CN101760438B - Broad-spectrum antifungal plant endophytic bacillus subtillis and application thereof - Google Patents

Abstract

The invention relates to bacillus subtillis R31 with broad-spectrum antagonism capacity on plant pathogenic fungi, and in particular provides a bacterial strain (CCTCC NO: M209261) of the Bacillus subtillis R31 as well as growth inhibition action thereof on the plant pathogenic fungi and especially capacity thereof on the biological prevention and control of banana vasicular wilt. The plant endophytic bacillus subtillis is proved to be new bacillus subtillis through obtained 16S rDNA and gyrA and gyrB gene segments as well as BLAST search and comparison on NCBI (National Center of Biotechnology Information) and has better flat antagonism activity on multiple plant pathogenic fungi, better antagonism capacity especially on various soil-borne and vascular bundle diseases, i.e. fusarium oxysporum, rhizoctonia solani, alternaria, and the like, and better prevention and control effect in greenhouses and fields.

Description

One broad-spectrum antifungal plant endophytic bacillus subtillis and application thereof

Technical field

The present invention relates to a strain has the wide spectrum antagonistic ability to plant pathogenic fungi Bacillus subtillis R31; Specifically provide Bacillus subtillis R31 (Bacillus subtilis R31) bacterial strain (CCTCC NO:M 209261) and to the growth-inhibiting effect of plant pathogenic fungi; Especially the ability aspect the banana blight biological control, it belongs to the category of biological pesticide, anti-disease microbial fertilizer.

Background technology

The due to illness harmful loss that causes of garden crops such as China's fruit, vegetables is very serious; Result according to the ornamental plant disease and pest generaI investigation of the 1984-1986 whole nation; On 1256 kinds of ornamental plants, find 5508 kinds of Plant diseasess; On average every kind of plant 4.4 kinds, the disease of garden crops such as vegetables, flowers, fruit tree takes place serious more than large cereal class farm crop.According to agricultural technology extension center, China Ministry of Agriculture whole nation statistics, China farm crop every year, due to illness the Chinese caterpillar fungus mouse was caused a large amount of losses, and wherein fruit, vegetables are far longer than grain (10%) and cotton (15%-20%) with a toll of more than 25%.Especially in recent years; Adjustment along with development of rural economy and agricultural structure; Increasing peasant is engaged in production and the cultivation of garden crop, because long-term crop rotation on limited soil, soil-borne disease, vascular bundle diseases have had a strong impact on agriculture sound development.

Along with the agricultural sustainable development implementation, people pay attention to the dangerous of chemical pesticide and harm gradually, therefore, meet the theme that environmental protection, health, efficient, safety, eco-friendly biological pesticide become current ecological agriculture research.The biological pesticide product has surpassed kind more than 100 in the world at present, but is microbial pesticide more than 90%.It will be microbial bactericide that second important commercial biological pesticide of scholarly forecast arranged.

Bacillus subtillis is a kind of gram positive bacterium that is distributed widely in the various different living environments; The born of the same parents of sprouting in can producing, at the surperficial ubiquity of soil and plant, still common a kind of endophyte in the plant materials simultaneously; To the person poultry harmless, free from environmental pollution.Its fast growth, nutritional needs are simple, be easy to survival on the surface of plant, grow surely and with breeding, and it is simple to produce the technology of Bacillus subtillis preparation, preparation stabilization is used the aspect, storage period is long, is a kind of ideal biological and ecological methods to prevent plant disease, pests, and erosion mikrobe.

Bacillus subtillis is mainly through five kinds of different approaches performance effects.At first; Bacillus subtillis can grow on host plant surely; Yi Youjin has studied the problem that Bacillus subtillis B-001 bacterial strain is grown through natural aperture and wound invasion host plant surely, and Mu Changqing etc. have studied Bacillus subtillis B-332 deciding on paddy rice and grown and the relation of preventing and treating rice blast.Secondly, Bacillus subtillis can produce antimicrobial substance through the non-ribosomal approach, like lipopeptide antibiotic, polypeptide antibiotics etc., like iturin family, surfactivity element, fragrant subtilyne etc. and the unknown cyclic peptide microbiotic of some results.Once more, Bacillus subtillis can also be adsorbed on the mycelia of pathogenic bacteria, and grows along with the growth of mycelia, produces the bacteriolyze material, cause the fungi protoplasma to reveal after, make cell walls or the membranolysis of fungi, cause cell phenomenons such as perforation, deformity to occur.Lin Fucheng produces bacteriolysis from the isolating subtilis S9 of sugarcane rhizosphere to dry thread Pyrenomycetes; Gao Xuewens etc. have carried out separation and purification to the outer material of born of the same parents that the bacillus subtilis B 2 bacterial strain produces, and suppress its sprouting thereby the antimicrobial substance of acquisition can make the conidium of fusarium graminearum that distortion takes place.Once more; Bacillus subtillis can produce system's resistance through inducing plant and play a role; The Li De congruence is carried out mutagenesis to Bacillus subtillis Bs-916; And find that Bs-916 and mutant strain excretory active substance thereof have the induction of resistance effect to paddy rice, make disease-resistant enzyme such as paddy rice body endoperoxide enzyme live and strengthen.Once more, Bacillus subtillis can have growth-promoting functions like discovery plant endogenesis Bacillus subtillis BS-2 such as Cai Xueqing to Plantula Brassicae chinensis, and show resistance against diseases through promoting that plant-growth reaches disease-resistant effect.

According to the summary of Jiang Lili etc., the existing 4 strain Bacillus subtillis biocontrol strains (QST713, MB1600, GB03 and FZB24) of the U.S. obtain EPA commercialization or limited commercialization production application permission.Wherein QST713 trade(brand)name Serenade obtains that the alimentary crop disease uses examining and approves, can prevent and treat the bacterium and the fungus-caused diseases such as Powdery Mildew, root rot, oidium, eqpidemic disease, gray mold of vegetables, cherry, tomato, grape and English walnut various crop through foliage applying.GB03 trade(brand)name Kodiak can directly be applied to soil or seed dressing is used to prevent the fungal disease by caused root of sickle-like bacteria, aspergillus tubigensis, Alternaria and Rhizoctonia and seed such as cotton, beans and peanut.MB1600 trade(brand)name Subtilex, the leaf portion and the root disease that are used to prevent and treat gray mold, Powdery Mildew and cause, FZB24 trade(brand)name Taegro by Rhizoctonia, Fusarium, Aspergillus ^TMBe applied to greenhouse or indoor cultivation sapling, shrub and decorative plant root and can prevent and treat root rot and the blight that sickle-like bacteria and rhizoctonia cause; Also can be used to prevent and treat tomato late blight, gray mold, wheat powdery mildew and plant Verticillium, can also be as the raising the output growth-promoting agent.China has succeeded in developing at present and the Bacillus subtillis commodity preparation that comes into operation has hundred anti-, wheat Fengning, line Qu Ning, inferior treasured etc.Hundred anti-effective constituents are subtilis B908; Mechanism of action is that nutrient competition, site are captured; Crop to easy trouble soil diseases such as tobacco, pseudo-ginseng, flowers, wheat, Chinese cabbages has good control effect, and the control effect of rice sheath blight disease is reached more than 70%.The wheat Fengning is the living body biological sterilant that utilizes Bacillus subtillis B3 bacterial strain to process, the main antimicrobial substance that produces of its diseases prevention mechanism.

Also have relevant work to carry out aspect the Bacillus subtillis control banana blight utilizing, but still be in bacterial strain screening, evaluation and indoor flat plate antagonistic activity and measure, minority has been carried out potted plant experiment, does not also have the report of field experiment.The just happy and auspicious king's center of percussion of grandson separates a strain from the banana rhizosphere soil have the Bacillus licheniformis C4 of control effect to banana blight, in the greenhouse, shows good preventive effect.Yin Xiaomin etc. separate in the banana caulo and obtain the Bacillus subtillis that a strain is numbered B215, and several kinds of banana pathogenic bacteria mycelial growths and spore germination to banana blight and other on flat board have the inhibition effect.The inhibition ability that Fu Yeqin etc. grow to banana blight at differing temps, pH value and substratum bottom fermentation liquid to living Bacillus subtillis BEB2 in the banana; And under the condition of artificial inoculation; Studied in 2 months BEB2 to the potted plant preventive effect of No. 4 physiological strains of banana blight, the preventive effect of inoculation BEB2 reaches 98% earlier.Sun Zhengxiang etc. separate acquisition one strain Bacillus subtillis S-1 from the banana rhizosphere soil; To comprising that a collection of pathogenic fungies such as banana blight, tomato wilt have the wide spectrum antagonistic activity; And measuring the righttest pH value of this bacterial strain is 7.0 ~ 8.0; 26 ~ 30 ℃ of the righttest culture temperature, the righttest substratum is PYTG or PDA.Yellow little light etc. has been measured the inhibition effect of biocontrol microorganisms such as Bacillus subtillis, Pseudomonas fluorescens to the banana blight bacteria mycelia.Yang Xiujuan etc. utilize rifampicin resistance bacillus T2WF and W10 bacterial strain carried out bacillus in the banana body with the research of rhizosphere colonization.Above-mentioned all relate in the Bacillus subtillis bacterial strain of banana blight biological control, still do not have bacterial strain and carried out daejeon prevention test, and lack the preventive effect of daejeon prevention test.

