CN107699526A - One plant of actinomycetes strain for preventing and treating gray mold and its application - Google Patents

One plant of actinomycetes strain for preventing and treating gray mold and its application Download PDF

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CN107699526A
CN107699526A CN201711157296.8A CN201711157296A CN107699526A CN 107699526 A CN107699526 A CN 107699526A CN 201711157296 A CN201711157296 A CN 201711157296A CN 107699526 A CN107699526 A CN 107699526A
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CN107699526B (en
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冯宝珍
李培谦
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Yuncheng University
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Abstract

The invention discloses one plant of actinomycetes strain for preventing and treating gray mold and its application.Entitled grey raw silk rings streptomycete (Streptomyces chungwhensis) LA 5 of weldering of the bacterial strain, China typical culture collection center is deposited on November 13rd, 2017, deposit number is CCTCC NO：M 2017683.The tunning of the actinomyces can suppress various plants disease fungus and bacterium, not only there is good inhibiting effect to fungies such as ash arrhizus bacteria, cucumber fusarium axysporum, cotton-wilt fusarium, scraping and printing unit, Rhizoctonia solani Kuhns, also there is certain bioactivity to pathogenetic bacterias such as cauliflower Bacteria erwinia, bacterial wilt of tomato bacterium.The strain culturing condition is simple, is easy to industrialized production, available for develop new and effective, wide spectrum, low toxicity, environmental protection biological pesticide, there is good development prospect.

Description

One plant of actinomycetes strain for preventing and treating gray mold and its application
Technical field
The invention belongs to crop disease control technical field, the actinomycetes strain of more particularly to one plant preventing and treating gray mold and It is applied.
Background technology
Crop gray mold is a kind of worldwide fungal disease as caused by Botrytis cinerea (Botrytis cinerea), complete State various regions generally occur.Its pathogen host range is quite varied, can infect including tomato, cucumber, capsicum, strawberry, grape Deng about 200 various crops, not only occur during plant strain growth seriously, and also occur in the storage after adopting, transportation Seriously.At present, ash arrhizus bacteria is one of most thorny issue in agricultural production to the resistance to the action of a drug of conventional sterilants.Secondly, closely The use of a little years a large amount of chemical pesticides causes the problems such as limited export of farm produce, the resistance to the action of a drug, environmental pollution, return ecological environment, Human health etc. causes serious threat.At present, it is always crop disease to develop new and effective, wide spectrum, low toxicity, the bactericide of environmental protection The difficult point and focus of evil preventing and treating.Therefore, the preventing and treating of biological source bactericide botrytis cinerea of alternative chemical bactericide is developed It is very urgent.
Actinomyces (Actinomycetes) are a kind of microorganisms with Important Economic value and practical value, and species is rich Richness, it is distributed widely in nature.Isolation of Soil Antagonistic actinomyces have the advantages that environmentally friendly, effect is lasting, with strong points, exhibition Reveal good application prospect.Therefore separation obtains the unwrapping wire for having antagonism to tomato gray mould bacterium from tomato rhizosphere soil Bacterium, the preventing and treating to the disease is significant, meets the sustainable development requirement of agricultural.
The content of the invention
The shortcomings that primary and foremost purpose of the present invention is to overcome prior art and deficiency, there is provided one plant of actinomyces, the actinomyces Zymotic fluid can suppress various plants disease fungus and bacterium, available for develop new and effective, wide spectrum, low toxicity, environmental protection biological agriculture Medicine.
It is still another object of the present invention to provide the preparation method of the tunning of described actinomyces.
A further object of the present invention is to provide obtains tunning by the fermentation process of described actinomyces.
Another object of the present invention is to provide described actinomyces and/or its tunning in terms of pathogen is suppressed Using.
The purpose of the present invention is achieved through the following technical solutions：
One plant of actinomyces, is named as grey raw silk rings streptomycete (Streptomyces chungwhensis) LA-5 of weldering, and preservation is compiled Number it is CCTCC NO：M 2017683, the Chinese Typical Representative training positioned at Wuhan, China Wuhan University is deposited on November 13rd, 2017 Support thing collection.
The bacterial strain is that isolated in the soil that Yuncheng City of Shanxi Pinglu County green house of vegetables gathers one plant of inventor is put Line bacterium.
