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’