In addition, Bacillus subtillis can produce various enzymes, microbiotic, tensio-active agent, be widely used with commercial, industrial production and agriculture prodn in (Fritze, 2004), in biocontrol of plant disease, also be widely used (Zhao Da etc., 2007; Jiang Lili etc., 2009).The kind of Bacillus subtillis group comprises Bacillus subtilis subsp.subtilis (Smith et al., 1964; Nakamura et al., 1999), Bacillus lichenifromis (Skerman et al., 1980); Bacillus atrophaeus (Nakamura, 1989), Bacillusmojavensis (Robert et al., 1994); Bacillus vallismortis (Roberts et al., 1996), Bacillus subtilis subsp.spizizenii (Nakamura et al., 1999); Bacillus sonorensis (Palmisano et al., 2001), Bacillus subtilis subsp.inaquosorum (Rooney et al., 2009); The sibship of these kinds is very near, can make a distinction through fatty acid compositional analysis, DNA-DNA hybridization analysis and restricted enzyme cutting analysis, but be difficult to make a distinction (Fritze through the phenotypic characteristic analysis; 2004, Wang, etal.; 2007), though 16S rDNA/RNA gene order be widely used in Bacteria Identification or make up the phyletic evolution relation of bacterium, between the very near classification monoid of sibship; Lost efficacy owing to the similarity between sequence is too high (Christensenet al., 1998), 16S rDNA sequence can not effectively be distinguished the bacterial classification of subtilis group.

Chun and Bae (2000) has analyzed the evolutionary relationship of Bacillus subtillis and close kind with the gene gyrA of coding DNA gyrase (gyrase) A subunit; Discovery can be distinguished the sibling species in the Bacillus subtillis group based on the phylogenetic analysis of gyrA Gene Partial sequence.Rooney etc. (2009) have measured the gyrA gene order of a large amount of bacillus subtilis strains; And the complicacy of having utilized the careful research of gyrA gene between the bacillus subtilis bacterial classification; There is very big difference in the different strains of finding Bacillus subtillis; Can be divided into different crowds based on gyrA, and, find and described the new subspecies Bacillussubtilis subsp.inaquosorum of subtilis according to the phylogenetic analysis that the gyrA gene is carried out; Till 2009, Bacillus subtillis is to have confirmed 3 subspecies.

The molecular evolution speed of the gene gyrB of coding DNA gyrase B subunit is greater than 16S rRNA gene; Thereby the gyrB gene also is fit to the evaluation of sibling species very much; At present at Rhodopseudomonas Pseudomonas (Yamamoto&Harayama; 1998), acinetobacter Acinetobacter (Yamomoto&Harayama, 1996; Yamamotoet al., 1999), Mycobacterium Mycobacterium (Kasai et al., 2000; Niemann et al.; 2000), salmonella Salmonella, Shigella Shigella, intestinal bacteria Escherichia coli (Fukushima et al.; 2002), Aeromonas Aeromonas et al., 2003; Hou Xiaoli etc.; 2006), nocardia belongs to Nocardiopsis (Yang Lingling etc.; 2007), anthrax bacillus-wax shape bacillus-Tribactur group Bacillus anthracis-cereus-thuringiensis group (La Duc et al.; 2004), Bacillus subtillis group Bacillus subtilis group (Wang et al.; 2007), used the gyrB gene in the evaluation of Bacillusamyloliquefaciens sibling species Bacillus velezensis (wang et al., 2008).Domestic existing investigator has carried out summary research (Li Xianmei etc., 2008 with regard to the gyrB gene in the application facet of differentiating the bacterium sibling species; Hao Yunjie and Han Suzhen, 2008).

Summary of the invention

The object of the present invention is to provide a plants endogenetic Bacillus subtillis.

Bacillus subtillis R31 bacterial strain provided by the invention (Bacillus subtilis R31); Chinese typical culture collection center (CCTCC), address have been preserved in on November 12nd, 2009: Chinese Hubei Province loujia hill belongs Wuhan University Chinese in the school typical culture collection center; Preserving number is CCTCC NO:M 209261.

This strains separation is gram-positive microorganism from the double-colored dendrobium blade (Dendrobiumchrysopterum) of Center of Agriculture Sciences & Research, Zhuhai City's plantation, and thalline is shaft-like, produces the brood cell; Catalase, the oxidase test positive have the ability of hydrolyzed starch, casein, tyrosine, urea, tween 80, can utilize Citrate trianion and propionic salt, and the V.P test is negative reaction; Have glycolysis-ability, nitrate reduction ability, have the ability of producing indoles, hydrogen sulfide, otan, do not produce PD, aerobic growth; Growth pH scope is wider, and pH5.0 ~ pH9.0 all can grow, the inclined to one side alkali well-grown of meta-acid; Meta-acid environment optimum growh pH value is 5.0, and alkali environment optimum growh pH is 9.5 partially, and optimum nitrogen source is the yeast extractive substance; Optimum carbon source is a sucrose, can not utilize oxysuccinic acid and glycocoll, can be 50 ℃ of growths; Can not grow for 55 ℃, can not grow for 10 ℃, can in 0.001% N,O-Diacetylmuramidase, 3% ~ 7% NaCl, grow.On the NA substratum, aged colony colour is golden yellow, smooth surface is than thickness, fold occurs sometimes.

Another object of the present invention is to provide this bacterial strain in the purposes that is used to suppress aspect the phytopathogen, especially the purposes aspect the control banana blight is is particularly prevented and treated the purposes that is caused the banana blight aspect by Fusarium oxysporum.

16S rDNA, gyrA and gyrB that above-mentioned bacterial strains provided by the invention passes through to obtain are because of fragment; And be proved to be a kind of new Bacillus subtillis through blast search with after comparing on the NCBI; Can have dull and stereotyped preferably antagonistic activity to the various plants pathogenic fungi; Especially soil-borne vascular bundle diseases such as various Fusarium oxysporums, dry thread Pyrenomycetes, Alternaria are had good antagonistic ability, and show good control effect in greenhouse and field; The little Chi Kan of sluice gate carries out the field experiment that the filling root is prevented and treated Brazilian any of several broadleaf plants blight, emperor any of several broadleaf plants blight and No. 1 resistance banana blight of Nong Ke in Zhuhai; 2 years integrated control effects of Brazilian any of several broadleaf plants blight are reached 91.71%; Preventive effect to No. 1 resistance any of several broadleaf plants of agricultural section reaches 82.71%, and the preventive effect of emperor any of several broadleaf plants is reached 85.07%.

Description of drawings

Figure 1A, Figure 1B are respectively R31 flat-plate bacterial colony front and back form;

Fig. 2 is a R31 thalline microscopic morphology;

Fig. 3 R31 growth curve;

Fig. 4 different nitrogen sources is to the influence of R31 growth;

The different carbon sources of Fig. 5 are to the influence of R31 growth;

The different pH of Fig. 6 are to the influence of R31 growth;

Fig. 7 is genome dna electrophoresis figure; (M:Marker DL100001: genomic dna)

Fig. 8 is 16S rDNA, gyrA and gyrB gene PCR amplification figure; (M:Marker DL10000 1:16SrDNA 2:gyrA 3:gyrB)

Fig. 9 is based on the bacterial strain R31 of gyrA gene order and the phylogenetic tree of relevant bacterial strain;

Figure 10 is based on the bacterial strain R31 of gyrB gene order and the phylogenetic tree of relevant bacterial strain.