The morphological feature of described actinomyces is：On Gause I culture medium, well-grown, bacterium colony after cultivating 10 days Flat and dark brown is presented, fibrillae of spores color black gray expandable produces coffee-like soluble pigment, and the bacterium colony back side is in brownish black；Scanning electricity Microscopic observation, spore are short cylindrical shape, and surface is smooth, chain life；About 0.6 μm of axial length, footpath is about 1.0 μm.
The physiological and biochemical property of described actinomyces：The bacterial strain can make gelatin liquefaction, can hydrolysis starch, can on cellulose Growth, produce hydrogen sulfide；Rhamnose, mannose, sucrose, D-Fructose, D- xyloses and D-Glucose can be utilized；Flesh can not be utilized Alcohol, arabinose, sorbierite, raffinose and mannitol.
The 16S rDNA gene expression characteristicses of described actinomyces：Its 16S rDNA sequence such as SEQ ID No.1 nucleotides sequence Shown in row, sequence length 1391bp.
Through morphological feature, cultural characteristic observation and molecular biology, (bacterial strain extracts DNA after culture, expands and determines 16S rDNA sequence, phylogenetic tree construction analysis) research identification, the bacterial strain is belonged into the grey raw silk rings streptomycete of weldering (Streptomyces chungwhensis)。
The preparation method of described actinomycete fermentation product, comprises the following steps：
(1) preparation of seed liquor：Actinomyces after activation are inoculated in fermentation medium, 160~180rpm is trained at 28 DEG C 48~60h is supported, obtains seed liquor；
(2) fermented and cultured：Fermentation medium, bottling amount 60mL/250mL, in 28 are inoculated in 10% (v/v) inoculum concentration DEG C, shaking table culture 7 days under the conditions of 180r/min, obtain described actinomycete fermentation product.
Described fermentation medium is：Soluble starch 20g, dusty yeast 1g, NaCl 0.5g, K2HPO40.5g, MgSO4 0.5g, FeSO40.01g, water 1000mL, pH 7.2~7.4.
The mode of described activation is preferably：Under the conditions of 28 DEG C~30 DEG C, in Gause I medium culture, until bacterium Silk and spore growth are ripe.
The formula of described Gause I culture medium is preferably：KNO31g, K2HPO40.5g, MgSO40.5g, NaCl 0.5g, FeSO40.01g, soluble starch 20g, agar powder 15g, distilled water 1000mL, pH 7.2~7.4；In 115~121 DEG C sterilizing 30min.
The tunning being prepared by described preparation method.
Described actinomyces and/or its tunning have the bioactivity for suppressing various plants pathogen.
Described phytopathogen includes plant pathogenic fungi and/or plant pathogenetic bacteria.
Described pathogen of crop includes ash arrhizus bacteria (Botrytis cinerea), cucumber fusarium axysporum (Fusarium oxysporum f.sp.cucumerinum), scraping and printing unit (Pythium aphanidermatum), capsicum Phytophthora (Phytophthora capsici), cotton wilt (Fusarium oxysporum f.sp.vasinfectum), Apple anthrax bacteria (Colletotrichum gloeosporioides), corn leaf spot bacterium (Mycosphaerella Maydis), Rhizoctonia solani Kuhn (Rhizoctonia solani), bacterial wilt of tomato bacterium (Pseudomanas Solanacearum), cauliflower Bacteria erwinia (Erwinia carotovora subsp.carotovora), the black shin of potato Germ (Erwinia carotovora subsp.atroseptica), bacillary gummosis germ (Pseudomonas Syringae pv.syringae) in one kind or at least two.
The application of described actinomyces and/or its tunning in plant disease microorganism bactericide is prepared.
A kind of microbial bactericide of controlling plant diseases, contain described actinomyces and/or its tunning.
The application method of described microbial bactericide is preferably to spray.
Described plant disease is preferably gray mold.
The present invention is had the following advantages relative to prior art and effect：
(1) present invention provides a kind of new actinomycetes strain LA-5.The bacterial strain has a broad antifungal spectrum, to various plants disease fungus And bacterium has certain inhibitory action.The bacterial strain fermentation liquor is not only to ash arrhizus bacteria, cucumber fusarium axysporum, cotton wilt The fungies such as bacterium, scraping and printing unit, Rhizoctonia solani Kuhn have good inhibiting effect, also to cauliflower Bacteria erwinia, bacterial wilt of tomato The pathogenetic bacterias such as bacterium have certain bioactivity.The bacterial strain efficient, wide spectrum, low toxicity, the microorganism of environmental protection can kill as development The biomaterial of microbial inoculum.
