CN102533593A - Burkholderia cepacia SD7 and culturing method and application thereof - Google Patents
Burkholderia cepacia SD7 and culturing method and application thereof Download PDFInfo
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Abstract
The invention discloses burkholderia cepacia SD7 and a culturing method and application thereof. The bacterial strain is preserved in China General Microbiological Culture Collection Center on October 24th, 2001, and the preservation number is CGMCC No.5384. Through research, the bacterial strain has strong inhibiting effect on rhizoctonia solani, collectotrichum musae, pyricularia grisea, gibberella zeae, fusarium oxysporum f. sp. cubense, peronophythora litchi, ustiago scitamimea syd, fulvia fulva, xanthomonas oryzaepv.oryzae, xanthomonas oryzae pv. Oryzicola, and the like, is broad-spectrum antagonistic bacteria, can be used for preventing and treating a plurality of plant diseases, particularly has better preventing and treating effect for banana fruit anthracnose and rice sheath blight, has the advantages of harmlessness to person and animals, environmental protection and safety, good environmental compatibility, and difficulty in becoming resistant, is good for business development, and has broad application prospect.
Description
Technical field
The invention belongs to plant protection (microbial pesticide) field, be specifically related to a kind of onion bulkholderia cepasea SD7 and cultural method and application.
Background technology
The Plant diseases that phytopathogen (comprising fungi, bacterium, virus etc.) causes has caused great financial loss to agriculture prodn.At present, the main method of important Plant diseasess such as control paddy rice, banana is to use sterilant.A large amount of uses of sterilant can increase the drug-fast risk of pathogenic bacteria, and can cause serious pollution to environment, and human and livestock health is also constituted a threat to.Biological control is a kind of new way to the friendly controlling plant diseases of environment.
Not only there is pathogenic bacteria around the plant, but also exists a large amount of nonpathogenic bacteria, the still antagonistic microbe of pathogenic bacteria that wherein has.These antagonistic microbes can suppress pathogenic bacteria through the mechanism such as antibiosis, competition effect, hyperparasitism and inducing plant generation system resistance with pathogenic bacteria, reach the purpose of controlling plant diseases.In addition, antagonistic microbe is environmentally safe, causes environmental pollution and to people and animals' murder by poisoning, antagonistic microbe is difficult for making pathogenic bacteria to produce resistance simultaneously unlike the chemical pesticide that kind, and some also has the effect that promotes plant-growth, and actual production technology is simple.Therefore, utilize antagonistic microbe to come the research of controlling plant diseases just more and more to receive attention both domestic and external.At present, people have been separated to multiplely has the antagonistic microbe of control effect in various degree to various different Plant diseasess, wherein some oneself through getting into practical stage, produced suitable social benefit and environmental benefit.
People to bulkholderia cepasea belong to (
Burkholderia) research more, mainly concentrate on the bacterial strain of using in this genuss and produce enzyme or be used to curb environmental pollution, like following patent documentation: the mikrobe and the application thereof of that decompose halogenated hydrocarbons of publication number 1195373 (Japan, the U.S. perfume (or spice) in middle mountain etc., open day 2004/03/24); The Hexose phosphate dehydrogenase and the method for producing this desaturase of publication number 1484703 (Japan, Zaochu Guangsi, open day 2004/03/24); The preparation method of the scyllo-inositol of publication number 1867676 (Japan, the mountain pass is with constitution etc., open day 2006/11/22); The method of the production lypase of publication number 101198695 (Germany, Bei Sailin etc., open day 2008/06/11); A kind of lead and cadmium tolerant bacteria of publication number 101153272 (China contains and to transfer etc., open day 2008/04/02) and be used for the plant restoration method of heavy metal pollution of soil; Publication number 102046778A (Japan, Gu Heyi control, increase field gentry I, open day 2011/05/04) the reduction plant materials in the bacterium of heavy metal content; A kind of heavy metal resistance plant growth-promoting bacteria preparation and the application process thereof of publication number 101671636 (China, Guo Junkang etc., open day 2010/03/17).All do not relate to the related application of phytopathogen biological control in the above document.