Embodiment

The separation and purification of embodiment 1:R31 and physico-chemical property research

(1) separation of R31, purifying and preservation

In the greenhouse in Center of Agriculture Sciences & Research, Zhuhai City orchid production base, gather healthy double-colored stem of noble dendrobium blade, tap water is rinsed well, and dry back 75% alcohol and handle 1min, again with 5% NaOCl processing 5min, rinsed with sterile water 4 times; After drying, blade is cut into the tissue block about 0.2cm * 0.5cm, is laid on NA (beef-protein medium) flat board that contains cycloheximide 50 μ g/mL with sterile scissors and tweezers; 28 ℃ of constant temperature culture, observe every day, in case find that having bacterium to grow promptly from otch chooses, cultivates in the dull and stereotyped purifying of new NA, and be stored in-20 ℃ and-80 ℃ with bacterium liquid form with 20% glycerine.The sterilized water 200 μ l that get the 4th rinsing again are applied to the NA flat board, do 3 repetitions, and equal conditions is cultivated, and observe and whether grow bacterium.In case find the long bacterium of flat board that sterilized water is coated with, the corresponding bacterial strain that then is separated on the separating plate is regarded as non-endophyte and abandons.

(2) physical and chemical property determining of R31

TP and required substratum:

(1) NA substratum (being used for culturing bacterium): Carnis Bovis seu Bubali cream 3g, peptone 5g, glucose 2.5g, agar powder 15g, zero(ppm) water 1000mL, pH 7.0.

(2) aerobism test (being used for Bacteria Identification): carry out at anaerobic jar, substratum is that NA is dull and stereotyped; Under the situation of anaerobic, the bacterium to be measured of picking fresh culture is rule in the NA flat board, 37 ℃ of cultivations; It is then negative not grow, otherwise positive.Test result is seen table 1, down together.

(3) oxidase test (being used for Bacteria Identification): buy the oxydase test paper, the bacterium lawn to be measured of picking fresh culture spreads upon on the test paper; It is positive that red person appears in the lawn of in 10s, smearing, and 10s～60s red person occurs and is time-delay reaction, and it is all negative not occur occurring red person more than red or the 60s.

(4) catalase test (being used for Bacteria Identification): preparation 3%～10% hydrogen peroxide, the bacterium to be measured one little ring of picking fresh culture is applied in and drips on the sheet glass that superoxol is arranged; Then positive if any the bubble generation, on the contrary negative.

(5) V.P test (being used for Bacteria Identification): culture medium prescription is peptone 5g, glucose 5g, potassium hydrogenphosphate 5g, water 1000mL, pH7.0～7.2, every pipe packing 4mL～5mL, 115 ℃ of sterilization 30min; Inoculate bacterium to be measured in this nutrient solution behind 37 ℃ of shaking culture 24h; Getting nutrient solution mixes with 40% sodium hydroxide equivalent mutually; Add a little creatine, redness then positive (need place the just red reaction of appearance of longer time sometimes) occurs like nutrient solution behind the 10min, on the contrary negative.

(6) the highest and minimum growth temperature test (being used for Bacteria Identification): the NA liquid culture is adopted in maximum growth temperature (being provided with 55 ℃, 50 ℃) test; The fresh liquid culture one little ring of picking bacterium to be measured changes in the NA nutrient solution; Place 55 ℃, 50 ℃ shaking bath shaking culture respectively; Observe growing state, other is provided with 37 ℃ of cultivations and is contrast; If continuous 3 culture transferrings growth is then thought and in this temperature, can be grown in this temperature, and is positive, otherwise negative.Minimum growth temperature (being provided with 10 ℃, 15 ℃) test adopts the NA solid plate to cultivate; The fresh liquid culture one little ring of picking bacterium to be measured is rule in the NA flat board; Place 10 ℃, 15 ℃ incubators then respectively, observe growing state, other is provided with 37 ℃ of cultivations and is contrast; It is then positive to grow, otherwise negative.

(7) to N,O-Diacetylmuramidase resistant proof (being used for Bacteria Identification): at the triangular flask of 100mL; Put into the aseptic 0.01mol/L HCL of 60mL～65mL; Add the 0.1g N,O-Diacetylmuramidase, the aseptic cotton of bottle top plug is boiled 20min on little fire; After being cooled to room temperature, supplying liquid volume with aseptic 0.01mol/L HCL and be N,O-Diacetylmuramidase liquid to 100mL; Get 1mL N,O-Diacetylmuramidase liquid and mix with the aseptic NA nutrient solution of 99mL, be sub-packed in sterile test tube, every pipe 2.5mL is containing 0.001% N,O-Diacetylmuramidase NA nutrient solution test tube and is not having in the NA nutrient solution test tube control tube of N,O-Diacetylmuramidase, and each inoculates 1 ring bacterium liquid, cultivates 5d～7d for 37 ℃; It is then positive to grow, otherwise negative.

(8) can be in the nutrient solution of pH5.7 growth test (being used for Bacteria Identification): get bacterium liquid one ring to be measured, be inoculated in the NA nutrient solution of pH5.7, inoculate common NA nutrient solution (pH7.2) simultaneously and compare, observe growing state after cultivating 1d～3d for 37 ℃; It is then positive to grow, otherwise negative.

(9) salt tolerant and need salt test (being used for Bacteria Identification): add NaCl at the NA nutrient solution, make the NaCl final concentration be respectively 3%, 5%, 7% and 10%, get bacterium liquid one ring to be measured, be inoculated in the above-mentioned substratum, growing state is observed in 37 ℃ of cultivations; It is then positive to grow, otherwise negative.

(10) produce acid test (being used for Bacteria Identification): the sugar and the alcohol of needs test comprise glucose, pectinose, wood sugar, N.F,USP MANNITOL and semi-lactosi; Culture medium prescription is Secondary ammonium phosphate 1.0g, Repone K 0.2g, sal epsom 0.2g, yeast extract paste 0.2g, sugar or alcohols 10.0g, dibromothymolsulfonphthalein 1% aqueous solution 3mL, zero(ppm) water 1000mL, pH7.0～7.2, test tube packing, 112 ℃ of sterilization 30min; The bacterium to be measured of picking fresh culture, 4 test tubes of percutaneous puncture-inoculation, wherein 2 draw the sterilization the capping of Vaseline Yellow Protopet 2A, about 0.5cm～1.0cm is thick, is stopped pipe with the secluding air; In addition 2 not seal the Vaseline Yellow Protopet 2A be open pipe; Simultaneously do contrast with stopped pipe and the open pipe do not inoculated; Cultivate observations behind 1d, 2d, 3d, 7d, the 14d for 37 ℃; It is positive that the substratum color becomes yellow person, otherwise negative, wherein has only open pipe to produce sour flavescence person and produce acid for oxidized form, and open pipe and stopped pipe all produce sour flavescence person and produce acid for fermented type.

(11) starch hydrolysis experiment (being used for Bacteria Identification): in preparation NA solid medium, add Zulkovsky starch, making its final concentration is 0.2%, 121 ℃ of sterilization 20min, falls dull and stereotyped subsequent use; The bacterium point to be measured of picking fresh culture is connected in the above-mentioned flat board, cultivates 2d～5d for 37 ℃, form obvious bacterium colony after; On flat board, drip iodine liquid, it is black-and-blue that flat board promptly is, and periphery of bacterial colonies is if any the nondiscoloration transparent circle; Then represent the starch hydrolysis, positive, still be black-and-blue then negative.

(12) Citrate trianion utilization experiment (being used for Bacteria Identification): culture medium prescription is sodium-chlor 1g, 7 Magnesium sulfate heptahydrate 0.2g, primary ammonium phosphate 0.5g, Trisodium Citrate 2g, zero(ppm) water 1000mL, 0.04% phenol red liquid 20mL; 121 ℃ of sterilization 20min fall dull and stereotyped subsequent use; The bacterium to be measured of picking fresh culture is rule in above-mentioned flat board, cultivates 3d～7d for 37 ℃, if substratum be an alkalescence (indicator becomes blue or pink) then positive, on the contrary negative.

(13) propionic salt utilization experiment (being used for Bacteria Identification): culture medium prescription is sodium-chlor 1g, 7 Magnesium sulfate heptahydrate 0.2g, primary ammonium phosphate 0.5g, Sodium Propionate 2g, zero(ppm) water 1000mL, 0.04% phenol red liquid 20mL, and 121 ℃ of sterilization 20min fall dull and stereotyped subsequent use; The bacterium to be measured of picking fresh culture is rule in above-mentioned flat board, cultivates 3d～7d for 37 ℃, if substratum be an alkalescence (indicator becomes blue or pink) then positive, on the contrary negative.

