CN102433282B - Bacillus subtilis NB12, as well as culture method and application thereof - Google Patents

Abstract

The invention discloses bacillus subtilis NB12, as well as a culture method and an application thereof. The strain with a preservation number of CGMCC No.5383 is preserved in China General Microbiological Culture Collection Center on October 24, 2011. Researches prove that: the strain has strong inhibition effects to rhizoctonia solani, colletotrichum musae, pyricularia grisea, fusarium graminearum, fusarium oxysporum f.sp.cubense, peronophythora litchi, ustilago scitaminea, fulvia fulva, xanthomonas oryzae and xanthomonas oryzae pv.oryzicola, is a broad-spectrum antagonistic strain, can be used for prevention and control of various plant diseases, has a particular good prevention and control effect to the rice sheath blight and damping off in seedling phase of cowpea, has the advantages of harmlessness to human and livestock, greenness and safety, good environment compatibility, difficult resistance generation and the like, can be used for commercial development, and has a wide application prospect.

Description

Subtilis NB12 and cultural method thereof and application

Technical field

The invention belongs to plant protection (microbial pesticide) field, be specifically related to a kind of subtilis NB12 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 production.Dry thread Pyrenomycetes ( Rhizoctonia solaniK ü hn) is a kind of economically very important plant pathogenic fungi.In China, occupied first of the paddy rice three large diseases by the microbial rice sheath blight disease of miliary damping-off.This germ also can cause the seedling blight of other a lot of plants (vegetables, fruit tree, forest, flowers etc.) except can causing the banded sclerotial blights such as paddy rice, corn.Mainly be to use chemical pesticide (sterilant) to the control of this disease at present, and mostly sterilant is the interior absorption medicament in single-acting site, life-time service can make pathogenic bacteria develop immunity to drugs, and pollutes the environment, and residual sterilant can have influence on food safety.

Not only there is pathogenic bacteria around the plant, but also exists a large amount of nonpathogenic bacteria, antagonistic microbe that wherein have or pathogenic bacteria.These antagonistic microbes can suppress pathogenic bacteria by the mechanism such as antibiosis, Competition, 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 chemical pesticide, and some also has the effect of Promoting plant growth, and actual production technique is simple.Therefore, utilize antagonistic microbe to come the research of controlling plant diseases just more and more to be subject to attention both domestic and external.At present, people have been separated to multiplely has the in various degree antagonistic microbe of prevention effect to various different Plant diseasess, wherein some oneself through entering practical stage, produced suitable social benefit and environmental benefit.

People to subtilis ( BacillusSpp.) research is more, this bacterium can produce various bioactivators, such as subtilyne, polymyxin, nystatin and linear gramicidins etc., pathogenic bacteria had obvious restraining effect, self can also synthesize α-amylase, lipase and cellulase etc., can be used for animal feedstuff additive, in digestive tube with animal body in digestive enzymes together play a role; This bacterial classification can also grow by stimulating plant simultaneously.

About subtilis ( Bacillussubtilis) report is also arranged aspect rice sheath blight disease and other disease preventing and treating, such as Chinese patent application 200410014484.1(publication number CN1590535) a kind of subtilis JA is disclosed, all inhibited to various plants pathogenic bacterias such as Rhizoctonia solani Kuhn, fusarium graminearum, withered germ of water-melon; Chinese patent application 200910212779.2(publication number CN101697737A) disclose and be used for the control rice blast as sterilant afterwards with subtilis and tricyclazole are composite, prevention effect is up to 84.67%; Chinese patent application 201010175797.0(publication number CN101857848A) a kind of biocontrol bacillus subtilis WJ-1 for rice sheath blight and microbial inoculum and application are disclosed, this microbial inoculum is obvious to rice sheath blight disease indoor living inoculation prevention effect, reaches as high as 95.74%; In addition, relevant document also has: Chen Zhiyi etc. and subtilis B-916 prevents and treats the field test of rice sheath blight disease. Chinese biological control, 1997; Chen Zhiyi etc. the research of Antagonistic Bacteria Against Rhizoctonia Solani B-916 culture condition and fermentating formula. southwestern agriculture journal, 1999; Chen Wei is good etc. antagonistic bacterium Bacillus subtilisA30 is to the restraining effect of rice pathogens. Zhejiang Agricultural Univ's journal, 1997; Xu Hanhong etc. the preliminary study of Bacillus subtilus BS04 bacterial strain fungicides. agricultural chemicals, 2007; Lixia ZHANG etc. the optimization of fermentation of bacillus subtilis substratum, Plant Pathology, 2005.