Aspect the biological control of Plant diseases; Discovery Burkholderia pyrrocinia JK-SH007 such as Ren Jiahong to dothiorella gregaria have control effect (Ren Jiahong etc. the separation and purification of Burkholderia pyrrocinia JK-SH007 antibacterial protein. microbiology circular, 2010-06-20; Ren Jiahong etc. the fermentation condition of Burkholderia pyrrocinia JK-SH007 and to the control effect of dothiorella gregaria. Chinese biological control, 2010-08-08; Ye Jianren etc. a kind of Burkholderia pyrrocinia and the application in the control dothiorella gregaria thereof. Chinese patent, 2009-10-14); Look into pass brave the discoverys onion bulkholderia cepasea Lu10-1 of grade can produce antibacterial substance (look into pass brave etc. the fermention medium and the fermentation condition optimization of onion bulkholderia cepasea Lu10-1 generation antibacterial substance. silkworm is science already, 2009-06-15).
Discovery bulkholderia cepasea such as Lin Shanhai have control effect (Lin Shanhai etc. to banana sigatoka leaf spot disease; The antagonism bulkholderia cepasea is to the biological control test of banana sigatoka leaf spot disease. Chinese Plants pathology meeting academic nd Annual Meeting collection in 2007,2007-08-01).
Summary of the invention
The objective of the invention is to various plant pathogenic fungis in the prior art and bacterial Plant diseases chemical agent control spinoff is big, the biological control medicine lacks defective, a kind of wide spectrum biocontrol strain that suppresses to comprise the various plants pathogenic bacteria of Glorosprium musarum Cookeet Mass and Rhizoctonia solani Kuhn is provided.
The present invention realizes above-mentioned purpose through following technical scheme:
Onion bulkholderia cepasea SD7, classification called after: onion bulkholderia cepasea
Burkholderia cepacia, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, address on October 24th, 2011: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number CGMCC No.5384.The 16S rDNA sequence of this bacterial strain is shown in SEQ ID NO:1.
The present invention also provides a kind of bacteria agent; This bacteria agent contains onion bulkholderia cepasea SD7 or contains onion bulkholderia cepasea SD7 fermented liquid (being nutrient solution) bacteria-free filtrate after filtering, and the supernatant that said bacteria-free filtrate preferably gets through centrifuging and taking with onion bulkholderia cepasea SD7 nutrient solution filters through biofilter and obtains.
The cultural method of this onion bulkholderia cepasea SD7; It is characterized in that fermentation condition is following: LB substratum (yeast extract powder 5 g, Tryptones 10 g, NaCl 10 g, agar 15～18 g, water 1000 mL; PH 6.0～7.0); Liquid amount 100 mL add the 1 mL seed bacterial strain in 2 d ages (being the every 100mL LB culture medium inoculated 1 mL seed bacterial strain in 2 d ages), under 25 ℃ of conditions, cultivate 3 d with 180 r/min.
The application of onion bulkholderia cepasea SD7 microbial inoculum in preparation control plant pathogenic fungi and/or bacterial Plant diseases.Said plant pathogenic fungi be Rhizoctonia solani Kuhn (
Rhizoctonia solani), Glorosprium musarum Cookeet Mass (
Colletotrichum musae), Pyricularia oryzae (
Pyricularia oryzae), fusarium graminearum (
Gibberella zeae), banana blight bacteria (
Fusarium oxysporumF. sp.
Cubense), peronophythora litchi (
Peronophythora litchii), the sugarcane black rot (
Ceratocystis adiposum) and the leaf muld of tomato bacterium (
Fulvia fulva); Said plant pathogenetic bacteria be rice leaf spot bacteria (
Xanthomonas oryzaePv.