(14) nitrate salt is reduced to nitrites test (being used for Bacteria Identification): the NA nutrient solution adds saltpetre 1g and is the nitrate salt liquid nutrient medium, pH7.0～7.6, and every pipe packing 4mL～5mL, 121 ℃ of sterilization 20min are subsequent use; Prepare Ge Lisishi reagent A liquid and B liquid in addition, A liquid is Sulphanilic Acid 0.5g, dilute acetic acid (about 10%) 150mL, and B liquid is a-naphthylamines 0.1g, zero(ppm) water 20mL, dilute acetic acid (about 10%) 150mL; Pentanoic reagent is dissolved in the 100mL vitriol oil for pentanoic 0.5g, uses the 20mL distilled water diluting; Bacterium to be measured is inoculated in the nitrate salt liquid nutrient medium, 37 ℃ of shaking culture 1d, 3d, 5d, other stays two pipes not inoculate and compares; Get 2 clean empty test tubes, pour the nutrient solution that a little cultivates 1d, 3d, 5d respectively into, respectively add A liquid and B liquid again, control tube is also done same processing; If solution becomes pink, rose, orange, brown etc. then represent that nitrite exists, and is that nitrate reduction is positive, occur like redfree; Then can add 1,2 pentanoic reagent; Be blue like solution, then represent to still have nitrate salt in the nutrient solution, and do not have the nitrite reaction again; Then represent the nitrate-free reductive action, negative; As not being blue reaction, the nitrite of expression nitrate salt and formation has been reduced to other material, so must be judged as the nitrate reduction positive.

(15) produce indole test (being used for Bacteria Identification): prepare the 1% tryptone aqueous solution, pH7.2～7.6, test tube packing, 115 ℃ of sterilization 30min; Other prepares following reagent---Paradimethylaminobenzaldehyde 8g, 95% ethanol 760mL, concentrated hydrochloric acid 160mL; Picking bacterium to be measured behind shaking culture 1d, 2d, 4d, the 7d, slowly adds 3mL～5mL mentioned reagent in the nutrient solution surface along test tube wall in above-mentioned nutrient solution, redness occurs at the liquid layer interface, and is promptly positive, otherwise negative.

(16) hydrogen sulfide production test (being used for Bacteria Identification): culture medium prescription is peptone 10g, sodium-chlor 5g, Carnis Bovis seu Bubali cream 10g, halfcystine 0.5g, zero(ppm) water 1000mL, pH7.0～7.4, test tube packing, 112 ℃ of sterilization 30min.It is wide in addition common filter paper to be cut into 0.5cm～1cm, and length is decided according to test tube and nutrient solution height; Plumbic acetate with 5%～10% soaks into paper slip, uses oven for drying then, and it is subsequent use to be put in the petridish sterilization.The bacterium to be measured of choosing fresh culture is inoculated in the above-mentioned nutrient solution, also uses the tampon jam-pack with 1 plumbic acetate paper slip of aseptic nipper gripping then, and it is hung in the pipe, and 37 ℃ leave standstill cultivation, behind 3d, 7d, 14d, observe respectively.Paper slip turns black then positive, and nondiscoloration is then negative.

(17) produce otan test (being used for Bacteria Identification): culture medium prescription is glycerine 10.0mL, yeast extract paste 1.0g, peptone 0.5g, lime carbonate 0.3g, potassium primary phosphate 0.3g, agar powder 1.8g; PH5.5～6.0; 121 ℃ of sterilization 15min fall dull and stereotyped subsequent use; Prepare colour developing liquid---pentanoic 2g, acetate 200mL, vitriol oil 20mL in addition.Choose the bacterium to be measured of fresh culture, in above-mentioned flat board, rule, behind 37 ℃ of cultivation 10d, pour colour developing liquid into, be advisable to cover bacterium colony, inspection is then positive as red haloing occurs in periphery of bacterial colonies in the 2h, otherwise negative.

(18) PD test (being used for Bacteria Identification): culture medium prescription is yeast extract paste 3g, sodium-chlor 5g, Sodium phosphate, dibasic 1g, DL-phenylalanine(Phe) 2g, agar 12g, zero(ppm) water 1000mL; PH7.0; The test tube packing is put into the inclined-plane behind 121 ℃ of sterilization 10min; Other prepares the liquor ferri trichloridi of 10% (W/V).The bacterium to be measured of picking fresh culture is rule in above-mentioned medium slant; Cultivate 4h or 8h～24h for 37 ℃, then with 4～5 liquor ferri trichloridis to the inclined-plane of growth bacterium, show the formation phenyl-pyruvic acid when green when producing in the water of condensation on the inclined-plane; Positive, on the contrary negative.

(19) casein hydrolysis test (being used for Bacteria Identification): get the 5g skim-milk, 1.5g agar is dissolved in 50mL zero(ppm) water respectively, 121 ℃ of sterilization 20min; After treating two kinds of liquid cooling to 45 ℃～50 ℃, be mixed even mutually and fall dull and stereotyped; The dull and stereotyped inversion spent the night so that the planar surface moisture drying, connects the bacterium to be measured of fresh culture then, 37 ℃ cultivate 1d, 3d, 5d after, whether the observation periphery of bacterial colonies transparent circle occurs, as it is then positive transparent circle to occur, otherwise negative.

(20) tyrosine hydrolysis test (being used for Bacteria Identification): 0.5g is suspended in 10mL zero(ppm) water with L-tyrosine, and 112.6 ℃ of sterilization 20min mix with the aseptic NA solid medium of 100mL then; Dull and stereotyped, after waiting to solidify, will test bacterium and be inoculated on the plate; Cultivate 7d～14d for 37 ℃; Whether the record tyrosine crystal is hydrolyzed and bleach, then positive like bleach, otherwise negative.

(21) urea decomposition test (being used for Bacteria Identification): culture medium prescription is peptone 1g, sodium-chlor 5g, glucose 1g, potassium primary phosphate 2g, 0.2% phenol red aqueous solution 6mL, agar 20g, zero(ppm) water 1000mL; PH6.8～6.9; The test tube packing; 115 ℃ of sterilization 30min, this moment, substratum was safran or little band pink; Be cooled to about 50 ℃, add 20% urea soln of filtration sterilization, making its final concentration is 2%, puts into the inclined-plane.The bacterium to be measured of picking fresh culture is rule on the inclined-plane, 37 ℃ of cultivations, and it is positive that substratum is pink person, the constant person of substratum color negative (negative findings will be observed 4d).

(22) soil temperature 80 degraded tests (being used for Bacteria Identification): culture medium prescription is peptone 10g, sodium-chlor 5g, 7 hydration calcium chloride 0.1g, agar 9g, zero(ppm) water 1000mL, pH7.4,121 ℃ of sterilization 20min; Treat that the substratum temperature is cooled to about 45 ℃, adding soil temperature 80 to its final concentration is 1%, falls dull and stereotyped then; The bacterium to be measured of picking fresh culture is rule in above-mentioned flat board, cultivates 7d for 37 ℃, and observe every day, has fuzzy haloing then positive like periphery of bacterial colonies, otherwise negative.

(23) strain culturing morphologic observation: this bacterial strain is on the NA substratum, and aged colony colour is golden yellow, smooth surface is than thickness, fold (seeing accompanying drawing 1) occurs sometimes.At microscopically, thalline is rod-short, gives birth among the brood cell, and thalline is about 2.06 μ m, wide about 0.78 μ m (seeing accompanying drawing 2).

(23) mensuration of R31 growth curve: the NA nutrient solution is loaded in the triangular flask; Tonburden is 40%, after the sterilization, and the R31 fresh seeds liquid of inoculation 0.1%; Gather the R31 nutrient solution behind 37 ℃, 180r/m shaking culture 12h, 24h, 36h, 48h, 72h, survey OD behind 10 times of the distilled water dilutings ₆₀₀, five repetitions are set, each repeats each time period and surveys three times, calculates average OD ₆₀₀, be X-coordinate with incubation time (h), OD ₆₀₀For ordinate zou is drawn growth curve.Referring to accompanying drawing 3.

(24) R31 is to the utilization of different nitrogen sources: the basic medium prescription is potassium primary phosphate 1.36g, Calcium dichloride dihydrate 0.005g, Sodium phosphate, dibasic 2.13g, glucose 10g, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.2g, iron vitriol 0.0005g, water 1000mL; Add various nitrogenous sources respectively; 116 ℃ the sterilization 30min (wait sterilized after; Calcium dichloride dihydrate dissolving separately also adds after the sterile filtration again), Carnis Bovis seu Bubali cream, peptone, yeast lixiviate powder, casein, saltpetre, ammonium chloride, add-on is 2%; Behind the R31 fresh seeds liquid of sterilization back inoculation 0.1%, 37 ℃, 180r/m shaking culture 24h, survey OD behind 10 times of the distilled water diluting nutrient solutions ₆₀₀, every kind of carbon source is provided with 3 repetitions, and each repeats to survey three times, calculates average OD ₆₀₀Referring to accompanying drawing 4.The different lowercase alphabet registrations certificates of column diagram top detect significant difference through the LSD method, P＜0.05, down together.