(2) there is the preparation condition of the zymotic fluid of bactericidal activity present invention also offers bacterial strain LA-5.Fermentation condition：28 DEG C, 180r/min, cultivate 7d；Culture medium forms：Soluble starch 20g, dusty yeast 1g, NaCl 0.5g, K2HPO40.5g, MgSO40.5g, FeSO40.01g, water 1000mL, pH 7.2~7.4.
(3) biocontrol actinomycetes LA-5 of the present invention is isolated from green house of vegetables tomato rhizosphere soil, compatible with soil ecology harmony, Be advantageous to give full play to the advantage of bacterial strain.The biocontrol actinomycetes strain culturing condition is simple, is easy to industrialized production, has good Development prospect.
Brief description of the drawings
Fig. 1 is actinomycetes strain LA-5 of the present invention colonial morphology figure.
Fig. 2 is actinomycetes strain LA-5 spores photo figure of the present invention；Wherein, A is the spore under light microscope Form, B are spore shape under ESEM.
Fig. 3 is actinomycetes strain LA-5 of the present invention to tomato gray mould bacterium flat board fungistatic effect figure.
Fig. 4 is actinomycetes strain LA-5 zymotic fluids of the present invention to tomato gray mould bacterium inhibition figure.
Fig. 5 is actinomycetes strain LA-5 of the present invention and systematic growth of the related strain according to 16S rDNA sequence constructs Tree graph.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited In this.
Used medium formula is as follows in embodiment：
Gause I culture medium：KNO31g, K2HPO40.5g, MgSO40.5g, NaCl 0.5g, FeSO40.01g, can Soluble starch 20g, agar powder 15g, distilled water 1000mL, pH 7.2~7.4；
Czapek's medium：Sucrose 30g, NaNO3 2g、K2HPO4 1g、FeSO40.01g, agar 15g, MgSO4·7H2O 0.5g, KCl 0.5g, distilled water 1000mL, pH 7.2~7.4；
Glucose asparagine agar medium：Glucose 10g, agar 15g, K2HPO40.5g, asparagine 0.5g, steam Distilled water 1000mL, pH 7.2~7.4；
Starch ammonium agar medium：(NH4)2SO4 2g、CaCO3 3g、K2HPO41g, starch 10g, MgSO41g, agar 15g, NaCL 1g, distilled water 1000mL；
Glucose yeast cream culture medium：Glucose 10g, yeast extract 10g, beef extract 4g, peptone 4g, NaCL 2.5g, steam Distilled water 1000mL；
PDA culture medium：Potato leaching juice (takes 200g potatos to be cut into small pieces, puts and boil 1 hour in 1000mL water, then mistake Leaching filtrate, then be diluted with water to cumulative volume 1000mL), agar 15g, glucose 10g, pH value 7.2~7.4；
Oat-agar cultures base：Oatmeal 20g, mark amount salting liquid 1mL (MgSO4·7H2O 0.1g、MnCl2·4H2O 0.1g、ZnSO4·7H2O 0.1g, distilled water 1000mL), agar 18%, be supplemented to 1000mL with distilled water；
NA culture mediums：Beef extract 3g, yeast extract 1g, peptone 5g, glucose 10g, agar 15g distilled water are settled to 1000mL, pH are 7.2~7.4.
The bacterial strain LA-5 of embodiment 1 separation and identification
1. the collection of pedotheque
Rhizosphere soil from Yuncheng Pinglu County greenhouse for planting vegetable collection tomato plot 5~10cm depths, Dispense and laboratory is taken back after marking, it is stand-by after natural air drying.
2. the separation of actinomyces
Separated using plate dilution method.Air-dried soil sample is ground with mortar, 10g is weighed and pours into equipped with 90mL sterilized waters Triangular flask in, 28 DEG C, 180r/min concussion 30min after stand 5 minutes, obtain stoste.Take stoste 1mL and dilute with 10 times of gradients Release, be configured to 10 respectively-2、10-3、10-4、10-5、10-6Suspension, each 0.2mL of suspension for drawing various concentrations is added to height Family name's No.1 culture medium (K containing 30mg/L2GrO7) on flat board, 28 DEG C of culture observations are placed in after even spread, picking is not after 5~7 days Same single bacterium colony line purifying.Bacterial strain after purification is transferred on Gause I slant medium and cultivated, 4 DEG C save backup, Isolate to obtain 15 plants of actinomyces.By the plate antagonism and Bactericidal test in embodiment 3, screening obtains having extensive antibacterial The actinomyces strain LA-5 of spectrum.