Oryzae) and xanthomonas oryzae pv. oryzicola (
X. oryzaePv.
Oryzicola).
Compared with prior art, the present invention has following beneficial effect:
Through experiment confirm, the aseptic ferment filtrate of onion bulkholderia cepasea SD7 of the present invention is 88.79% to the inhibiting rate of Rhizoctonia solani Kuhn mycelia, and the inhibiting rate that sclerotium is sprouted is 90%.Isolated experiment shows that the preventive effect to rice sheath blight disease reaches 90.0%, and the preventive effect of rice sheath blight disease is 53.7% to seedling stage.Simultaneously, the indoor biocontrol effect to banana anthrax also reaches 52.12%.Therefore; This bacterial strain is a kind of wide spectrum biocontrol strain that the various plants pathogenic bacteria is all had the strongly inhibited effect; And have good to person poultry harmless, green safety, environment compatibility, be not easy to produce advantages such as resistance, can commercialize, have a extensive future.
Description of drawings
Fig. 1. the cultivation form of SD7 bacterial strain on the LB substratum.
Fig. 2. the 16S rDNA sequence of SD7 bacterial strain and the phylogenetic tree of close bacterial strain sequence.
Fig. 3. onion bulkholderia cepasea SD7 is to the control effect of banana anthrax; Wherein 1 for only inoculating the control group CK1 of anthrax bacteria; 2 ~ 5 inoculate the experimental group of anthrax bacteria after handling again for the bacteria-free filtrate of onion bulkholderia cepasea SD7, and 6～7 for only adding the control group CK2 of onion bulkholderia cepasea SD7 bacteria-free filtrate.
Fig. 4. onion bulkholderia cepasea SD7 fermented liquid is to the preventive effect experimental result of rice sheath blight disease, and wherein 1 is the SD7 sterile liquid, and 2 is SD7 bacterium liquid, and 3 is the LB nutrient solution, and 4 is jingganmycin, and 5 are the sterilized water contrast.
Embodiment
Screening and the evaluation of embodiment 1 onion bulkholderia cepasea SD7
1, the screening of bacterial strain
(1) collection of pedotheque
Soil sample is gathered in this experiment from orchard, Agricultural University Of South China farm; According to 5 samplings of employings such as the landform of gathering ground; Sampling depth is 15 cm below the arable layer or the face of land, the amount of getting 200～250 g, 5 sample uniform mixing that gather in each place; The polyethylene bag that seals of packing into and sterilizing is taken back the laboratory and is separated at once.
(2) preparation of sample suspension
Pedotheque is added fierce concussion in the aqua sterilisa.Sample: water=1:9 (quality and volume ratio) adds like: 10 g soil that the gained suspension concentration is 10 in the triangular flask that 90 mL aqua sterilisas are housed
-1Every kind of sample prepared 5 test tubes that the 9mL aqua sterilisa is housed by 10 times of gradient dilution methods, is 10 with above-mentioned concentration
-1Suspension-s be diluted to 10
-2～10
-6Series concentration suspension-s; Promptly from No. 1 test tube, draw 1 mL suspension-s and put into No. 2 test tubes (filling 9 mL aqua sterilisas), fully shake mixing, from No. 2 test tubes, draw 1 mL suspension-s again and put into No. 3 test tubes (filling 9 mL aqua sterilisas); The rest may be inferred, till the 6th test tube.
(3) separation method
Adopt LB substratum (yeast extract powder 5 g, Tryptones 10 g, NaCl 10 g, agar 15～18 g, water 1000 mL).Draw the dilution solution 0.5mL of above-mentioned difference respectively with pipettor, drip by on culture medium flat plate (3 flat boards/extent of dilution), even with the L type glass rod coating of sterilization; Dry slightly; Perform mark and date, petridish is inverted, and in 25～28 ℃ of incubators, cultivates 2～3 d; The single bacterium colony that grows on the flat board is chosen to the new substratum purifying to be cultivated; Bacterial strain (being called bacterial strain to be measured) to all being separated to is numbered, and then its antagonistic activity is carried out primary dcreening operation and multiple sieve, to obtain this patent bacterial strain.