(25) R31 is to the utilization of different carbon sources: the basic medium prescription is ammonium sulfate 2.0g, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.2g, SODIUM PHOSPHATE, MONOBASIC 0.5g, Calcium dichloride dihydrate 0.1g, potassium hydrogenphosphate 0.5g; Water 1000mL; Add various carbon sources respectively, 116 ℃ of sterilization 30min (wait sterilize after, separately also adding again after the sterile filtration of dissolving of Calcium dichloride dihydrate); Supply examination carbon source fructose, glucose, sucrose, glycocoll, N.F,USP MANNITOL, l-arginine, oxysuccinic acid, add-on is 2%; Behind the R31 fresh seeds liquid of sterilization back inoculation 0.1%, 37 ℃, 180r/m shaking culture 24h, survey OD behind 10 times of the distilled water diluting nutrient solutions ₆₀₀, every kind of carbon source is provided with 3 repetitions, and each repeats to survey three times, calculates average OD ₆₀₀Referring to accompanying drawing 5.

(26) pH is to the influence of R31 growth: with hydrochloric acid and sodium hydroxide the NA medium pH value is adjusted to 4.0,5.0,6.0,7.0,8.0,8.5,9.0,9.5,10.0,10.5,11.0 respectively; After the conventional sterilization; The pH value of above-mentioned NA nutrient solution becomes 4.15,5.13,6.11,7.23,7.83,8.30,8.65,8.99,9.29,9.56,9.78 respectively; Behind inoculation 0.1% the R31 fresh seeds liquid, 37 ℃, 180r/m shaking culture 24h, survey OD behind 10 times of the distilled water diluting nutrient solutions ₆₀₀, every kind of pH is provided with 3 repetitions, and each repeats to survey three times, calculates average OD ₆₀₀Referring to accompanying drawing 6.

Table 1R31 physiological and biochemical property

Annotate: "-" expression is negative, and "+" expression is positive.

(3) utilize the utilization of carbon source result of Biolog Bacteria Identification systems measurement

The strain broad-spectrum antifungal biological and ecological methods to prevent plant disease, pests, and erosion bacillus R31 bacterial strain that the application separates from dendrobium Center of Agriculture Sciences & Research, Zhuhai City agricultural microbiology laboratory has carried out the Biolog evaluation, and sample is sent to Guangdong Province microbiological analysis inspection center and identifies.Biolog fails to provide qualification result accurately, but the data of detailed relevant bacterial strain utilization of carbon source is provided, and the result is referring to table 2.

The R31 bacterial strain metabolic capacity that table 2, Biolog identification systems provide

Annotate: "+" expression has metabolic capacity, and "-" representes not have metabolic capacity, down together.

(4) result and the utilization of carbon source result that utilize API biochemical identification system to identify

France API identification systems are that application is the widest in the world wide; Generally acknowledged by the microbiologist; The application has also carried out the API biochemical identification to the strain broad-spectrum antifungal biological and ecological methods to prevent plant disease, pests, and erosion bacillus R31 bacterial strain that separate from dendrobium in Center of Agriculture Sciences & Research, Zhuhai City agricultural microbiology laboratory, and sample is sent to Guangdong Province microbiological analysis inspection center and identifies.API identifies that the result that provides reaches 97.2% for the R31 bacterial strain to the coincidence rate of 48 kinds of utilization of carbon source situation and Bacillus subtillis.The result of bacterial strain utilization of carbon source is referring to table 3.

The R31 metabolic capacity that table 3, API identification systems provide

(5) Molecular Identification of utilizing 16S rDNA, gyrA gene and gyrB gene to carry out

The application has carried out 16S rDNA gene order, gyrA gene order and gyrB gene order clonal analysis and phylogenetic tree structure to Center of Agriculture Sciences & Research, Zhuhai City's agricultural microbiology laboratory separation from a strain broad-spectrum antifungal biological and ecological methods to prevent plant disease, pests, and erosion bacillus R31 of dendrobium, through phylogenetic analysis.The separation of R31, purifying and preservation are with present embodiment (one) joint.

The extraction of 1 bacterial strain DNA

Genomic dna uses the MiNiBEST Bacterial Genomic DNA Extraction KitVer.2.0 bacterial genomes DNA extraction test kit of TaKaRa company to extract, and can obtain the ideal genomic dna.

216S rDNA gene order pcr amplification

Adopt the bacterium universal primer

27F:5 '-AGAGTTTGATCCTGGCTCAG-3 ' and

1513R：5’-ACGGCTACCTTGTTACGACTT-3’

Carry out the pcr amplification of 16S rDNA.PCR condition: 95 ℃ of 5min, 94 ℃ of 1min, 56 ℃ of 2min, 72 ℃ of 2min, 30 circulations, 72 ℃ of 10min.

Reclaiming test kit (Gel Extraction Kit) with OMEGA company gel reclaims amplified fragments; To reclaim product is connected on the pMD19-T carrier; Transformed E .coli DH5 α chooses transformant after bacterium colony PCR check defines the goal gene insertion, shakes bacterium; Protect the glycerine kind, send Invitrogen biotechnology ltd Guangzhou Branch to measure the base sequence of goal gene.

3gyrA gene order pcr amplification

According to the report of Chun and Bae 2000 at " Antonie van Leeuwenhoek " magazine the 78th volume 123-127 page or leaf; Design of amplification primers f (5 ' CAGTCAGGAAATGCGTACGTCCTT-3 ') and r (5 '-CAAGGTAATGCTCCA GGCATTGCT-3 ') amplification gyrA gene, 95 ℃ of 5min, 94 ℃ of 1min; 62.3 ± 1 ℃ of (optimizing the back) 1min; 72 ℃ of 2min, 30 circulations, 72 ℃ of 10min.

The clone of amplified production and sequence measurement are with clone and the order-checking of 16S rDNA.

4gyrB gene order pcr amplification

Design degenerated primer UP-1S and UP-2Sr according to Yamamoto and Harayama1995 at the report of " Applied Environmental Microbiology " magazine 61 volume 1104-1109 pages or leaves.UP-1S:5 '-GAAGTCATCATGACCGTTCTGCA (TC) GC (TCAG) GG (TCAG) GG (TCAG) AA (AG) TT (TC) GA-3 '; UP-2Sr:5 '-AGCAGGGTACGGATGTGCGAGCC (AG) TC (TCAG) AC (AG) TC (TCAG) GC (AG) TC (TCAG) GTCAT-3 '; The gyrB gene that is used to increase, 95 ℃ of preparatory sex change 5min, 94 ℃ of 1min; 55 ℃-62 ℃ annealing (optimizing the back) 1min; 72 ℃ are extended 2min, 30 circulations, 72 ℃ of 10min.

The clone of amplified production and order-checking are with clone and the order-checking of 16S rDNA.

The phylogenetic evolution analysis of 5 sequences

Remove and obtain primer fragment in the gene order; Residue sequence is logined NCBI (www.ncbi.nlm.nih.gov) carry out online BLAST homology search, be chosen at the similarity of publishing on " International Journal of Systematic and EvolutionaryMicrobiology " magazine and carry out phylogenetic analysis above 90% sequence.When wherein utilizing the gene constructed systematic evolution tree of gyrA, with the gyrA gene order of the lower Bacillus vallismortis NRRL B-14890 bacterial strain of similarity as outer crowd.When utilizing the gene constructed systematic evolution tree of gyrB, with the lower close kind of gyrB gene order of similarity among the Blast result as outer crowd.Sequence adopts earlier ClustralX 2.0 to carry out the multiple sequence comparison, then with the MEGA4.0 software package adopt the Neighbour-joining method (Kimura ' s 2-parameter model, bootstrap1000) the method constructing system is grown and is set.

The clone result of 616S rDNA, gyrA and gyrB gene order

Utilize genome to extract the genomic dna that test kit obtains R31; Electrophoresis detection genomic dna molecular weight greater than 10000bp (shown in accompanying drawing 7, M:Marker DL100001: genomic dna), the 16S rDNA of the genomic dna pcr amplification R31 that utilize to extract; GyrA and gyrB gene are (shown in accompanying drawing 8; M:Marker DL100001:16SrDNA 2:gyrA 3:gyrB), wherein about gyrA gene 1000bp, about gyrB gene 1200bp.The 16S rDNA gene fragment length 1544bp that the order-checking back obtains, gyrA gene fragment length 1025bp, gyrB gene fragment length 1259bp.

