CN113930355A - Biocontrol streptomyces weinmannii W7 from termites and application thereof - Google Patents
Biocontrol streptomyces weinmannii W7 from termites and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
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Abstract
The invention discloses a termite-derived Streptomyces biocontrol strain W7 with a preservation number of GDMCC No: 61902, the preservation date is 2021, 8 and 30 days, and the preservation unit is Guangdong province microorganism culture preservation center. Also discloses application of the termite-derived biocontrol streptomyces W7 and a biocontrol product thereof in preventing and controlling phytopathogen. The obtained biocontrol streptomyces W7 has good antibacterial effect, stable control effect, wider antibacterial spectrum, better development prospect and great biological control potential, for example, antibacterial active components in fermentation extracts of the biocontrol streptomyces W7 can be further developed and applied to control of various plant diseases as biological pesticides through technologies such as separation and purification and the like; compared with chemical pesticides, the biocontrol streptomyces strain W7 and biocontrol products thereof such as fermentation extracts have the advantages of no residue, low toxicity, environmental friendliness and the like, and better accord with the current green environmental protection concept.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and relates to a termite-derived biocontrol streptomyces W7 strain and application thereof.
Background
Termite (Termitit), a Blattaria (Blattaria) Formicidae (Termitidae), also known as Lijiangyi, Solenopsis invicta and the like, is a social insect and a world-wide occurrence of omnivorous pests. Termites live widely in tropical and subtropical areas, and are also found in temperate areas. China is mainly distributed in south regions, wherein a large amount of China is distributed in Yunnan, Guangdong and Hainan, and only a few China are distributed in North China and northeast China. Termites are not only distributed widely in regions, but also have a wide range of damage. Termites have been reported to be able to cause damage to important substances in building construction, garden trees, hydraulic engineering, traffic facilities, telecommunication equipment, and the like. The termite can produce about 200 eggs within one year, the reproductive rate of the termite is increased in a geometric speed as a mature colony, and once the external climatic conditions are proper, the male termite and the female termite with the wings can find a mate for mating. The loss of no less than 1.5 hundred million RMB caused by termite damage in architecture, communication, garden, road and the like in Hainan province each year seriously affects the crop planting production and the economic benefit.
In the natural environment, the nesting and foraging activities of the termites are mainly performed on soil and rotten wood, and the termites have few nutrient substances and are easy to invade by environmental pathogenic microorganisms. Termites therefore rely on natural products produced by the gut's dense diverse microbial flora for nutrition and to form defense systems. Actinomycetes are an important group of these flora. A large amount of symbiotic actinomycetes are contained in intestinal tracts, nests and soil around the nests of the higher termites and the lower termites.
Actinomycetes are one of the main sources of biologically active compounds, the most well known being metabolites with antibiotic activity, with about 70% of the natural antibiotics being derived from actinomycetes. The antibacterial agent is prepared to make the greatest contribution to human. The actinomyces symbiosis of termites is a special environment microorganism which is relatively less researched, is one of novel natural product sources, and the research is still in the initial stage. The cathelicidium formosanum and the like discover a symbiotic actinomycete strain BY02 of odontotermes formosanum, and have the inhibiting effect on apple rot bacteria, poplar canker bacteria and tomato early blight bacteria.
Fungal infection is the first cause of plant diseases, which can lead to a great reduction in yield of crops. At present, in actual production, a chemical method is still an indispensable measure for controlling plant diseases. However, the wide use of chemical pesticides is extremely harmful to the ecological environment, the pesticide resistance of crops can be enhanced, and the chemical pesticide residues also have influence on human health. With the increasing awareness of environmental protection, the search for an environment-friendly and efficient prevention and treatment method becomes a research and development target of all countries in the world. Microbial pesticides have the characteristics of high safety to crops, environmental friendliness, difficulty in generating drug resistance and the like, and are widely concerned. Scientists in China also put forward a plurality of research directions, including living microbial pesticides and metabolite pesticides. Actinomycetes are the first discovered microorganisms with biological control action, such as abamectin, jinggangmycin and peptones, and play an important role in controlling plant diseases and insect pests. Streptomyces occupies an important position in actinomycetes. The plant diseases are controlled by utilizing the inhibition effect of the symbiotic actinomycetes from the termites on the pathogenic fungi of the tropical crops, and the fungi are controlled by the fungi, so that the environment-friendly concept is met, and the purpose of high-efficiency control is achieved.