(4) antimicrobial primary dcreening operation short of money and multiple sieve
Adopt dull and stereotyped face-off culture method, detect the bacteriostatic activity of above-mentioned various bacterial strain to be measured pathogenic bacterias such as Rhizoctonia solani Kuhn and Glorosprium musarum Cookeet Masses.Beat the mycelia piece of cut-off footpath 5 mm at the colony edge of pathogenic bacterias such as cultured Rhizoctonia solani Kuhn and Glorosprium musarum Cookeet Mass with punch tool; Be seeded in the dull and stereotyped central authorities of PDA; To the various inoculation to be measured of activatory 2 ~ 3cm place around the mycelia piece of pathogenic bacterias such as Rhizoctonia solani Kuhn or Glorosprium musarum Cookeet Mass again; 4 bacterial strains to be measured of each PDA plating are provided with the contrast of not inoculating bacterial strain to be measured, and every processing repeats for 3 times.Cultivate 3 ~ 5 d down at 25 ~ 28 ℃, measure the antibacterial bandwidth between the pathogenic bacterias such as bacterial strain to be measured and Rhizoctonia solani Kuhn or Glorosprium musarum Cookeet Mass then, as the strong and weak index of antagonistic activity.
Through above-mentioned primary dcreening operation and multiple sieve, obtain a strain multiple pathogenic bacterias such as Rhizoctonia solani Kuhn and Glorosprium musarum Cookeet Mass are had the bacterial strain of strongly inhibited effect, be numbered SD7, preserve subsequent use.
, the SD7 bacterial strain morphology and Molecular Identification
(1) morphological observation
On the LB substratum, cultivate 48 h, bacterium colony is circular, and oyster white is translucent, cement, and the edge is more neat, along with incubation time increases, the bacterium colony thickening, median rise, and its yeast culture thing is than thickness (Fig. 1); Liquid LB cultivates and can enrich muddiness of growth, bacterium liquid.
(2) molecular biology and Physiology and biochemistry are identified
Through the cloning and sequencing of PCR reaction and specific fragment, obtain the 16S rDNA sequence (SEQ ID NO:1) of SD7 bacterial strain:
With the 16S rDNA sequence of SD7 bacterial strain with Blast software and having logined of from GenBank, searching for
Burkholderia cepacia(DQ387437),
Burkholderia vietnamiensis(AY741147),
Burkholderia anthina(AJ420880),
Burkholderia cenocepacia(FJ810079),
Burkholderia multivorans(AB222021),
Paenibacillus daejeonensis(NR025170) 16S rDNA sequence is carried out homology relatively, and constructing system is grown tree (Fig. 2).
According to " uncle's Jie Shi bacteriology handbook and " common bacteria system identification handbook " record the Physiology and biochemistry proterties of SD7 bacterial strain, and experimental result is (table 1) as follows.
The Physiology and biochemistry proterties of table 1 SD7 bacterial strain
| The Physiology and biochemistry proterties | Reaction |
| Denitrification | – |
| Citrate trianion utilizes | + |
| Leucine utilizes | + |
| Tartrate utilizes | – |
| Catalase | + |
Annotate :+expression is positive; – representes to be negative
Analyze through form, Physiology and biochemistry proterties and 16S rDNA sequence alignment, the SD7 identification of strains is the onion bulkholderia cepasea the most at last
Burkholderia cepaciaThe present invention is called for short onion bulkholderia cepasea SD7.