3. bacterial strain LA-5 identification
(1) morphological feature is observed
Bacterial strain LA-5 well-grown (being shown in Table 1) on most of culture mediums, the bacterium colony flat on Gause I culture medium, Dark brown, fibrillae of spores color black gray expandable, culture produce coffee-like soluble pigment (Fig. 1) after 10 days, and the bacterium colony back side is in brownish black；Light Learn under microscope that base silk is not broken, aerial hyphae forms fibrillae of spores, fibrillae of spores is straight, flexible；Observed under ESEM, spore is Short cylindrical shape, surface is smooth, chain life；About 0.6 μm of axial length, footpath is about 1.0 μm (Fig. 2).
The cultural characteristic of the LA-5 bacterial strains of table 1
Note:+++ represent that growth is fine, ++ well-grown is represented ,+represent to grow
(2) physiological and biochemical property
Gelatin liquefaction, starch water with reference to Lechevalie method (Lechevalier et al, 1980) measure bacterial strain The characteristics such as the solidification of solution, milk is with peptonizing, cellulose hydrolysis, utilization of carbon source.
The bacterial strain can make gelatin liquefaction, can hydrolysis starch, can grow on cellulose, produce hydrogen sulfide；Sandlwood can be utilized Sugar, mannose, sucrose, D-Fructose, D- xyloses and D-Glucose；Inositol, arabinose, sorbierite, raffinose can not be utilized And mannitol.
(3) Molecular Identification is carried out to bacterial strain LA-5 using 16S rDNA gene sequencing methods
16S rDNA sequencings commission Shanghai Sheng Gong bioengineering limited company is carried out.
Specific sequence measurement is as follows：Genomic DNA is extracted from new fresh thalli with lysozyme Method, using universal primer (27F And 1492R) 16S rDNA amplifications are carried out, PCR primer is sequenced afterwards after testing, and 16S rDNA sequences are as follows：
GCTCCTCCCCACAAGGGGGTTGGGCCACCGGCTTCGGGTGTTACCGACTTTCGTGACGTGACGGGCGGT GTGTACAAGGCCCGGGAACGTATTCACCGCAGCAATGCTGATCTGCGATTACTAGCAACTCCGACTTCATGGGGTCG AGTTGCAGACCCCAATCCGAACTGAGACAGGCTTTTTGAGATTCGCTCCGCCTCGCGGCTTCCCAGCTCATTGTACC TGCCATTGTAGCACGTGTGCAGCCCAAGACATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCGAGTTG ACCCCGGCAGTCTCCTGTGAGTCCCCATCACCCCGAAGGGCATGCTGGCAACACAGAACAAGGGTTGCGCTCGTTGC GGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCACCCGACCACAAGGGGGGCCG TATCTCTACGGCTTTCCGGGCGATGTCAAGCCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAGCCACATGCTCCG CTGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGAACTTAATGCG TTAGCTGCGGCACCGACGACGTGGAATGTCGCCAACACCTAGTTCCCACCGTTTACGGCGTGGACTACCAGGGTATC TAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTAATGGCCCAGAGATCCGCCTTCGCCACCGGTGTTC CTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCGATCTCCCCTACCACACTCTAGCCTGCCCGTATCGA CTGCAGACCCGGGGTTAAGCCCCGGGCTTTCACAACCGACGTGACAAGCCGCCTACGAGCTCTTTACGCCCAATAAT TCCGGACAACGCTTGCGCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGCGCTTCTTCTGCAGGTACCG TCACTTGCGCTTCTTCCCTGCTGAAAGAGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAG GCTTTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCC GGTCGCCCTCTCAGGCCGGCTACCCGTCGTCGCCTTGGTGAGCTTCTACCTCACCAACTAGCTGATAGGCCGCGGGC TCATCCTGCACCGCCGGAGCTTTCAACCTTCTCCCATGCGAGAGAAAGTGTCATCCGGTATTAGACCCCGTTTCCAG GGCTTGTCCCAGAGTGCAGGGCAGATTGCCCACGTGTTACTCACCCGTTCGCCACTAATCCACCCCGAAGGGCTTCA TCGTTCGACTGCA
Bacterial strain LA-5 16S rDNA complete nucleotide sequences overall length is 1391bp.Gained sequence is submitted to GenBank numbers BLAST analyses and comparison are carried out according to storehouse, finds to belong to streptomyces with the higher bacterial strain of LA-5 homologys, chooses 12 typical bacterium The 16Sr DNA sequence dnas software building phylogenetic trees of MEGA 6.0 of strain.As a result show, bacterial strain LA-5 is with welding grey raw silk rings strepto- Bacterium Streptomyces chungwhensis belong to same branch (Fig. 5).