7 phylogenetic analysis based on gyrA gene Blast result

Be depicted as based on the bacterial strain R31 of gyrA gene order and the phylogenetic tree of relevant bacterial strain like accompanying drawing 9.According to the result who grows tree, find to utilize the gyrA gene can identify accurately that R31 is Bacillus subtillis, and can obtain the sibship of R31 and known kind.Phylogenetic tree with gyrA gene order comparison result structure; Wherein B.subtilissubsp.inaquosorum 2 bacterial strain gyrA gene fragment order and R31 similarity lower (94%); But evolutionary relationship for complete each subspecies of demonstration Bacillus subtillis; Still select these 2 sequences of publishing to analyze, found to utilize the phylogenetic tree of gyrA gene series structure, can be good at reacting the sibship between different Bacillus subtillis bacterial strains.

8 phylogenetic analysis based on gyrB gene Blast result

Be depicted as based on the bacterial strain R31 of gyrB gene order and the phylogenetic tree of relevant bacterial strain like accompanying drawing 10.According to the result who grows tree, find to utilize the gyrB gene can identify accurately that also R31 is Bacillus subtillis, when growing tree, selected a plurality of genes series similaritys to reach 90% different strains as outer crowd with gyrB gene order comparison result constructing system.The bacterial strain of having measured the gyrB gene owing to Bacillus subtillis in the Bacillus subtillis group is more, thereby the gyrB gene can show well that the sibship between R31 and other bacterial strain is far and near.

Embodiment 2:R31 is to the antagonistic effect of different sickle-like bacteria cause of disease bacterium

(1) isolation and purification of different sickle-like bacteria cause of disease bacterium

Separate the source: in the Zhuhai City and surrounding cities gather typical banana blight, celery verticillium, dendrobium leaf spot diseased tissues, carry out pathogenic bacteria and separate, be used to test the antagonistic activity of R31 to different sickle-like bacteria cause of disease bacterium.

Isolation and purification method: with clear water diseased tissues is cleaned up, dry up, cut the diseased tissues of the strong intersection of disease and be cut into suitable size with sterile razor blade; Under the aseptic situation with 70% alcohol surface sterilization 30s; Using available chlorine again is that 5% NaOCl handles 5min, and rinsed with sterile water 4 times is cut into the tissue block about 0.2cm * 0.5cm with the diseased tissues after the surface sterilization with sterile scissors and tweezers; Drying up diseased tissues piece surface-moisture is placed in the sterile petri dish; Add little P DA (potato dextrose agar contains the Vetstrep of 300ug/mL) substratum then, substratum is mixed with the diseased tissues piece; After treating culture medium solidifying, add the above-mentioned PDA substratum of one deck again, add one deck after the culture medium solidifying again, form three layers of culture medium flat plate.At last flat board is placed 27 ℃ of incubators; Observe every day; When having treated that mycelia grows and passes three layers of substratum and grows to the outermost layer media surface from diseased tissues, the picking mycelia in expand in SNA (SpeziellerNahrstoffarmer Agar, sickle-like bacteria the preserves special culture media) substratum numerous after; The picking mycelia is in the SNA slant medium again, treats that mycelia covers with the inclined-plane and be placed in 4 ℃ of refrigerators and preserve.In addition with the dull and stereotyped mycelia of cultivating after the expansion of SNA substratum is numerous of PDA; After treating that mycelia is covered with whole plate, add an amount of sterilized water, scrape mycelia gently with the aseptic inoculation pin; Spore in the mycelia is come off; Form spore suspension, draw this suspension-s and 40% glycerine, be stored in-20 ℃ and reach-80 ℃ according to 1: 1 mixed.

Isolate 16 strain target sickle-like bacteria altogether, go out 1 strain target sickle-like bacteria, go out 1 strain target sickle-like bacteria from banana blight tissue (adhering to the different banana varieties in Zhuhai City and surrounding cities banana planting district separately) from dendrobium leaf spot separate tissue from celery verticillium separate tissue.

It is pathogenic that tieback is measured isolate: in strict accordance with Koch's Postulates (Koch ' s Postulate) above-mentioned strain separated is carried out pathogenic mensuration.Banana blight and celery verticillium separate tissue bacterium are coiled seedling (5cm～10cm is high) as the tieback object with the anosis banana seedlings of health (15cm is high, 4～5 leaves), celery cave respectively; The spore suspension of preparation isolate, concentration is 10 ⁶Individual/mL, artificial mechanism damage banana seedlings and celery shoot root portion tissue place the spore suspension of corresponding isolate to soak 30min respectively the banana seedlings and the celery seedling of hindering the root processing then; At last with postvaccinal banana seedlings and celery transplantation of seedlings to the flowerpot that sterile soil is housed; Treat that plant occurs gathering diseased tissues and separation behind the typical disease symptom, compare separating the bacterial strain that obtains and the inoculating strain that sets out, thereby confirm whether starting strain is pathogenic bacteria.Dendrobium leaf spot separate tissue bacterium with the healthy anosis blade of dendrobium (seedling age is 1～2 month) as the tieback object; Before the inoculation, blade is all done surface sterilization with 75% alcohol, treats to inoculate after the alcohol volatilization fully; Earlier cause slight wound in leaf surfaces with aseptic banister brush, again with inoculating needle picking one fritter 7 days isolate bacterium piece of cultivation on PDA, left-hand thread on wound, the bagging 24h that preserves moisture; Supply examination dendrobium seedling to place the greenhouse to cultivate, 26 ℃～29 ℃ of daytimes, relative humidity about 80～90%; Treat that plant occurs gathering diseased tissues and separation behind the typical disease symptom, compare separating the bacterial strain that obtains and the inoculating strain that sets out, thereby confirm whether starting strain is pathogenic bacteria.

Through above-mentioned separation and pathogenic mensuration, and separation acquisition 16 pathogen strain bacterium from the geographic banana blight tissue of difference (Fusarium oxysporum Cuba specialized form, Fusarium oxysporum Schl.f.cubense (E.F.Smith) Snyder etHansen, FOC); Numbering is respectively: FOC-zh (connecting the gulf), FOC-zh (No. 2, north, crane state), FOC-zj (being prone to), FOC-zh (he), FOC-zh (emperor), FOC-zh (Xinxiang), FOC-zh (Liao); FOC-zh (Aug. 1st), FOC-zh (nine enclose 1), FOC-zh (nine enclose 2); FOC-zh (Pingtang), FOC-zh (No. 1, north, crane state), FOC-zh (chicken); FOC-zh (east) is white, and is fragrant withered 4, FOC-ts; Separation acquisition 1 pathogen strain bacterium from celery verticillium tissue (Fusarium oxysporum celery specialized form, Fusarium oxysproum f.sp.Apii, FOA); From dendrobium leaf spot tissue, separate obtaining 1 pathogen strain fungi (Fusarium moniliforme Sheld), be numbered B10b.

(2) R31 is to the antagonistic activity of above-mentioned fungi, and the result sees shown in the table 4

The inoculation diameter is the pathogenic bacteria bacterium piece of 5mm in the dull and stereotyped central authorities of PDA, meets R31 apart from bacterium piece 3cm place's point, and 28 ℃ of constant temperature culture 6d observe the antibacterial nothing that has, and measures size, and the contrast that is treated to that does not meet R31 is set.Antibacterial band is big more, and the expression fungistatic effect is strong more.

Embodiment 3:R31 is to the antagonistic activity of other plant pathogenic fungi, and the result sees shown in the table 5

Method when giving birth to the survey antagonistic activity is with embodiment 2; Wherein Alternaria, curvularia lunata, mango blossom-end rot, watermelon blight etc. are respectively the plant pathogenic fungi that Zhuhai City true green technology ltd gives, the plant pathogenic fungi that dry thread Pyrenomycetes is given by doctor Yi Runhua of Guangdong Ocean University.Antibacterial band is big more, and the expression fungistatic effect is strong more.

Table 4R31 is to the antagonistic activity of different host source pathogenic fungi sickle-like bacteria (Fusarium spp.)

Table 5R31 is to the dull and stereotyped antagonistic activity of several plant pathogenic fungi

Embodiment 4:R31 is to the biological control research of banana blight

(1) to the antagonism mechanism of different banana blight bacteria strains

R31 has good antagonistic effect (seeing embodiment 2) to the different pathogenic strainss of banana blight bacteria; Through microscopic examination, after R31 handled, different pathogenic strainss were compared with contrast; Considerable change all takes place in mycelia and conidium, and main the realization is: coarse, the lopsided swelling of mycelia; Spore deformity, intermediate expansion, two ends are hung and are contracted etc.