Disclosure of Invention
The invention aims to provide a termite-derived Streptomyces sp W7 strain and a biocontrol product thereof, wherein the termite-derived Streptomyces sp W7 strain and the biocontrol product thereof have antibacterial effects on various phytopathogens.
The second purpose of the invention is to provide the Streptomyces sp W7 derived from the termites and the application of the biocontrol product thereof in preventing and controlling phytopathogens.
It is a final object of the invention to provide a method of biological control.
The first object of the present invention can be achieved by the following technical solutions: a Streptomyces sp (Streptomyces sp.) W7 strain derived from termites, with the preservation number of GDMCC No: 61902, the preservation date is 2021, 8 and 30 days, and the preservation unit is Guangdong province microorganism culture preservation center.
The inventor collects termites in a park of tropical agricultural science institute of china in Hainan province, separates to obtain multiple actinomycetes, screens 1 antagonistic strain capable of effectively inhibiting multiple plant pathogenic fungi by a plate confronting method, and numbers the antagonistic strain as Streptomyces sp W7 (W7) in the embodiment of the invention.
The invention also provides a biocontrol product for controlling phytopathogens, which comprises the Streptomyces sp W7 derived from the termites and/or the fermentation extract of the Streptomyces sp W7 derived from the termites.
The second object of the present invention can be achieved by the following technical solutions: the application of the termite-derived biocontrol streptomyces sp W7 or the biocontrol product in preventing and treating phytopathogen.
Preferably, the plant pathogenic bacteria comprise one or more of dragon fruit rot, small leaf dragon boat leaf spot disease, small leaf dragon boat pseudoplectania pilosula leaf spot disease, dragon boat flower colletotrichum gloeosporioides, asparagus stem blight disease, pepper colletotrichum gloeosporioides, pepper corynebacterium parvum, papaya ulcer disease, coconut stem rot disease, mango colletotrichum gloeosporioides, papaya leaf spot disease, papaya colletotrichum gloeosporioides, banana colletotrichum cowpea ring rot disease, cowpea colletotrichum blight disease and peronospora litchii.
Preferably, the fermentation extract is prepared by the following method:
(1) activating the Streptomyces sp W7 by a plate, inoculating the activated Streptomyces sp W7 into a TSB culture medium, and carrying out shaking culture at the rotating speed of 140-180 rpm at the temperature of 26-30 ℃ for 3-5 d;
(2) then inoculating the mixture into a liquid culture medium, and carrying out shaking culture at the temperature of 26-30 ℃ and the rotating speed of 140-180 rpm for 12-15 days to obtain a fermentation liquid;
(3) extracting the fermentation liquor with ethyl acetate, and concentrating the ethyl acetate extract under reduced pressure to dryness to obtain crude extract, i.e. fermentation extract.
Preferably, the TSB medium in step (1) is prepared according to the following ratio: in 1L of water, 17-19 g of tryptone, 3-4 g of soybean papain hydrolysate (commercially available), 4-5 g of sodium chloride, 2-3 g of dipotassium hydrogen phosphate and 2-3 g of glucose, and the pH value is 7.0-7.3.
More preferably, the TSB medium in step (1) is prepared according to the following ratio: in 1L of water, tryptone 17g, soybean papain hydrolysate (commercially available) 3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, and pH 7.3.
More preferably, in step (1), the culture is performed under shaking at a rotation speed of 160rpm at 28 ℃ for 3-5 days.
Preferably, the liquid culture medium is inoculated according to the inoculation amount of 8-12% (volume percentage content) in the step (2).
