonies by using a scribing method until obtaining pure actinomycete cultures; a strain QY-3 with high inhibitory activity to colletotrichum gloeosporioides is screened by taking colletotrichum gloeosporioides as a target strain and adopting a confronting culture method.
Compared with the prior art, the invention has the beneficial effects that:
the streptomyces debarkii QY-3 has broad-spectrum activity of resisting plant pathogenic fungi, has obvious inhibiting effect on plant pathogenic bacteria such as colletotrichum gloeosporioides, banana fusarium oxysporum, wheat gibberellic disease, cucumber fusarium oxysporum, dragon fruit fester bacteria, apple ring rot bacteria, mango stem rot bacteria, guava pseudoplectania pilosus and the like, particularly has good preventing and treating effect on colletotrichum gloeosporioides, and is a biocontrol actinomycete strain with great development potential.
Drawings
FIG. 1 shows the colony morphology (A) and spore filament morphology (B) of Streptomyces debarkensis QY-3;
FIG. 2 shows the in-dish antagonistic activity of Streptomyces debarkensis QY-3 against colletotrichum gloeosporioides;
FIG. 3 shows the results of the stability determination of the Streptomyces debarkensis QY-3 fermentation broth;
FIG. 4 shows the effect of Streptomyces debarkii QY-3 fermentation broth on controlling anthracnose-infected rubber leaves.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
Example 1 isolation and screening of Streptomyces debaryensis (Streptomyces decenanensis) QY-3
Taking glacier surface soil to obtain glacier sediment, taking 10g of glacier sediment sample, fully grinding, adding the glacier sediment sample into a triangular flask filled with 100mL of sterile water and glass beads, oscillating the solution on a shaking table at 100rpm for 20min, taking supernatant, performing 10-fold gradient dilution to obtain a diluent, taking 0.1mL of the diluent, uniformly coating the diluent on a Gao's I culture medium flat plate, putting the flat plate into a constant-temperature incubator at 28 ℃ for culturing for 5-10d, selecting actinomycete colonies, and further separating and purifying the actinomycete colonies by using a scribing method until obtaining an actinomycete strain pure culture; a strain QY-3 with high inhibitory activity to colletotrichum gloeosporioides is screened by taking colletotrichum gloeosporioides as a target strain and adopting a confronting culture method.
The biological characteristics of the Streptomyces debaryensis (Streptomyces decenanensis) QY-3 are as follows:
the length of the 16S rDNA sequence (shown in a sequence table) of the streptomyces debarkensis QY-3 effective fragment is 1449bp, and the accession number submitted to a GenBank database is as follows: MG 751325. The sequence similarity search of the effective species in the EzTaxon database was carried out, and the Streptomyces debarkensis QY-3 was found to be highly related to the strain of Streptomyces and to be an actinomycete of Streptomyces, and the Streptomyces debarkensis QY-3 was most similar to Streptomyces decenensis DAS-139(T), with a similarity of 99.65%, a similarity of 99.59% to Streptomyces scabieiNRRL B-16523(T), a similarity of 99.52% to Streptomyces europeisacesaceieieii KACC 20186(T), and a similarity of 97.83-99.00% to other Streptomyces.
The Streptomyces debarkii QY-3 is cultured on a Gaoshi I plate for 7d at 28 ℃, and the colony morphology is observed to be circular, flat and white, a circle of villus and micro-folds are arranged at the periphery, and the spore silk morphology is spiral two-stage recurrent (figure 1).
The streptomyces debarkensis QY-3 is inoculated in different solid culture media, cultured for 7-15 days at 28 ℃ and observed for growth conditions, and the culture characteristics are shown in table 1.
TABLE 1 cultivation characteristics of Streptomyces dekaempferi QY-3
The physiological and biochemical reactions and the carbon source utilization conditions of the streptomyces debarkensis QY-3 are respectively determined, and the results are shown in tables 2 and 3. The results show that the streptomyces delbrueckii QY-3 can hydrolyze starch, liquefy gelatin, can not utilize cellulose, can reduce nitrate, can enable milk to generate peptonization and solidification, and can not generate pigment and H2S (Table 2). In terms of carbon source utilization, it can utilize glucose, raffinose, D-galactose, D-fructose, rhamnose, and can not utilize arabinose, mannitol, inositol, sucrose, maltose, D-xylose (Table 3).