Grey raw silk rings streptomycete (Streptomyces chungwhensis) LA-5 is welded into bacterial strain LA-5 names, the bacterial strain in It is preserved on November 13rd, 2017 and is positioned at the Chinese Typical Representative biological deposits center of Wuhan, China Wuhan University, deposit number CCTCC NO：M 2017683.
The fermentation of the LA-5 bacterial strains of embodiment 2
(1) above-mentioned bacterial strains are inoculated in Gause I slant medium, cultivated under the conditions of 28 DEG C~30 DEG C, until mycelia It is ripe with spore growth；
(2) mycelia on inclined-plane and spore are accessed into fermentation medium, 160~180rpm cultures, 48~60h is obtained at 28 DEG C To actinomyces LA-5 seed liquors；
Described fermentation medium forms：Soluble starch 20g, dusty yeast 1g, NaCl 0.5g, K2HPO40.5g, MgSO40.5g, FeSO40.01g, water 1000mL, pH 7.2~7.4.
(3) actinomyces LA-5 fermentation：Inoculum concentration 10% (v/v), bottling amount 60mL/250mL, 28 DEG C, 180r/min, training 7d is supported, concentration is made as 108CFU/mL LA-5 bacterial strain fermentation liquors, the same step of fermentation medium (2).
The bacterial strain LA-5 of embodiment 3 Antibacterial Activity
Using flat board opposite culture method, bacterial strain LA-5 is made into antagonism measure to disease fungus, first in PDA culture medium flat board Disease fungus 7mm bacteria cakes to be measured are accessed in center, and uniformly point connects bacterial strain actinomyces bacteria cake at the 2cm of bacterium colony periphery, is trained at 26 DEG C Support 3 days after check fungistatic effect, test result indicates that bacterial strain LA-5 to botrytis cinerea (bacterial strain document " Wang Meiqin, He Yunchun, Liu Huiping, wait diseases prevention Mechanism Study [J] the Ecology in China of endogenous cycles shape bacillus Jcxy8 to graw mold of tomato Agriculture journal, 2010,18 (1):Disclosed in 98-101. "), (bacterial strain is in document for cucumber fusarium axysporum “FocVel1influences asexual production,filamentous growth,biofilm formation, and virulence in Fusarium oxysporum f.sp.cucumerinum.Frontiers in Plant Science,2015,6:Disclosed in 312. "), (bacterial strain is in document " Jiashi's muskmelon samping off biological and ecological methods to prevent plant disease, pests, and erosion unwrapping wire for scraping and printing unit The screening Journal of Northwest Sci Tech University of Agriculture and Forestry (natural science edition) of bacterium, 2009,37 (05):Disclosed in 144-148 "), capsicum epidemic disease (bacterial strain is in document " Characterization of necrosis-inducing NLP proteins in for mould Phytophthora capsici.BMC Plant Biology,2014,14(1):Disclosed in 126 "), cotton wilt (bacterium In document, " it is big that the biocontrol microorganisms disease prevention growth-promoting to cotton wilt mixed with chemical fertilizer and bactericide acts on northwests agriculture and forestry science and technology for strain Learn journal (natural science edition), 2016,44 (07):Disclosed in 165-172. "), (bacterial strain is in document for apple anthrax bacteria " genetic transformation and transformant the mirror Scientia Agricultura Sinicas of agriculture bacillus mediated apple anthrax bacteria, 2013,46 (09):1799- Disclosed in 1807. "), (in document, " bacillus amyloliquefaciens B9601-Y2 is to corn leaf spot for the bacterial strain for corn leaf spot bacterium Preventing and treating effect Maize Sciences, 2017,25 (02):Disclosed in 130-135. "), (bacterial strain is in " one plant of document for Rhizoctonia solani Kuhn The separation identification of anti-maize sheath blight endogenetic bacteria and its disease-resistant growth-promoting functions microbiologies circular, 2008, (08):1240- Disclosed in 1245. ") there is preferable antagonism；By taking tomato gray mould bacterium as an example, fungistatic effect is as shown in Figure 3.