More preferably, the liquid culture medium is inoculated in the step (2) in an amount of 10% (by volume).
Preferably, the liquid culture medium in step (2) is prepared according to the following proportion: per 1L of water, 18-20 g of soluble starch, 4-5 g of peptone, 3-4 g of malt extract (commercially available), 3-4 g of yeast extract (commercially available), 10-13 g of glucose, CaCO3 0.5～1g，pH7.0～7.3。
More preferably, the liquid medium in step (2) is prepared according to the following ratio: per 1L water, 20g soluble starch, 5g peptone, 3g malt extract (commercially available), 3g yeast extract (commercially available), 10g glucose, CaCO31g，pH7.0。
Preferably, in the step (2), the shaking culture is carried out at the rotating speed of 160rpm at the temperature of 28 ℃ for 12-15 days.
Preferably, the volume ratio of the ethyl acetate to the fermentation liquor in the step (3) is 0.9-1.1: 1.
More preferably, the volume ratio of the ethyl acetate to the fermentation liquor in the step (3) is 1: 1.
The last object of the present invention can be achieved by the following technical solutions: a biological control method adopts the Streptomyces sp W7 and/or the fermentation extract of the Streptomyces sp W7 to carry out biological control on plants.
Preferably, the plant comprises one or more of dragon fruit, dragon boat flower, asparagus, pepper, mango, banana, coconut, pawpaw, cowpea and litchi.
Compared with the prior art, the invention has the following advantages:
(1) the Streptomyces biocontrol Streptomyces (Streptomyces sp.) W7 obtained by the invention has good antibacterial effect, stable control effect, wider antibacterial spectrum, better development prospect and large biological control potential, and can be further developed and applied to control various plant diseases as a biological pesticide by separating and purifying antibacterial active components in a fermentation extract of the Streptomyces biocontrol Streptomyces;
(2) compared with chemical pesticides, the biocontrol streptomyces strain W7 and biocontrol products thereof such as fermentation extracts have the advantages of no residue, low toxicity, environmental friendliness and the like, and better accord with the current green environmental protection concept.
Drawings
FIG. 1 is a colony morphology of actinomycetes W7 obtained by screening in example 2 (cultured for 7 days);
FIG. 2 is a phylogenetic tree of strain W7 and related strains from example 3;
FIG. 3 shows the inhibitory effect of the viable strain W7 on mango colletotrichum in example 4;
FIG. 4 shows the inhibitory effect of the fermented extract of strain W7 in example 7 on mango anthracnose at 40 ℃ and 50 ℃ in a water bath at 60 ℃ for 1 hour.
Detailed Description
Example 1 Strain screening
Collecting termite samples from the park of Chinese tropical agricultural science institute of Okinawa, Haikou, China, selecting Gao's synthetic culture medium I, separating and screening by adopting a multi-concentration gradient dilution coating method, observing and selecting strains conforming to the colony morphology of actinomycetes every day, transferring the strains to an ISP2 culture medium, separating and purifying, and storing at-20 ℃ in a glycerol tube.
The Gao's synthetic No. one culture medium is prepared from the following components in percentage by mass and volume: 20g/L of soluble starch, 0.5g/L of NaCl, FeSO4 0.01g/L，KNO3 1g/L，K2HPO4 0.5g/L，MgSO40.5g/L, agar 15g/L, final pH 7.3. + -. 0.2.
The ISP2 culture medium is prepared from the following components in percentage by mass and volume: 4g/L of yeast extract powder, 10g/L of malt extract powder, 4g/L of glucose, 20g/L of agar and pH7.2 +/-0.2.