(2) the greenhouse control of banana blight is tested

Utilize R31 that banana wilt germina number-four biological strain (FOC-zj among the embodiment 2 (being prone to) bacterial strain) is carried out greenhouse biological control test, supplying examination banana seedlings kind is Brazilian any of several broadleaf plants (susceptible variety, anosis tissue cultured seedling potted plant growth to the about 15cm of plant height, 6～7 leaves).The inoculation of banana blight bacteria is adopted and is hindered the root inoculation method,, irritates then and executes 10 the slight scratch of plant root with pocket knife ⁶The banana blight bacteria spore suspension 50mL of individual/mL; Pour into 50 times of diluents of R31 fermented liquid with the mode of irritating root then, 50mL is irritated in every strain, and every separated 7d uses once, uses altogether 7 times.The final data statistics is: any of several broadleaf plants seedling death rate of the onset of control treatment 1 (inoculation pathogenic bacteria) is 50.00%, and any of several broadleaf plants seedling death rate of the onset of control treatment 2 (not connecing pathogenic bacteria) is 0, and any of several broadleaf plants seedling that R31 handles (inoculation pathogenic bacteria) death rate of the onset is 23.33%.

(3) to the field controling test of banana blight

Utilize R31 that banana blight is carried out field biological control test, the test place is banana planting ground, the little Chi Kan of Zhuhai City's sluice gate village, and supplying the examination banana variety is Brazilian any of several broadleaf plants (banana; Susceptible variety), (banana of agricultural section; Disease-resistant variety), emperor any of several broadleaf plants (dwarf banana, disease-resistant variety), be newly and plant seedlings.The R31 use-pattern is 40 times of dilutions of NA fermented liquid liquid irrigating root, and 4 jin of diluents are irritated in every strain, irritates once in every month.

(1) R31 irritates root and handles the control test to Brazilian any of several broadleaf plants banana blight

Test period is from year July in March, 2008 to 2009, and the place is the little Chi Kan of Zhuhai sluice gate village, crosses over 2 batches, and wherein first batch from year October in March, 2008 to 2008, and second batch from year September in August, 2008 to 2009 (inhaling bud for first batch).Test is provided with sees table 6, and experimental result is seen table 7 and table 8.First batch of test, 82.40%, the second batch of test of control effect, control effect 94.35% (table 7), 2 take turns integrated control test control effect 91.71% (table 8).

Table 6 test period and test plant

The every pilot survey result that takes turns of table 7

Annotate: owing to the CK in the test handles when investigate in December, 2008, inhale the bud sickness rate and abandoned above 50%, therefore second batch of CK sickness rate is by 60.00% estimation.

Table 8 two-wheeled is tested total investigation result

In the control banana blight, the output that the filling root of R31 is handled Brazilian any of several broadleaf plants does not have influence: first batch data show that the banana production of the plant that contrast and R31 filling root are handled is about 50 jin/strain; Second batch data show that the banana production of the plant that contrast and R31 filling root are handled is about 64 jin/strain.

(2) R31 irritates root and handles the control test to a banana blight of agricultural section

Test period is from year October in March, 2008 to 2008, and the place is the little Chi Kan of Zhuhai sluice gate village.Test is provided with and investigation result is seen table 9.Control effect to No. 1 blight of agricultural section behind the R31 filling root reaches 82.71%.

Table 9 test is provided with and investigation result

(3) R31 irritates root and handles the control test to emperor any of several broadleaf plants banana blight

Test period is from year September in September, 2008 to 2009, and emperor any of several broadleaf plants planting any of several broadleaf plants seedling is interplanted in the original dead vacancy of first batch of Brazilian any of several broadleaf plants.The test place is the little Chi Kan of Zhuhai sluice gate village, and test is provided with sees table 10, and test-results is seen table 11.

Table 10 test is provided with

Table 11 pilot survey result

Sequence table

< 110>Center of Agriculture Sciences & Research, Zhuhai City

< 120>one broad-spectrum antifungal plant endophytic bacillus subtillis and application thereof

<160>9

<210>1

<211>1513bp

< 212>16S rDNA gene fragment

< 213>bacillus order, Bacillaceae, Bacillus, Bacillus subtillis

<220>9bp-1501bp

< 223>this sequence encoding product is ribosomal RNA-16S

<400>1

9 A GAGTTTGATC CTGGCTCAGG ACGAACGCTG GCGGCGTGCC TAATACATGC

60 AAGTCGAGCG GACAGATGGG AGCTTGCTCC CTGATGTTAG CGGCGGACGG GTGAGTAACA

120 CGTGGGTAAC CTGCCTGTAA GACTGGGATA ACTCCGGGAA ACCGGGGCTA ATACCGGATG

180 GTTGTTTGAA CCGCATGGTT CAAACATAAA AGGTGGCTTC GGCTACCACT TACAGATGGA

240 CCCGCGGCGC ATTAGCTAGT TGGTGAGGTA ACGGCTTACC AAGGCGACGA TGCGTAGCCG

300 ACCTGAGAGG GTGATCGGCC ACACTGGGAC TGAGACACGG CCCAGACTCC TACGGGAGGC

360 AGCAGTAGGG AATCTTCCGC AATGGACGAA AGTCTGACGG AGCAACGCCG CGTGAGTGAT

420 GAAGGTTTTC GGATCGTAAA GCTCTGTTGT TAGGGAAGAA CAAGTACCGT TCGAATAGGG

480 CGGTACCTTG ACGGTACCTA ACCAGAAAGC CACGGCTAAC TACGTGCCAG CAGCCGCGGT

520 AATACGTAGG TGGCAAGCGT TGTCCGGAAT TATTGGGCGT AAAGGGCTCG CAGGCGGTTT

580 CTTAAGTCTG ATGTGAAAGC CCCCGGCTCA ACCGGGGAGG GTCATTGGAA ACTGGGGAAC

640 TTGAGTGCAG AAGAGGAGAG TGGAATTCCA CGTGTAGCGG TGAAATGCGT AGAGATGTGG

700 AGGAACACCA GTGGCGAAGG CGACTCTCTG GTCTGTAACT GACGCTGAGG AGCGAAAGCG

760 TGGGGAGCGA ACAGGATTAG ATACCCTGGT AGTCCACGCC GTAAACGATG AGTGCTAAGT

820 GTTAGGGGGT TTCCGCCCCT TAGTGCTGCA GCTAACGCAT TAAGCACTCC GCCTGGGGAG

880 TACGGTCGCA AGACTGAAAC TCAAAGGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT

940 GTGGTTTAAT TCGAAGCAAC GCGAAGAACC TTACCAGGTC TTGACATCCT CTGACAATCC

1000?TAGAGATAGG ACGTCCCCTT CGGGGGCAGA GTGACAGGTG GTGCATGGTT GTCGTCAGCT

1060?CGTGTCGTGA GATGTTGGGT TAAGTCCCGC AACGAGCGCA ACCCTTGATC TTAGTTGCCA

1120?GCATTCAGTT GGGCACTCTA AGGTGACTGC CGGTGACAAA CCGGAGGAAG GTGGGGATGA

1180?CGTCAAATCA TCATGCCCCT TATGACCTGG GCTACACACG TGCTACAATG GACAGAACAA

1240?AGGGCAGCGA AACCGCGAGG TTAAGCCAAT CCCACAAATC TGTTCTCAGT TCGGATCGCA

1300?GTCTGCAACT CGACTGCGTG AAGCTGGAAT CGCTAGTAAT CGCGGATCAG CATGCCGCGG

1360?TGAATACGTT CCCGGGCCTT GTACACACCG CCCGTCACAC CACGAGAGTT TGTAACACCC

1420?GAAGTCGGTG AGGTAACCTT TTAGGAGCCA GCCGCCGAAG GTGGGACAGA TGATTGGGGT

1480?GAAGTCGTAA CAAGGTAGCC GT

<210>2

<211>20bp

<212>DNA

< 213>artificial sequence

<220>

< 223>primer

<400>2

5’-AGAGTTTGATCCTGGCTCAG-3’

<210>3

<211>21bp

<212>DNA

< 213>artificial sequence

<220>

< 223>primer

<400>3

5’-ACGGCTACCTTGTTACGACTT-3’

<210>4

<211>1025bp

< 212>gyrA gene fragment

< 213>bacillus order, Bacillaceae, Bacillus, Bacillus subtillis

<220>42bp-1066bp

< 223>this gene order coded product A albumen (subunit A) that is dna gyrase gyrase