TABLE 2 physiological and biochemical reactions of Streptomyces dekaempferi QY-3
Note: "+" is positive and "-" is negative
TABLE 3 carbon Source utilization by Streptomyces dekaempferi QY-3
Note: "+" is positive and "-" is negative
Example 2: determination of inhibitory activity of Streptomyces debarkensis QY-3 on pathogenic bacteria
The fermentation medium used in the present invention: 20g of millet, 3g of peptone, 20g of glucose and CaCO32g, NaCl 2.5g, water 1000mL, pH: 7.2-7.4, the culture conditions are 28 ℃, and the shaking culture is carried out at 180rpm for 7 d.
The plate confrontation test is utilized to determine the inhibition activity of the streptomyces debarkensis QY-3 on the colletotrichum gloeosporioides, and as shown in figure 2, the streptomyces debarkensis QY-3 has stronger antagonistic activity on the colletotrichum gloeosporioides.
The streptomyces dirichiana QY-3 is subjected to fermentation culture, thalli are removed through filtration to obtain a fermentation liquid supernatant, and the inhibitory activity of the streptomyces dirichiana QY-3 on different plant pathogenic fungi is tested. As can be seen from Table 4, the fermentation broth of Streptomyces dekaki QY-3 has broad-spectrum antifungal activity, especially has strong inhibitory activity against pathogenic bacteria of the genus anthrax, such as 87.0% and 88.9% for the inhibitory activity against Colletotrichum gloeosporioides and Colletotrichum bananas, respectively.
TABLE 4 inhibitory Activity of Streptomyces dekaempferi QY-3 on different plant pathogenic fungi
Example 3: temperature and pH stability determination of Streptomyces dekaempferi QY-3 fermentation liquor
The Streptomyces debarkii QY-3 fermentation liquor is treated for 1 hour at different temperatures and different pH values, and the inhibitory activity of the fermentation liquor on colletotrichum gloeosporioides is determined. As shown in FIG. 3, the Streptomyces dekaempferi QY-3 fermentation broth has good temperature and pH stability.
Example 4: prevention and treatment effect of Streptomyces debarkii QY-3 fermentation liquor on anthracnose-infected rubber leaves
Respectively soaking the newly collected rubber tree leaves in sterile water, chlorothalonil (1000 mu g/mL) and strain QY-3 fermentation liquor for 1h, taking out, placing in a shade, airing, manufacturing wounds on the leaves by using a sterilization needle, inoculating colletotrichum gloeosporioides fungus cakes on the wounds, carrying out moisture preservation culture at 28 ℃ for 3-5d, and observing the disease condition of the leaves. As shown in figure 4, the Streptomyces dekaempferi QY-3 fermentation liquor has a good prevention and treatment effect on rubber leaves infected with anthracnose, the prevention and treatment effect reaches 70 percent and is remarkably higher than that of chlorothalonil (1000 mu g/mL).
The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. It will be apparent to those skilled in the art that a number of simple derivations or substitutions can be made without departing from the inventive concept.