1, onion bulkholderia cepasea SD7 is to the preventive and therapeutic effect of banana anthrax
(1) supplies examination banana anthracnose bacteria strain
The Glorosprium musarum Cookeet Mass that is used to inoculate (
C. musae) be that separation and purification obtains from the banana of morbidity, and preserve by this laboratory.
(2) onion bulkholderia cepasea SD7 is to the preventive and therapeutic effect of banana anthrax
The onion bulkholderia cepasea SD7 bacterial strain seed in 2d age is by per 100 mL LB substratum (yeast extract powder 5g, Tryptones 10 g, NaCl 10 g, agar powder 15～18 g, water 1000 mL; PH 7.0～7.2) the ratio inoculation enlarged culturing of inoculation 1 mL seed; Culture condition is: rotating speed 180 r/min, 25 ℃, cultivate 3d after; Supernatant is collected in centrifugal back, filters with biofilter, obtains bacteria-free filtrate.Choose the banana of ripe degree very likely, carry out surface sterilization with 70% alcohol after, respectively in upper, middle and lower the punching of three positions as well.Only inoculate the Glorosprium musarum Cookeet Mass spore suspension of 20 μ L in control group (CK1) hole; The bacteria-free filtrate that adds 20 μ L onion bulkholderia cepasea SD7 in the experimental group hole adds the Glorosprium musarum Cookeet Mass spore suspension of 20 μ L again behind the natural air drying; Only inoculate onion bulkholderia cepasea SD7 bacterium liquid (purpose is to confirm whether it causes a disease to banana) in control group (CK2) hole.28 ℃ of cultivations of preserving moisture, observations behind 5 d is measured the scab size.10 repetitions of every processing.
The method of calculation of control effect:
Control effect (%)=[lesion area of (lesion area of the lesion area ﹣ experimental group of CK1)/CK1] * 100
Control effect: onion bulkholderia cepasea SD7 is 52.12% (Fig. 3) to the preventive effect of banana anthrax.
2, onion bulkholderia cepasea SD7 bacterial strain is to the control experiment of rice sheath blight disease
(1) Rhizoctonia solani Kuhn strains tested
The strong virulence bacterial strain that the Rhizoctonia solani Kuhn strains tested GD-118 that this experiment is used preserves for this laboratory is (referring to document: Yang Yingqing etc.; The optimization of preparation of Rhizoctonia solani Kuhn protoplastis and regeneration condition; Hua Zhong Agriculture University's journal; In October, 2010, the 29th the 5th phase of volume: 546-551).
(2) paddy rice excised leaf experiment
The clip paddy rice becomes the strain blade, gets the long leaf section of middle part 10 cm as experiment material, carries out following processing respectively:
SD7 sterile liquid group: soak leaf section 10min with the SD7 bacteria-free filtrate, bacteria-free filtrate is that 25 ℃ of onion bulkholderia cepasea SD7 fermented liquids processes of cultivating 72 h are centrifugal, and supernatant filters the sterile liquid of gained with biofilter;
SD7 bacterium liquid group: press 10 with 25 ℃ of onion bulkholderia cepasea SD7 fermented liquids of cultivating 72 h
10The concentration of CFU/mL is soaked leaf section 10min;
LB nutrient solution control group: the blank LB nutrient solution with sterilization soaks leaf section 10min;
The jingganmycin control group: using concentration is the jingganmycin solution soaking leaf section 10min of 40 μ g/mL;
Sterilized water control group (CK): soak leaf section 10min with sterilized water.
More than 20 leaf sections of each component other places reason, the leaf section after handling neatly is placed in place mat to be had in the plastic tub of 2 layers of sterile gauze, in leaf section middle part, wherein CK does not inoculate the GD-118 bacterial strain with the inoculated by hypha block of the Rhizoctonia solani Kuhn strains tested GD-118 of 5 mm.All handle with the preservative film sealing and preserve moisture, place 28～30 ℃, illumination condition to handle 3 d " Invest, Then Investigate " incidence of leaf numbers and sick level down.Scab grade scale reference literature (Zhou Erxun etc., the virulence of Water in Guangdong Province Rhizoctonia solani Kuhn and fusion crowd research, guangdong agricultural science, 1999 the 5th phases: method 36-38).The area that promptly accounts for blade with scab is divided into 5 grades with incidence: not falling ill is 0 grade; Be 1 grade 0～1/8 (not comprising 1/8), and be 2 grades 1/8～1/4 (not comprising 1/4); Be 3 grades 1/4～1/2 (not comprising 1/2); Be 4 grades 1/2～3/4 (not comprising 3/4); 3/4 and more than be 5 grades.