Then the inhibitory action using filter paper strip method measure LA-5 bacterial strain fermentation liquors to tomato gray mould bacterium.By 50mL fermentations 7 My god, 108CFU/mL LA-5 bacterial strain fermentation liquors (embodiment 2 is made) are placed in 50mL centrifuge tubes, in 8000r/min centrifuging and takings Clearly, it is then degerming with (0.22 μm) suction filtration of miillpore filter, without fermented liquid is obtained, it is standby to be placed in 4 DEG C of preservations in sterilization container.With hair Zymotic fluid infiltrates aseptic filter paper bar, is symmetrically attached to after fully drying in PDA culture medium, culture dish home position inoculating tomato botrytis cinerea Bacteria cake, control is used as using sterilized water.Found after 3 days, experimental group bacterium colony is long and narrow, growth retardation, and control group has no significant effect (figure 4)。
Determination of activity for pathogenetic bacteria：Strains tested includes bacterial wilt of tomato bacterium, and (in document, " tomato is blue or green for the bacterial strain The screening Plant Pathologies of rot Endophytic antagonistic bacteria, 2003, (04):Disclosed in 364-367. "), cauliflower Bacteria erwinia (bacterial strain document " the screening Plant Pathologies of Erwinia Carotovora Pv. carotovora carrot subspecies motility mutant, 2000,(02):Disclosed in 176-180. "), (bacterial strain is in the document " disease of Guangdong blakleg of potato for blakleg of potato bacterium Original identification Plant Pathologies, 2015,45 (05):Disclosed in 449-454. "), bacillary gummosis germ (Pseudomonas Syringae pv.syringae, the bacterial strain " Xu Li, Wang Jiawei, Chen Xin, wait point of sweet cherry gummosis pathogens in document Son identification and pathogenic detection [J] Plant Pathologies, 2015,45 (4):Disclosed in 350-355. ", that is, correspond in the document Bacterial strain P.syringae pv.syringae).Draw in the NA culture mediums of 50 DEG C or so of 1mL pathogenetic bacterias suspension addition and shake It is even, pour into culture dish rapidly, treat culture medium solidifying.Sterile lotion will be dipped after diameter about 6mm filter paper sterilizing, dry patch Filter paper 5 can be pasted on culture medium, in each ware, is repeated 3 times, not add the filter paper of any filtrate to be used as blank control. 30 DEG C of cultures, 1~2d is cultivated, the inhibition zone and record size that observation edge clear occurs, test result indicates that LA-5 zymotic fluids To having certain inhibitory activity (table 2) for examination pathogenetic bacteria.
The actinomyces LA-5 zymotic fluid fungistatic effects of table 2
The bacterial strain LA-5 of embodiment 4 bioactivity and its application
1. using mycelial growth rate method (Mu Liyi, 1994), will trained in 26 DEG C of incubators for examination disease fungus bacterial strain Support after being cultivated 5~7 days on base flat board, beaten with card punch in colony edge and take diameter 7mm bacteria cakes.Take above-mentioned without fermented liquid according to 1:19 ratio is added in 50 DEG C of PDA culture medium, and culture dish is poured into mixing.Pathogen bacteria cake is inoculated with after cooling, to be not added with sending out It is control for examination disease fungus to be grown on the PDA plate of zymotic fluid.Each processing is repeated 4 times, and 26 DEG C are cultivated 5 days, treat control group When bacterium colony covers with flat board, colony diameter is measured with crossing method, according to formula A, the average mycelial growth that B calculates bacterial strain suppresses Rate, as a result as shown in table 3.
(A) colony diameter (mm)=measurement diameter-bacteria cake diameter
(B) mycelial growth inhibition rate=[(control colony diameter-processing colony diameter)/compare colony diameter] ×
100%
The LA-5 bacterial strain fermentation liquors of table 3 are to 8 kinds of disease fungus fungistatic effects
2. bacterial strain LA-5 zymotic fluids are to the prevention effect of graw mold of tomato
Prevention effect of the LA-5 zymotic fluids to graw mold of tomato is determined using rules of organization.