Summary of the invention

The object of the invention is to, provide a strain to have to suppress the wide spectrum Antagonistic Fungi bacterial strain of the various plants pathogenic bacteria that comprises Rhizoctonia solani Kuhn and fresh kidney beans seedling blight bacterium.

The present invention is achieved through the following technical solutions above-mentioned purpose:

Subtilis NB12, the Classification And Nomenclature of this bacterial strain are subtilis Bacillus subtilis, 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.5383.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 subtilis NB12 or contains subtilis NB12 fermented liquid (being nutrient solution) bacteria-free filtrate after filtering, and the supernatant that described bacteria-free filtrate preferably gets through centrifuging and taking with subtilis NB12 nutrient solution filters through biofilter and obtains.

This subtilis ( B. subtilis) cultural method of NB12, fermentation (cultivation) condition is as follows: substratum is LB(yeast extract powder 5 g, Tryptones 10 g, NaCl 10 g, agar 15～18 g, water 1000 mL) or BPY substratum (extractum carnis 5g, yeast extract paste 5g, glucose 5g, peptone 10g, NaCl 5g, agar 20g distilled water 1000ml, transfer pH to 7.2), under 30 ℃, rotating speed 180 r/min conditions, cultivate 48h and get final product.Preferred initial pH value is 7.0,40 mL/250 mL(seed/substratum) liquid amount.

Subtilis ( B. subtilis) application of NB12 in the microbial Plant diseases microbial inoculum of preparation control pathogenic.Described phytopathogen be dry thread Pyrenomycetes ( 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), Fulvia fulva ( Fulvia fulva), rice leaf spot bacteria ( Xanthomonas oryzaePv. Oryzae) and xanthomonas oryzae pv. oryzicola ( X. oryzaePv. Oryzicola).

Subtilis ( B. subtilis) NB12 the control dry thread Pyrenomycetes ( R. solani) application in the Plant diseases that causes, especially better to rice sheath blight disease and fresh kidney beans seedling blight prevention effect.

Compared with prior art, the present invention has following beneficial effect:

The subtilis that the present invention announces ( B. subtilis) the NB12 bacterial strain is higher to the preventive effect of rice sheath blight disease, particularly the sprouting for the Rhizoctonia solani Kuhn sclerotium has obvious restraining effect, and this has no report in document in the past.Sclerotium is the important sources that rice sheath blight disease just infects, and the sprouting that suppresses sclerotium can be controlled the generation of this disease effectively, and this is one of characteristics of the present invention.Simultaneously, subtilis of the present invention ( B. subtilis) the NB12 bacterial strain also has good restraining effect to other various plants pathogenic bacteria, and is different with the controlling object of disclosed other bacillus subtilis strain of Chinese patent, especially can be used in prevent and treat on the vegetables seedling blight ( R. solani), be with a wide range of applications and be worth.This has no report in document in the past.