Experimental result: onion bulkholderia cepasea SD7 fermented liquid preventive effect is up to 90.0% (Fig. 4).
(3) seedling stage rice sheath blight disease control experiment
Susceptible rice varieties force is educated No. 3 vernalization of round-grained rice be sowed in the loaf case of white, every basin 6 rows, 10 basins are broadcast in every row's 10 strains altogether.When rice seedling length during to 3 leaves, 1 heart, adopt the grain inoculum to inoculate, be about to paddy kernel and be tiled on the PDA culture plate that is connected to bacterial strain GD-118 mycelia piece, treat that mycelia is covered with grain after, 2 paddy that carry disease germs of base portion inoculation of every young plant.Spray processing next day, establish 4 groups altogether, SD7 bacterium liquid group spraying concentration is 10
10Bacterium liquid 100 mL of CFU/mL; SD7 bacteria-free filtrate group sprays sterile liquid, and (25 ℃ of onion bulkholderia cepasea SD7 fermented liquids of cultivating 72 h are through centrifugal; Supernatant filters gained with biofilter) 100 mL; Jingganmycin control group spraying concentration is jingganmycin solution 100 mL of 40 μ g/mL, selects one group to be left intact as negative control in addition.Spray the back and paddy rice is covered, wait to contrast the sickness rate and relative scab height of falling ill about 10 d, and calculate sick level and disease index to appropriate level " Invest, Then Investigate " rice seedling with plastics film.The method of disease classification (9 grades) standard employing Tang Fang (reference: Tang Fang. the virulence differentiation of Rhizoctonia solani Kuhn and genetic diversity research. Guangzhou: Agricultural University Of South China's master thesis. 2009:1 – 89); Disease index calculate method with reference to Fang Zhongda (reference: Fang Zhongda. plant disease research method (third edition). Beijing: Chinese agriculture press, 1998:11 – 12).The calculation formula of sick level and disease index is following:
Sick level=(scab height ÷ plant height) * 9 grades
Disease index=[∑ (diseased plant numbers at different levels * corresponding progression)/investigation total strain number * highest level value] * 100
Control effect: onion bulkholderia cepasea SD7 fermented liquid reaches 53% (table 2) to the preventive effect of rice sheath blight disease.
Table 2 onion bulkholderia cepasea SD7 fermented liquid is to the control effect of rice sheath blight disease
| Treatment group | Sickness rate (%) | Disease index | Preventive effect (%) |
| SD7 bacterium liquid group | 76.74 | 22.38 d | 53.73 |
| SD7 bacteria-free filtrate group | 94.44 | 24.86 c | 48.60 |
| The jingganmycin control group | 67.39 | 14.99 e | 69.00 |
| The sterilized | 100 | 48.37 a | 0 |
Annotate: The data DuncanShi duncan's new multiple range method (DMRT) carries out significance of difference analysis in the table, and to be illustrated on 5% level (P=0.05) difference not remarkable with having same letter person behind the column data.
, onion bulkholderia cepasea SD7 is to the restraining effect of various plants pathogenic fungi
(1) strains tested
Plant pathogenic fungi that this experiment is used and pathogenetic bacteria all are the bacterial strain of being preserved by this laboratory.