First group of experiment：Choose that diameter about 5cm, size be similar, tamato fruit 30 of healthy growth, it is small to be equally divided into 3 Group, respectively first with LA-5 bacterial strains without fermented liquid, 2% 1500 times of liquid of (active ingredient) propamidine aqua, 50% (active ingredient) 4000 times of liquid of fludioxonil wettable powder as protective agent to tomato spraying treatment, spray amount with just uniformly, do not produce dropping liquid It is advisable.With 10 after drying6Individual/mL tomato gray mould bacterium spore suspension inoculation minimally invasive wound of fruit, inoculum concentration are uniform to spray Not producing dropping liquid is advisable.
Second group of experiment：Reverse inoculation and spraying order, is first inoculated with 106Individual/mL pathogens spore suspension is after 24 hours The zymotic fluid and agricultural chemicals for spraying above-mentioned same concentration using sterilized water as control (CK), set three as treatment group, two groups of experiments Individual repetition, measured after 5 days and record lesion area, calculate preventive effect.As a result as shown in table 4, show bacterial strain LA-5 zymotic fluids to kind Solanum cinerea has preferable prevention effect.
Protection and treatment preventive effect of the LA-5 zymotic fluids of table 4 to tomato gray mould bacterium
Shown according to the present embodiment result, bacterial strain LA-5 of the present invention passes through liquid fermentation and culture, in its zymotic fluid Containing the material for suppressing common crops pathogen, to botrytis cinerea and part soil-borne fungus, (such as cucumber fusarium axysporum, melon and fruit corruption is mould Bacterium, Rhizoctonia solani Kuhn, cucumber fusarium axysporum) percentage mycelial inhibition more than 80%, can significantly mitigate the generation of disease, be expected to out Hair turns into a kind of microbial reagent of new preventing and treating corps diseases.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.
Sequence table
<110>Yuncheng Univercity
<120>One plant of actinomycetes strain for preventing and treating gray mold and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1391
<212> DNA
<213>Weld grey raw silk rings streptomycete (Streptomyces chungwhensis)
<400> 1
gctcctcccc acaagggggt tgggccaccg gcttcgggtg ttaccgactt tcgtgacgtg 60
acgggcggtg tgtacaaggc ccgggaacgt attcaccgca gcaatgctga tctgcgatta 120
ctagcaactc cgacttcatg gggtcgagtt gcagacccca atccgaactg agacaggctt 180
tttgagattc gctccgcctc gcggcttccc agctcattgt acctgccatt gtagcacgtg 240
tgcagcccaa gacataaggg gcatgatgac ttgacgtcgt ccccaccttc ctccgagttg 300
accccggcag tctcctgtga gtccccatca ccccgaaggg catgctggca acacagaaca 360
agggttgcgc tcgttgcggg acttaaccca acatctcacg acacgagctg acgacagcca 420
tgcaccacct gtcacccgac cacaaggggg gccgtatctc tacggctttc cgggcgatgt 480
caagccttgg taaggttctt cgcgttgcgt cgaattaagc cacatgctcc gctgcttgtg 540
cgggcccccg tcaattcctt tgagttttag ccttgcggcc gtactcccca ggcggggaac 600
ttaatgcgtt agctgcggca ccgacgacgt ggaatgtcgc caacacctag ttcccaccgt 660
ttacggcgtg gactaccagg gtatctaatc ctgttcgctc cccacgcttt cgctcctcag 720
cgtcagtaat ggcccagaga tccgccttcg ccaccggtgt tcctcctgat atctgcgcat 780
ttcaccgcta caccaggaat tccgatctcc cctaccacac tctagcctgc ccgtatcgac 840
tgcagacccg gggttaagcc ccgggctttc acaaccgacg tgacaagccg cctacgagct 900
ctttacgccc aataattccg gacaacgctt gcgccctacg tattaccgcg gctgctggca 960
cgtagttagc cggcgcttct tctgcaggta ccgtcacttg cgcttcttcc ctgctgaaag 1020
aggtttacaa cccgaaggcc gtcatccctc acgcggcgtc gctgcatcag gctttcgccc 1080
attgtgcaat attccccact gctgcctccc gtaggagtct gggccgtgtc tcagtcccag 1140
tgtggccggt cgccctctca ggccggctac ccgtcgtcgc cttggtgagc ttctacctca 1200
ccaactagct gataggccgc gggctcatcc tgcaccgccg gagctttcaa ccttctccca 1260
tgcgagagaa agtgtcatcc ggtattagac cccgtttcca gggcttgtcc cagagtgcag 1320
ggcagattgc ccacgtgtta ctcacccgtt cgccactaat ccaccccgaa gggcttcatc 1380
gttcgactgc a 1391

Claims (10)

1. one plant of actinomyces, it is characterized in that：It is named as grey raw silk rings streptomycete (Streptomyces chungwhensis) LA- of weldering 5, deposit number is CCTCC NO：M 2017683, it is deposited on November 13rd, 2017 in Wuhan, China Wuhan University State's Type Tissue Collection.