Through experiment confirm, subtilis of the present invention ( B. subtilis) NB12 fermented liquid (microbial inoculum) all shows good prevention effect to rice sheath blight disease and fresh kidney beans seedling blight, wherein the preventive effect of Detached Rice Leaves banded sclerotial blight reached 86.2%, this germ sclerotium Germination suppression rate is reached 84.52%, preventive effect to the pot rice banded sclerotial blight is 57.51%, is 65.38% to the preventive effect of fresh kidney beans seedling blight.This shows that this biocontrol bacteria NB12 bacterial strain has huge application prospect aspect the control dry thread Pyrenomycetes disease.The preparation method of its microbial inoculum is ripe; can be developed into microbial pesticide; it is the substitute of chemical pesticide; can be used for the control of the dry thread Pyrenomycetes disease such as rice sheath blight disease and other Plant diseases; residual and pollute, preserve the ecological environment significant to reducing chemical pesticide; be the developing direction that green agriculture and Organic farming are advocated, meet the requirement of China's modern agricultural development.Therefore, in industrial production, only need better simply microbial fermentation and preparation equipment, just can carry out large-scale promotion and use, its market outlook are wide, will bring huge ecological benefits, Social benefit and economic benefit.

Description of drawings

Fig. 1. the phylogenetic tree of the sequence of bacterial strain NB12 and close bacterial strain sequence.

Fig. 2. subtilis NB12 wherein inoculates the GD-118 contrast for not preventing and treating contrast to the prevention effect of rice sheath blight disease, and CK is normal paddy rice contrast.

Embodiment

Screening and the evaluation of embodiment 1 subtilis NB12

1, the screening of bacterial strain

(1) collection of pedotheque

This experiment gathers soil sample from orchard, Agricultural University Of South China farm, according to 5 methods samplings of the employings such as the landform that gathers ground, each some sampling depth is following 15 cm in arable layer or earth's surface, taken amount 200～250 g, 5 samples that each place gathers evenly mix, the sealing polyethylene bag 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), add such as: 10 g soil that the gained suspension concentration is 10 in the triangular flask that 90 mL aqua sterilisas are housed ^-1, every 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 be diluted to 10 ^-2～10 ^-6Series concentration suspension, namely from No. 1 test tube, draw 1 mL suspension and put into No. 2 test tubes (filling 9 mL aqua sterilisas), fully shake mixing, from No. 2 test tubes, draw 1 mL suspension again and put into No. 3 test tubes (filling 9 mL aqua sterilisas), the rest may be inferred, until 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 respectively the dilution solution 0.5mL of above-mentioned difference with pipettor, drip on culture medium flat plate (each extent of dilution is done 3 repetitions), L-type glass rod coating with sterilization is even, slightly dry, perform mark and date, culture dish is inverted, in 25～28 ℃ of incubators, cultivate 2～3 d, the single bacterium colony that grows on the flat board is chosen to the new substratum purifying to be cultivated, the bacterial strain (being called bacterial strain to be measured) that all is separated to is numbered, then its antagonistic activity is carried out primary dcreening operation and multiple sieve, to obtain this patent bacterial strain.

(4) primary dcreening operation of Antagonistic Fungi and multiple sieve

Adopt dull and stereotyped face-off culture method, detect above-mentioned various bacterial strain to be measured to the bacteriostatic activity of the pathogenic bacterias such as dry thread Pyrenomycetes.Beat the mycelia piece of cut-off footpath 5 mm at the colony edge of the pathogenic bacterias such as cultured dry thread Pyrenomycetes with punch tool, be seeded in the dull and stereotyped central authorities of PDA, inoculation various to be measured that again will be activated 2 ~ 3cm place around the mycelia piece of the pathogenic bacterias such as dry thread Pyrenomycetes, 4 bacterial strains to be measured of each PDA plating, the contrast of not inoculating bacterial strain to be measured is set, and every processing repeats for 3 times.At 25 ~ 28 ℃ of lower 3 ~ 5 d that cultivate, then measure the antibacterial bandwidth between the pathogenic bacterias such as bacterial strain to be measured and dry thread Pyrenomycetes, as the index of antagonistic activity power.

Through above-mentioned primary dcreening operation and multiple sieve, obtain a strain the multiple pathogenic bacterias such as dry thread Pyrenomycetes are had the bacterial strain of strongly inhibited effect, be numbered NB12, save backup.