Sequence listing
<110> university of Hainan
<120> Streptomyces debaryensis QY-3 strain and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1449
<212> DNA
<213> Streptomyces dejordiensis QY-3(Streptomyces decenanensis)
<400> 1
gacgaacgct ggcggcgtgc ttaacacatg caagtcgaac gatgaaccac ttcggtgggg 60
attagtggcg aacgggtgag taacacgtgg gcaatctgcc cttcactctg ggacaagccc 120
tggaaacggg gtctaatacc ggatacgaca ctctcgggca tccgatgagt gtggaaagct 180
ccggcggcga aggatgagcc cgcggcctat cagcttgttg gtgaggtaat ggctcaccaa 240
ggcgacgacg ggtagccggc ctgagagggc gaccggccac actgggactg agacacggcc 300
cagactccta cgggaggcag cagtggggaa tattgcacaa tgggcgaaag cctgatgcag 360
cgacgccgcg tgagggatga cggccttcgg gttgtaaacc tctttcagca gggaagaagc 420
gaaagtgacg gtacctgcag aagaagcgcc ggctaactac gtgccagcag ccgcggtaat 480
acgtagggcg cgagcgttgt ccggaattat tgggcgtaaa gagctcgtag gcggtctgtc 540
gcgtcggatg tgaaagcccg gggcttaacc ccgggtctgc attcgatacg ggcagactag 600
agtgtggtag gggagatcgg aattcctggt gtagcggtga aatgcgcaga tatcaggagg 660
aacaccggtg gcgaaggcgg atctctgggc cattactgac gctgaggagc gaaagcgtgg 720
ggagcgaaca ggattagata ccctggtagt ccacgccgta aacggtggga actaggtgtt 780
ggcgacattc cacgtcgtcg gtgccgcagc taacgcatta agttccccgc ctggggagta 840
cggccgcaag gctaaaactc aaaggaattg acgggggccc gcacaagcag cggagcatgt 900
ggcttaattc gacgcaacgc gaagaacctt accaaggctt gacatacacc ggaaacggcc 960
agagatggtc gcccccttgt ggtcggtgta caggtggtgc atggctgtcg tcagctcgtg 1020
tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttgttctgtg ttgccagcat 1080
gcccttcggg gtgatgggga ctcacaggag actgccgggg tcaactcgga ggaaggtggg 1140
gacgacgtca agtcatcatg ccccttatgt cttgggctgc acacgtgcta caatggcagg 1200
tacaatgagc tgcgaagccg tgaggcggag cgaatctcaa aaagcctgtc tcagttcgga 1260
ttggggtctg caactcgacc ccatgaagtc ggagttgcta gtaatcgcag atcagcattg 1320
ctgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacgtcacg aaagtcggta 1380
acacccgaag ccggtggccc aaccccttgt gggagggagc tgtcgaaggt gggactggcg 1440
attgggacg 1449
Claims (4)
1. Streptomyces debarkii (Streptomyces deccanensis) QY-3 is characterized in that the strain number is QY-3, and the strain is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 14467.
2. Use of the streptomyces debarkensis QY-3 according to claim 1, characterized in that the streptomyces debarkensis QY-3 is used for inhibiting phytopathogenic fungi; the plant pathogenic fungi are one or more of anthrax pathogenic bacteria, banana fusarium wilt, wheat gibberellic disease, cucumber fusarium wilt, dragon fruit fusarium, dragon fruit canker, apple ring rot, mango stem rot or guava pestalotiopsis; the anthrax pathogenic bacteria are colletotrichum gloeosporioides or banana gloeosporioides.
3. A fermentation broth, prepared from Streptomyces dekaki QY-3 of claim 1.
4. The fermentation broth according to claim 3, obtained by: fermenting and culturing streptomyces debarkii QY-3, and filtering to remove thalli to obtain fermentation liquor; the fermentation culture medium adopted by the fermentation culture is as follows: 20g of millet, 3g of peptone, 20g of glucose and CaCO32g of NaCl, 2.5g of NaCl and 1000mL of water, pH7.2-7.4, under the conditions of 28 ℃ and shaking culture at 180rpm for 7 d.
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| CN110628687B (en) * | 2019-10-24 | 2022-07-29 | 广西科学院 | Streptomyces 5017 and application thereof in antagonism of phytopathogens |
| CN112725250B (en) * | 2021-03-10 | 2022-07-26 | 扬州大学 | Streptomyces lavendulae strain 2-1-2F-1 and application of biomass thereof in prevention and treatment of vegetable soil-borne diseases |
| CN113930355B (en) * | 2021-09-15 | 2023-03-28 | 中国热带农业科学院热带生物技术研究所 | Biocontrol streptomycete W7 from termite and application thereof |
| CN113999794B (en) * | 2021-11-12 | 2023-05-23 | 中国热带农业科学院热带生物技术研究所 | Streptomyces strain and application thereof |
| CN115125157B (en) * | 2021-12-30 | 2023-04-18 | 中国热带农业科学院南亚热带作物研究所 | Streptomyces for antagonizing pathogenic microorganisms of crops and application thereof |
| CN115812739A (en) * | 2022-06-15 | 2023-03-21 | 福建省农业科学院植物保护研究所 | Streptomyces wetland bacterial strain 13-3 and application of fermentation liquor thereof in prevention and treatment of corn stalk rot |
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