2. the tunning preparation method of the actinomyces described in claim 1, it is characterised in that comprise the following steps：
(1) preparation of seed liquor：Actinomyces after activation are inoculated in fermentation medium, 160~180rpm cultivates 48 at 28 DEG C ~60h, obtains seed liquor；
(2) fermented and cultured：Fermentation medium is inoculated in 10% (v/v) inoculum concentration, bottling amount 60mL/250mL, in 28 DEG C, Shaking table culture 7 days under the conditions of 180r/min, obtain described actinomycete fermentation product.
3. the tunning preparation method of actinomyces according to claim 2, it is characterised in that described fermentation medium For：
Soluble starch 20g, dusty yeast 1g, NaCl 0.5g, K2HPO40.5g, MgSO40.5g, FeSO40.01g, water 1000mL, pH 7.2~7.4.
4. the tunning preparation method of actinomyces according to claim 2, it is characterised in that the mode of described activation For：
By described actinomyces under the conditions of 28 DEG C~30 DEG C, in Gause I medium culture, until mycelia and spore growth It is ripe.
A kind of 5. tunning, it is characterised in that：
It is prepared by the tunning preparation method described in any one of claim 2~4.
6. the application of the actinomyces and/or its tunning described in claim 1, it is characterised in that：
Described application is the application in plant disease microorganism bactericide is prepared.
7. the application of actinomyces according to claim 6 and/or its tunning, it is characterised in that：
Described plant disease is by the microbial plant disease of pathogenic.
8. the application of actinomyces according to claim 7 and/or its tunning, it is characterised in that：
Described phytopathogen includes plant pathogenic fungi and/or plant pathogenetic bacteria.
A kind of 9. microbial bactericide of controlling plant diseases, it is characterised in that：
The microbial bactericide of described controlling plant diseases contains the actinomyces and/or its tunning described in claim 1.
10. the microbial bactericide of controlling plant diseases according to claim 9, it is characterised in that：
The application method of described microbial bactericide is spraying.
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CN108728378A (en) * 2018-05-30 2018-11-02 四川大学 One plants endogenetic actinomyces and its application
CN111363696A (en) * 2020-02-25 2020-07-03 东北农业大学 Streptomyces and screening method and application thereof
CN115927038A (en) * 2022-07-06 2023-04-07 西北农林科技大学 Streptomyces strain and application thereof in prevention and treatment of plant pathogenic fungi
CN116376708A (en) * 2022-12-05 2023-07-04 青岛农业大学 Cladosporium fungus and application thereof

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108728378A (en) * 2018-05-30 2018-11-02 四川大学 One plants endogenetic actinomyces and its application
CN111363696A (en) * 2020-02-25 2020-07-03 东北农业大学 Streptomyces and screening method and application thereof
CN111363696B (en) * 2020-02-25 2023-05-23 东北农业大学 Streptomyces, screening method and application thereof
CN115927038A (en) * 2022-07-06 2023-04-07 西北农林科技大学 Streptomyces strain and application thereof in prevention and treatment of plant pathogenic fungi
CN115927038B (en) * 2022-07-06 2024-04-30 西北农林科技大学 Streptomyces strain and application thereof in plant pathogenic fungi control
CN116376708A (en) * 2022-12-05 2023-07-04 青岛农业大学 Cladosporium fungus and application thereof
CN116376708B (en) * 2022-12-05 2024-06-07 青岛农业大学 Cladosporium fungus and application thereof

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