Example 2 Actinomycetes Activity measurement against plant pathogenic fungi
Transferring all the separated actinomycetes into a PDA culture medium for culture, taking mango colletotrichum gloeosporioides as an indicator bacterium after each actinomycetes produces enough spores, and culturing and determining the bacteriostatic activity of the actinomycetes by a plate confronting method: preparing mango colletotrichum gloeosporioides cake with the diameter of 7mm, inoculating the mango colletotrichum gloeosporioides cake in the center of a PDA (PDA) plate (with the diameter of 90mm), taking an actinomycete zone which is 2cm away from the upper part and the lower part of the cake and culturing for 3 days, taking a mango colletotrichum gloeosporioides plate which is not inoculated with actinomycetes as a control group, culturing at the constant temperature of 28 ℃ until the control group grows to be full of the whole plate, measuring, and repeating each treatment for 3 times. The diameter of the bacterial colony is measured by adopting a cross method, and the bacteriostasis rate of the bacterial strain to mango colletotrichum is calculated.
The bacteriostatic rate (%) - (control pathogen growth diameter-pathogen block diameter) - (treatment pathogen growth diameter-pathogen block diameter) ]/(control pathogen growth diameter-pathogen block diameter) × 100%.
According to the results, the bacterial inhibition spectrum of the strain W7 with the strongest antagonistic activity is used as an entry strain for determination.
The actinomycete strain W7 grows well in ISP2 medium, and when cultured at 28 deg.c for 1-3 days, the colony is smooth and no spore is produced, and after 5 days, spore begins to produce, and after 7 days, the color of the spore pile is near white, and the colony morphology (7 days) is shown in FIG. 1.
Example 3 molecular characterization of Strain W7
Actinomycetes W7 is inoculated to an ISP2 solid plate, cultured for 5d at 28 ℃, and a single colony is picked to ISP2 for secondary purification.
Total DNA was extracted using TSINGKE DNA extraction kit (general purpose). The 16S rRNA amplification was performed using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3').
The total volume of the PCR amplification reaction system is 50 mu L, and the PCR amplification reaction system comprises 45 mu L of 1 Xgold medal mix DNA polymerase, 2 mu L of primer 27F, 1492R2 mu L and 1 mu L of DNA template.
The amplification conditions are pre-denaturation 98 ℃, 2min, denaturation 98 ℃, 10s, annealing 56 ℃, 10s, extension 72 ℃, 10s and 35 cycles; final extension 72 ℃ for 5 min.
0.8% agarose gel was used to detect the fragments obtained after PCR reaction (voltage 300V, time 12min), the electrophoresis results were observed using a gel imager, and the positive results were submitted to Kunming division, Biotech, Inc., Beijing Ongzhike.
The 16S rRNA gene sequence (shown as SEQ ID NO: 1) of the strain is as follows:
gatgaaccac ttcggtgggg attagtggcg aacgggtgag tacacgtggg caatctgccc 60
ttcactctgg gacaagccct ggaaacgggg tctaataccg gatacaaccg ctgaccgcat 120
ggtcgggcgg tggaaagctc cggcggtgaa ggatgagccc gcggcctatc agcttgttgg 180
tgaggtaatg gctcaccaag gcgacgacgg gtagccggcc tgagagggcg accggccaca 240
ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat attgcacaat 300
gggcgaaagc ctgatgcagc gacgccgcgt gagggatgac ggccttcggg ttgtaaacct 360
ctttcagcag ggaagaagcg aaagtgacgg tacctgcaga agaagcgccg gctaactacg 420
tgccagcagc cgcggtaata cgtagggcgc aagcgttgtc cggaattatt gggcgtaaag 480
agctcgtagg cggcttgtca cgtcggttgt gaaagcccga ggcttaacct cgggtctgca 540
gtcgatacgg gcaggctaga gtgtggtagg ggagatcgga attcctggtg tagcggtgaa 600
atgcgcagat atcaggagga acaccggtgg cgaaggcgga tctctgggcc attactgacg 660
ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc cacgccgtaa 720
acggtgggaa ctaggtgttg gcgacattcc acgtcgtcgg tgccgcagct aacgcattaa 780
gttccccgcc tggggagtac ggccgcaagg ctaaaactca aaggaattga cgggggcccg 840
cacaagcagc ggagcatgtg gcttaattcg acgcaacgcg aagaacctta ccaaggcttg 900
acatacaccg gaaagcatta gagatagtgc cccccttgtg gtcggtgtac aggtggtgca 960
tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct1020
tgttctgtgt tgccagcatg cccttcgggg tgatggggac tcacaggaga ctgccggggt1080
caactcggag gaaggtgggg acgacgtcaa gtcatcatgc cccttatgtc ttgggctgca1140
cacgtgctac aatggcaggt acaatgagct gcgaaaccgt gaggtagagc gaatctcaaa1200
aagcctgtct cagttcggat tggggtctgc aactcgaccc catgaagtcg gagttgctag1260