(2) to the restraining effect of various plants pathogenic fungi
With the inoculating needle of sterilization in the PDA culture medium flat plate on one side, with onion bulkholderia cepasea SD7 inoculation simultaneously with Pyricularia oryzae (
P. oryzae), fusarium graminearum (
G. zeae), banana blight bacteria (
F. oxysporumF. sp.
Cubense), the banana anthrax-bacilus (
C. musae), lichee frost epidemic disease mould (
P. litchii), the sugarcane black rot (
C. adiposum) and the leaf muld of tomato bacterium (
C. fulvum) wait pathogenic fungi mycelium inoculation in the cultivation (in the flat board corresponding cultivate a kind of pathogenic bacteria and onion Burkholder SD7 stands facing each other) that stands facing each other of PDA culture medium flat plate the other side; Put in the biochemical incubator and cultivate 2～3 d down, observe antibacterial situation and inhibition zone size (table 4) at 28 ℃.
(3) to the restraining effect of two kind of plant pathogenetic bacterias
With the inoculating needle of sterilization with onion bulkholderia cepasea SD7 inoculation in the dull and stereotyped center of PSA, cultivate bacterium colony under 37 ℃ and be about 5 mm sizes (24～48 h).Cover at petridish and to add 3.0 mL chloroforms, left-hand thread petridish 2 h are with killing bacteria.Ventilating kitchen take out the imitative back of dechlorination in petridish, pour into about 200 μ L rice leaf spot bacterias (
X. oryzaePv.
Oryzae) or xanthomonas oryzae pv. oryzicola (
X. oryzaePv.
Oryzicola) bacterium liquid (seeing table 4) such as bacterium, evenly be laid on media surface, on super clean bench, dry up, cultivate about 24 h down for 28 ℃ then, observe antibacterial situation and inhibition zone size (table 4).
Table 4 onion bulkholderia cepasea SD7 is to the inhibition effect of various plants pathogenic fungi and bacterium
| Pathogenic bacteria | SD7 bacterial strain inhibition zone radius (cm) |
| Glorosprium musarum Cookeet Mass ( Cm) | 1.60 ± 0.35 |
| Pyricularia oryzae ( Po) | 0.93 ± 0.15 |
| Fusarium graminearum ( Gz) | 1.18 ± 0.44 |
| Banana blight ( Foc) | 2.20 ± 0.16 |
| Lichee frost epidemic disease mould ( Pl) | 1.06 ± 0.20 |
| The sugarcane black rot ( Ca) | 0.54 ± 0.08 |
| Leaf muld of tomato ( Cf) | 1.15 ± 0.13 |
| Paddy rice bacterial leaf spot bacterium ( Xooe) | 1.72 ± 0.20 |
| Xanthomonas oryzae pv. oryzicola ( Xooa) | 1.38 ± 0.35 |
In sum; Onion bulkholderia cepasea SD7 all has antagonistic action to multiple important plant pathogenic fungi and bacterium; For wide spectrum antagonism bacterium, can be used for preventing and treating plurality of plant diseases, especially preferable to the control effect of banana anthrax and rice sheath blight disease.