<400>4

42 CAGTCAGG AAATGCGTAC GTCCTTCTTG GATTATGCAA TGAGCGTTAT CGTGTCCCGT

100 GCTCTTCCAG ATGTTCGAGA CGGTTTAAAA CCGGTTCATA GACGGATTTT GTATGCAATG

160 AATGATTTAG GCATGACAAG TGACAAGCCT TATAAAAAAT CCGCGCGTAT CGTTGGAGAA

220 GTTATCGGGA AATACCACCC GCACGGTGAT TCAGCGGTAT ATGAATCCAT GGTCAGAATG

280 GCTCAGGATT TCAACTACCG TTATATGCTC GTTGACGGTC ACGGAAACTT CGGTTCTGTT

340 GACGGAGACT CAGCGGCGGC CATGCGTTAT ACAGAAGCAA GAATGTCTAA AATCTCAATG

400 GAGATTCTTC GTGACATCAC AAAAGACACA ATCGATTACC AGGATAACTA TGACGGGTCA

460 GAAAGAGAAC CTGTCGTTAT GCCTTCAAGG TTCCCGAATC TGCTCGTGAA CGGTGCTGCC

520 GGCATTGCGG TAGGTATGGC AACAAACATT CCTCCGCACC AGCTGGGAGA AATCATTGAC

580 GGTGTACTTG CTGTCAGTGA GAATCCGGAC ATTACAATTC CAGAGCTTAT GGAAGTCATT

640 CCAGGGCCTG ATTTCCCGAC CGCGGGTCAA ATCTTGGGAC GCAGCGGTAT CCGGAAAGCA

700 TACGAATCAG GCCGAGGCTC TATCACGATC CGGGCAAAAG CTGAGATCGA ACAAACATCT

760 TCGGGTAAAG AAAGAATTAT CGTTACAGAG TTACCTCACC AAGTAAATAA GGCGAAATTA

820 ATTGAGAAAA TTGCAGATCT CGTAAGGGAC AAAAAGATAG AGGGTATCAC AGATCTGCGT

880 GATGAGTCAG ATCGTACAGG TATGAGAATT GTCATTGAAA TCAGACGCGA CGCCAATGCA

940 AATGTCATCT TAAACAATCT GTACAAACAA ACTGCTCTAC AAACATCTTT TGGCATCAAC

1000?CTGCTTGCAC TTGTTGATGG CCGGCCGAAA GTTTTAACTC TTAAGCAATG CCTGGAGCAT

1060?TACCTTG

<210>5

<211>24bp

<212>DNA

< 213>artificial sequence

<220>

< 223>primer

<400>5

5‘-CAGTCAGGAAATGCGTACGTCCTT-3’

<210>6

<211>24bp

<212>DNA

< 213>artificial sequence

<220>

< 223>primer

<400>6

5’-CAAGGTAATGCTCCA?GGCATTGCT-3’

<210>7

<211>1259bp

< 212>gyrB gene fragment

< 213>bacillus order, Bacillaceae, Bacillus, Bacillus subtillis

<220>280bp-1538bp

< 223>this gene order coded product is dna gyrase B albumen gyrase (subunit B)

<400>7

280 GAAGTCATCA TGACCGTTCT GCACGCCGGA GGCAAGTTCG ACGGAAGCGG TATAAAGTAT

340 CCGGAGGATT ACACGGTGTA GGTGCGTCTG TCGTAAACGC ACTATCAACA GAGCTTGATG

400 TGACGGTTCA CCGTGACGGT AAAATTCACC GCCAAACCTA TAAACGCGGA GTTCCGGTTA

460 CAGACCTTGA AATCATTGGC GAAACGGATC ATACAGGAAC GACGACACAT TTTGTCCCGG

520 ACCCTGAAAT TTTCTCAGAA ACAACCGAGT ATGATTACGA TCTGCTTGCT AACCGCGTGC

580 GTGAATTAGC CTTTTTAACA AAGGGTGTAA ACATCACGAT TGAAGATAAA CGTGAAGGAC

640 AAGAGCGCAA AAATGAATAC CATTACGAAG GCGGAATTAA AAGTTATGTA GAGTATTTAA

700 ACCGCTCTAA AGAGGTTGTC CATGAAGAGC CGATTTACAT TGAAGGCGAA AAGGACGGCA

760 TTACGGTTGA AGTGGCTTTG CAATACAATG ACAGCTACAC AAGCAACATT TACTCGTTTA

820 CAAACAACAT TAACACGTAC GAAGGCGGTA CCCATGAAGC TGGCTTCAAA ACGGGCCTGA

880 CTCGTGTTAT CAACGATTAC GCCAGAAAAA AAGGGCTTAT TAAAGAAAAT GATCCAAACC

940 TAAGCGGAGA TGACGTAAGG GAAGGGCTGA CAGCGATTAT TTCAATCAAA CACCCTGATC

1000?CGCAGTTTGA GGGCCAAACG AAAACAAAGC TGGGCAACTC AGAAGCACGG ACGATCACCG

1060?ATACGTTATT TTCTACGGCG ATGGAAACAT TTATGCTGGA AAATCCAGAT GCAGCCAAAA

1120?AAATTGTCGA TAAAGGCTTA ATGGCGGCAA GAGCAAGAAT GGCTGCGAAA AAAGCCCGTG

1180?AACTAACACG TCGTAAGAGT GCTTTGGAAA TTTCAAACCT GCCCGGTAAG TTAGCGGACT

1240?GCTCTTCAAA AGATCCGAGC ATCTCCGAGT TATATATCGT AGAGGGTGAC TCTGCCGGAG

1300?GATCTGCTAA ACAAGGACGC GACAGACATT TCCAAGCCAT TTTGCCGCTT AGAGGTAAAA

1360?TCCTAAACGT TGAAAAGGCC AGACTGGATA AAATTCCTTC TAACAACGAA GTTCGCTCTA

1420?TGATCACAGC GCTCGGCACA GGCATTGGGG AAGACTTCAA CCTTGAGAAA GCCCGTTACC

1480?ACAAAGTTGT CATTATGACG GACGCGGACG TCGATGGCTC GCACATCCGT ACCCTGCT

<210>8

<211>35bp

<212>DNA

< 213>artificial sequence

<220>

< 223>primer

<400>8

5’-GAAGTCATCATGACCGTTCTGCA(TC)GC(TCAG)GG(TCAG)GG(TCAG)AA(AG)TT(TC)GA-3’

<210>9

<211>38bp

<212>DNA

< 213>artificial sequence

<220>

< 223>primer

<400>9

5’-AGCAGGGTACGGATGTGCGAGCC(AG)TC(TCAG)AC(AG)TC(TCAG)GC(AG)TC(TCAG)GTCAT-3’

Claims (6)

1. a plants endogenetic Bacillus subtillis R31 (Bacillus subtilis R31), preserving number is CCTCC N0:M209261.

2. a plants endogenetic Bacillus subtillis R31 according to claim 1 is characterized in that this bacterial strain is a gram-positive microorganism, and thalline is shaft-like, produces the brood cell, catalase, the oxidase test positive.

3. a plants endogenetic Bacillus subtillis R31 according to claim 1 is characterized in that this bacterial strain has the ability of hydrolyzed starch, casein, tyrosine, urea, tween 80, can utilize Citrate trianion and propionic salt; The V.P test is negative reaction, has glycolysis-ability, nitrate reduction ability, has the ability of producing indoles, hydrogen sulfide, otan, does not produce PD; Aerobic growth, growth pH scope is wider, and pH5.0～pH9.0 all can grow, the inclined to one side alkali well-grown of meta-acid; Meta-acid environment optimum growh pH value is 5.0, and alkali environment optimum growh pH is 9.5 partially, and optimum nitrogen source is the yeast extractive substance; Optimum carbon source is a sucrose, can not utilize oxysuccinic acid and glycocoll, can be 50 ℃ of growths; Can not grow for 55 ℃, can not grow for 10 ℃, can in 0.001% N,O-Diacetylmuramidase, 3%～7% NaCl, grow.

4. a plants endogenetic Bacillus subtillis R31 according to claim 1 is characterized in that, this bacterial strain is on the NA substratum, and aged colony colour is golden yellow, smooth surface is than thickness, fold occurs sometimes.

5. the application of any described plants endogenetic Bacillus subtillis R31 aspect the inhibition plant pathogenic fungi among the claim 1-4.

6. any described plants endogenetic Bacillus subtillis R31 causes the application aspect the banana blight in control by Fusarium oxysporum among the claim 1-4.

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