taatcgcaga tcagcattgc tgcggtgaat acgttcccgg gccttgtaca caccgcccgt1320
cacgtcacga aagtcggtaa cacccgaagc cggtggccca acccc 1365。
performing BLAST similarity comparison on the obtained sequences at an NCBI website, selecting a 16S rRNA gene sequence of a strain with the similarity of more than 98 percent as a reference object, performing multi-sequence comparison in MEGA-X software, performing cluster analysis and constructing a phylogenetic tree (the phylogenetic tree analysis result of the strain W7 and related strains is shown in figure 2) by an adjacency method, evaluating the phylogenetic matrix according to a Kimura two parameter model, and setting a self-development value for 1000 times for evaluating the stability of the topological structure of the phylogenetic tree. Identified as Streptomyces sp through molecular biology research, and the preservation number is as follows: GDMCC No: 61902, date of deposit: 30/08/2021, depository: guangdong province microorganism strain preservation center, preservation address: "building 5", Guangdong province, microbial research institute of Guangdong province, Zhou 100, Jie 59, Ministry of Guangzhou, Guangzhou.
EXAMPLE 4 actinomycete W7 antibiogram determination
The method is characterized in that the agricultural protective environment for the pathogenic bacteria of the dragon fruit rot fungus G.persicaria, the leptospora carotovora Phyllotheca calipitansis, the leptospora spinosa Clavipedunculata Clavictoria, the Colletotrichum gloeosporioides Coletotrichum Auricularia, the Phomopsis asparagi Phomopsis Asparagi, the Colletotrichum capsici Colestotrichum sp, the corynebacterium capsici Corynebacterium cassiacola, the cowpea ring spot pathogen Corynespora sp, the cowpea Pythium aphanidermatum Pythium (Edson) Fitzp, the coconut stem rot Thielaemia paramyxa, the mango C.gloeosporides, the L.theobroma, the phyllospora carotovora, the phytophthora carotovora C.gloeosporioides, the L.theobroma, the phytophthora carotovora, the phytophthora carotovora candida, the Colletotrichum and the phytophthora carotochyta.
Determining the antibacterial spectrum of actinomycetes W7 by adopting a plate confronting method, transferring actinomycetes W7 into a PDA culture medium for culture, and culturing and determining the antibacterial activity of the actinomycetes by adopting the plate confronting method by taking the plant pathogenic bacteria as indicator bacteria after the strains produce enough spores: preparing a bacterial cake with the diameter of 7mm for each plant pathogenic strain, inoculating the bacterial cake in the center of a PDA (personal digital assistant) plate (the diameter of 90mm), taking an actinomycete zone which is cultured for 3 days at a position 2cm away from the upper part and the lower part of the bacterial cake, taking a plant pathogenic bacteria plate which is not inoculated with actinomycete as a control group, culturing at the constant temperature of 28 ℃ until the control group grows to be full of the plate, measuring, and repeating the treatment for 3 times. And measuring the colony diameter by adopting a cross method, and calculating the bacteriostasis rate of the strain to each pathogenic fungus. The bacteriostatic rate (%) - (control pathogen growth diameter-pathogen block diameter) - (treatment pathogen growth diameter-pathogen block diameter) ]/(control pathogen growth diameter-pathogen block diameter) × 100%. The bacteriostatic ratio for each plant pathogenic fungus is shown in the following table 1:
TABLE 1 antimicrobial Spectrometry of Actinomycetes W7
As can be seen from the data in Table 1, the viable bacteria of the strain W7 have certain bacteriostatic effects on the tested 16 plant pathogenic fungi, wherein the viable bacteria of the strain W7 have the strongest inhibitory effect on Colletotrichum musae of banana anthracnose, and the inhibition rate reaches 78.26 +/-0.23%; secondly, the stem rot fungus Thielavirosis paradoxa, the leaf dragon boat pestalotiopsis cladospora and the mango anthracnose fungus C. gloeosporioides (the inhibition effect of the viable bacteria of the strain W7 on the mango anthracnose fungus is shown in figure 3), and the inhibition rates are all more than 68%; the inhibition effect on phytophthora litchi litchii and Phomopsis asparagi is the weakest, and the inhibition rates are respectively 18.18 +/-1.02% and 16.28 +/-1.18%.