SEQUENCE?LISTING
< 110>Agricultural University Of South China
< 120>onion bulkholderia cepasea SD7 and cultural method thereof and application
<130>
<160> 1
<170> PatentIn?version?3.3
<210> 1
<211> 1486
<212> DNA
<213> 16S?rDNA
<400> 1
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gcccggcgaa?agccggatta?ataccgcata?cgatctacgg?atgaaagcgg?gggaccttcg 180
ggcctcgcgc?tatagggttg?gccgatggct?gattagctag?ttggtggggt?aaaggcctac 240
caaggcgacg?atcagtagct?ggtctgagag?gacgaccagc?cacactggga?ctgagacacg 300
gcccagactc?ctacgggagg?cagcagtggg?gaattttgga?caatgggcga?aagcctgatc 360
cagcaatgcc?gcgtgtgtga?agaaggcctt?cgggttgtaa?agcacttttg?tccggaaaga 420
aatccttggc?tctaatacag?ycgggggatg?acggtaccgg?aagaataagc?accggctaac 480
tacgtgccag?cagccgcggt?aatacgtagg?gtgcaagcgt?taatcggaat?tactgggcgt 540
aaagcgtgcg?caggcggttt?gctaagaccg?atgtgaaatc?cccgggctca?acctgggaac 600
tgcattggtg?actggcaggc?tagagtatgg?cagagggggg?tagaattcca?cgtgtagcag 660
tgaaatgcgt?agagatgtgg?aggaataccg?atggcgaagg?cagccccctg?ggccaatact 720
gacgctcatg?cacgaaagcg?tggggagcaa?acaggattag?ataccctggt?agtccacgcc 780
ctaaacgatg?tcaactagtt?gttggggatt?catttcctta?gtaacgtagc?taacgcgtga 840
agttgaccgc?ctggggagta?cggtcgcaag?attaaaactc?aaaggaattg?acggggaccc 900
gcacaagcgg?tggatgatgt?ggattaattc?gatgcaacgc?gaaaaacctt?acctaccctt 960
gacatggtcg?gaatcctgaa?gagattcggg?agtgctcgaa?agagaaccgg?cgcacaggtg 1020
ctgcatggct?gtcgtcagct?cgtgtcgtga?gatgttgggt?taagtcccgc?aacgagcgca 1080
acccttgtcc?ttagttgcta?cgcaagagca?ctctaaggag?actgccggtg?acaaaccgga 1140
ggaaggtggg?gatgacgtca?agtcctcatg?gcccttatgg?gtagggcttc?acacgtcata 1200
caatggtcgg?aacagagggt?tgccaacccg?cgagggggag?ctaatcccag?aaaaccgatc 1260
gtagtccgga?ttgcactctg?caactcgagt?gcatgaagct?ggaatcgcta?gtaatcgcgg 1320
atcagcatgc?cgcggtgaat?acgttcccgg?gtcttgtaca?caccgcccgt?cacaccatgg 1380
gagtgggttt?taccagaagt?ggctagtcta?accgcaagga?ggacggtcac?cacggtagga 1440
ttcatgactg?gggtgaagtc?gtaacatggt?agccgtatcg?gaaggt 1486
Claims (6)
1. onion bulkholderia cepasea SD7 was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number CGMCC No.5384 on October 24th, 2011.
2. the said onion bulkholderia cepasea of claim 1 SD7, the 16S rDNA sequence that it is characterized in that this bacterial strain is shown in SEQ ID NO:1.
3. a bacteria agent is characterized in that containing claim 1 or 2 said onion bulkholderia cepasea SD7 or contains onion bulkholderia cepasea SD7 fermented liquid bacteria-free filtrate after filtering.
4. the enlarged culturing method of claim 1 or 2 said onion bulkholderia cepasea SD7 is characterized in that culture condition is following: every 100mL LB substratum adds the 1mL seed bacterial strain in 2 day age, under 25 ℃ of conditions, cultivates 3 d with 180 r/min;
The LB culture medium prescription is: yeast extract powder 5g, Tryptones 10g, NaCl 10g, agar powder 15～18g, water 1000 mL, pH 6.0～7.0.
5. claim 1 or the 2 said onion bulkholderia cepasea SD7 application in the Plant diseases microbial inoculum that preparation control plant pathogenic fungi and/or plant pathogenetic bacteria cause.
6. application according to claim 5 is characterized in that said plant pathogenic fungi is Rhizoctonia solani Kuhn, Glorosprium musarum Cookeet Mass, Pyricularia oryzae, fusarium graminearum, banana blight bacteria, peronophythora litchi, sugarcane black rot or leaf muld of tomato bacterium; Said plant pathogenetic bacteria is rice leaf spot bacteria or xanthomonas oryzae pv. oryzicola.
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