Example 5 Strain W7 antagonism persistence
Transferring the strain W7 into a PDA culture medium for culture, after sufficient spores are produced, culturing and determining the bacteriostatic activity of actinomycetes by a plate confronting method by taking mango colletotrichum as an indicator: preparing mango colletotrichum gloeosporioides cake with diameter of 7mm, inoculating actinomycetes bands for 3d at the position 2cm away from the upper and lower sides of the cake in the center of a PDA (personal digital assistant) plate (diameter of 90mm), taking mango colletotrichum gloeosporioides plate without actinomycetes as a control group, culturing at constant temperature of 28 ℃ for 7d, 15d, 30d and 40d, and repeating each treatment for 3 times. The calculated bacteriostatic rate for each period is shown in table 2 below:
TABLE 2 bacterial strain W7 bacteriostasis rates in different confrontation culture times
Note: the data in the table were compared to the same row, with the difference in lower case letters after the data indicating a significant difference at a level of P < 0.05.
As can be seen from the data in Table 2, the viable bacteria of the strain W7 have strong inhibition effect on the tested mango anthracnose pathogen within 40 days of confrontation, the inhibition rate is more than 67%, wherein the inhibition effect is strongest when the confronting culture is carried out for 7 days, and the inhibition rate reaches 72.73 +/-0.35%; after 30 days of opposite culture, the inhibition effect is obviously reduced compared with that of 15 days, but the bacteriostasis rate is still higher than 67%. The result shows that the antagonistic action of the viable bacteria of the strain W7 on the mango colletotrichum has good continuity.
Example 6 fermentation of Actinomycetes W7 and pretreatment of fermentation sample thereof
Activating Streptomyces sp.W7 by a plate, inoculating the activated Streptomyces sp.W7 into a TSB culture medium, and carrying out shake culture at 28 ℃ and 160rpm for 3 d; inoculating into 10L liquid fermentation culture medium according to 10% (volume percentage content), and performing shake culture at 28 deg.C and 160rpm for 15d to obtain fermentation liquid; extracting the fermentation broth with equal volume of ethyl acetate (1L), repeatedly extracting for 3 times, mixing the ethyl acetate extracts for 3 times, and concentrating under reduced pressure at 50 deg.C to obtain extract (2.3g), which is actinomycete W7 fermented extract.
The TSB culture medium is prepared from the following components in parts by weight and volume: 17g/L tryptone, 3g/L soybean papain hydrolysate, 5g/L sodium chloride, 2.5g/L dipotassium hydrogen phosphate, 2.5g/L glucose and pH 7.3 +/-0.2.
The liquid fermentation medium is prepared from the following components in percentage by weight and volume: 20g/L of soluble starch, 5g/L of peptone, 3g/L of malt extract, 3g/L of yeast extract, 10g/L of glucose and CaCO31g/L， pH7.0。
Example 7 Heat stability of fermented extract of Strain W7 against phytopathogens
Preparing the fermented extract into sample solution with concentration of 500 μ g/mL, and respectively placing in water bath at 40 deg.C, 50 deg.C and 60 deg.C for 1 h. The bacteriostatic activity of actinomycetes is determined by a flat filter paper sheet method by taking mango colletotrichum gloeosporioides as an indicator bacterium: preparing mango colletotrichum gloeosporioides cake with the diameter of 7mm, inoculating the mango colletotrichum gloeosporioides cake in the center of a PDA (PDA) plate (the diameter of 90mm), placing a filter paper sheet with the diameter of 8mm at a position 2cm away from the upper, lower, left and right sides of the cake, taking sterile water and DMSO as negative controls, taking a mango colletotrichum gloeosporioides plate without inoculating actinomycetes as a control group, repeating each treatment for 3 times, and carrying out inverted culture at the constant temperature of 28 ℃ for 10 days.
The calculated inhibition rate at each temperature is shown in table 3 below:
TABLE 3 bacteriostasis rates of fermentation extracts treated at different temperatures
The inhibitory effect of the strain W7 fermentation extract on mango colletotrichum gloeosporioides after 1h of water bath treatment at 40 ℃, 50 ℃ and 60 ℃ is shown in FIG. 4.
As can be seen from Table 3 and FIG. 4, the fermentation extract of the strain W7 has the strongest bacteriostatic effect on mango colletotrichum at room temperature, and the inhibition rate reaches 69.40 +/-1.54%; the 3 fermentation extracts treated at high temperature have strong bacteriostatic effect on mango anthracnose pathogen, wherein the bacteriostatic rate of the fermentation extracts treated at the maximum treatment temperature of 60 ℃ for 1 hour on the mango anthracnose pathogen also reaches 56.72 +/-1.23%, which shows that the mango anthracnose pathogen resistant activity of the fermentation extracts has good thermal stability and can play a stable control role in field biological control of plant pathogenic fungi.
It should be noted that the above-mentioned embodiments are only illustrative and not restrictive, and any modifications or changes within the meaning and range of equivalents to the technical solutions of the present invention by those skilled in the art should be considered to be included in the protection scope of the present invention.
Sequence listing
<110> research institute of tropical biotechnology of Chinese tropical academy of agricultural sciences
<120> termite-derived biocontrol streptomyces W7 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1365
<212> DNA
<213> Streptomyces biocontrol (Streptomyces sp)
<400> 1
gatgaaccac ttcggtgggg attagtggcg aacgggtgag tacacgtggg caatctgccc 60
ttcactctgg gacaagccct ggaaacgggg tctaataccg gatacaaccg ctgaccgcat 120
ggtcgggcgg tggaaagctc cggcggtgaa ggatgagccc gcggcctatc agcttgttgg 180
tgaggtaatg gctcaccaag gcgacgacgg gtagccggcc tgagagggcg accggccaca 240
ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat attgcacaat 300
gggcgaaagc ctgatgcagc gacgccgcgt gagggatgac ggccttcggg ttgtaaacct 360
ctttcagcag ggaagaagcg aaagtgacgg tacctgcaga agaagcgccg gctaactacg 420
tgccagcagc cgcggtaata cgtagggcgc aagcgttgtc cggaattatt gggcgtaaag 480
agctcgtagg cggcttgtca cgtcggttgt gaaagcccga ggcttaacct cgggtctgca 540
gtcgatacgg gcaggctaga gtgtggtagg ggagatcgga attcctggtg tagcggtgaa 600
atgcgcagat atcaggagga acaccggtgg cgaaggcgga tctctgggcc attactgacg 660
ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc cacgccgtaa 720
acggtgggaa ctaggtgttg gcgacattcc acgtcgtcgg tgccgcagct aacgcattaa 780
gttccccgcc tggggagtac ggccgcaagg ctaaaactca aaggaattga cgggggcccg 840
cacaagcagc ggagcatgtg gcttaattcg acgcaacgcg aagaacctta ccaaggcttg 900
acatacaccg gaaagcatta gagatagtgc cccccttgtg gtcggtgtac aggtggtgca 960
tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1020
tgttctgtgt tgccagcatg cccttcgggg tgatggggac tcacaggaga ctgccggggt 1080
caactcggag gaaggtgggg acgacgtcaa gtcatcatgc cccttatgtc ttgggctgca 1140
cacgtgctac aatggcaggt acaatgagct gcgaaaccgt gaggtagagc gaatctcaaa 1200
aagcctgtct cagttcggat tggggtctgc aactcgaccc catgaagtcg gagttgctag 1260
taatcgcaga tcagcattgc tgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1320
cacgtcacga aagtcggtaa cacccgaagc cggtggccca acccc 1365
Claims (9)
1. A Streptomyces sp (Streptomyces sp.) W7 strain derived from termites is characterized in that: the preservation number is GDMCC No: 61902, the preservation date is 2021, 8 and 30 days, and the preservation unit is Guangdong province microorganism culture preservation center.
2. A biocontrol product for controlling phytopathogens, which is characterized in that: comprising a fermentation extract of Streptomyces bio-control (Streptomyces sp.) W7 of termite origin as defined in claim 1 and/or of Streptomyces bio-control (Streptomyces sp.) W7 of termite origin as defined in claim 1.
3. Use of a Streptomyces sp biocontrol product as defined in claim 1 or 2 for controlling phytopathogens.
4. Use according to claim 3, characterized in that: the plant pathogenic bacteria comprise one or more of Pityrosporum ovale, Aleuritomyces parvulus, Aleuritomyces solani, Aleuritomyces capsici, Aleuritomyces papyrifera, Aleuritomyces papulosa, rotten germs of coconut stems, Aleuritomyces mango, Aleuritomyces chaenomyces, Aleuritomyces chaeta, Aleuritomyces cinerea, Aleuritomyces sinensis and Phytophthora litchi.
5. The biocontrol article for controlling phytopathogens of claim 2, wherein: the fermented extract is prepared by the following method:
(1) activating a plate of the Streptomyces biocontrol strain (Streptomyces sp.) W7 of claim 1, inoculating the plate into a TSB culture medium, and performing shaking culture at the rotating speed of 140-180 rpm at the temperature of 26-30 ℃ for 3-5 days;
(2) then inoculating the mixture into a liquid culture medium, and carrying out shaking culture at the temperature of 26-30 ℃ and the rotating speed of 140-180 rpm for 12-15 days to obtain a fermentation liquid;
(3) extracting the fermentation liquor with ethyl acetate, and concentrating the ethyl acetate extract under reduced pressure to dryness to obtain crude extract, i.e. fermentation extract.
6. The biocontrol article for controlling phytopathogens of claim 5, wherein: preparing the TSB culture medium in the step (1) according to the following proportion: 17-19 g of tryptone, 3-4 g of soybean papain hydrolysate, 4-5 g of sodium chloride, 2-3 g of dipotassium hydrogen phosphate, 2-3 g of glucose and pH 7.0-7.3 in 1L of water.
7. The biocontrol article for controlling phytopathogens of claim 5, wherein: the liquid culture medium in the step (2) is prepared according to the following proportion: in every 1L of water, 18-20 g of soluble starch, 4-5 g of peptone, 3-4 g of malt extract, 3-4 g of yeast extract, 10-13 g of glucose and CaCO3 0.5～1g，pH7.0～7.3。
8. A biological control method is characterized in that: biocontrol of plants is carried out using a fermented extract of Streptomyces biocontrol (Streptomyces sp.) W7 as defined in claim 1 and/or Streptomyces biocontrol (Streptomyces sp.) W7 as defined in claim 1.
9. The method of claim 8, wherein: the plant comprises one or more of dragon fruit, dragon boat flower, asparagus, pepper, mango, banana, coconut, pawpaw, cowpea and litchi.
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