, the NB12 bacterial strain morphology, molecular biology and Physiology and biochemistry identify

(1) Morphological Identification of NB12 bacterial strain

The NB12 bacterial strain is observed after the LB substratum is cultivated 3 d, the circular or irregular shape of its single bacterium colony, and oyster white, surface irregularity has the accordion projection, and is opaque, tarnish, cement is difficult for emulsification; In the LB liquid nutrient medium, slight muddy, look dark, and the surface forms complete white cement film.

(2) molecular biology identification of NB12 bacterial strain

Through Cloning and sequencing, the 16S rDNA sequence length that obtains the NB12 bacterial strain is 1044bp, and its sequence is shown in SEQ ID NO:1.

With the 16S rDNA sequence of NB12 bacterial strain with having logged among Blast software and the GenBank Bacillus subtilis(FJ595878.1), Bacillus amyloliquefaciens(FJ641035.1), Bacillus polyfermenticus(EF178464.1), Bacillus pumilus(EF178456.1), Bacillus licheniformis(FJ713021.1) and Bacillus velezensis(AB245422.1) 16SrDNA sequence is carried out homology relatively, phylogenetic tree construction (Fig. 1) and similarity factor table (table 1).

The comparison of 16S rDNA sequence similarity coefficient between table 1 bacterial strain NB12 and other different strains

	1	2	3	4	5	6	7
								1	**	99.7	100	98.7	99.8	99.9	97.7
2		**	99.9	98.9	99.6	99.7	97.6
								3			**	100	100	99.9	97.6
4				**	99.7	99	97.6
								5					**	99.7	97.7
6						**	97.6
								7							**

Annotate: 1. Bacillus amyloliquefaciensFJ641035.1; 2. Bacillus licheniformisFJ713021.1; 3. Bacillus polyfermenticusEF178464.1; 4. Bacillus pumilusEF178456.1; 5. Bacillus subtilisFJ595878.1; 6. Bacillus velezensisAB245422.1; 7. NB12 bacterial strain.

Can be found out by phylogenetic tree (Fig. 1) and similarity factor table (table 1), this bacterial strain with Bacillus amyloliquefaciensFJ641035.1 and Bacillus subtilisThe homology of FJ595878.1 is the highest, is 97.7%.

(3) Physiology and biochemistry of NB12 bacterial strain is identified

According to " uncle Jie Shi bacteriology handbook is with " common bacteria system identification handbook, the Physiology and biochemistry experimental result that records subtilis NB12 bacterial strain is as shown in table 2.

The Physiological-biochemical Characters of table 2 bacterial strain NB12

Physiological-biochemical Characters	Reaction
		Catalase	+
Starch Hydrolysis	+
		Citrate trianion utilizes	+
The V-P experiment	+
		N.F,USP MANNITOL produces acid	+
Lecithinase	–
		Gelatin hydrolysate	+
5 ℃ of growths	–

Annotate :+expression is positive; – represents to be negative

Analyze through form, physio-biochemical characteristics and 16S rDNA sequence alignment, the most at last bacterial strain NB12 be accredited as subtilis ( Bacillus subtilis).

Embodiment 2 subtilis NB12 are to the restraining effect of phytopathogen

1, subtilis NB12 is to the control experiment of Rice Seedlings banded sclerotial blight

(1) Rhizoctonia solani Kuhn strains tested

The strong Pathogenicity Strains that this experiment is preserved for this laboratory for the rice sheath blight disease bacteria strain GD-118 that tries.

(2) control of Rice Seedlings banded sclerotial blight test

The vernalization of susceptible rice varieties Wu-Yu-Geng 3 is 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 flat board that is connected to bacterial strain GD-118 mycelia piece, after mycelia is covered with grain, 2 paddy that carry disease germs of the base portion of every young plant inoculation.Spray processing next day, 4 groups are set altogether, NB12 bacterium liquid group spraying concentration is 10 ¹⁰The bacterium liquid 100mL of CFU/mL, NB12 bacteria-free filtrate group sprays bacteria-free filtrate, and (medium centrifugal of cultivating under 30 ℃, rotating speed 180 r/min conditions behind the 48h is collected supernatant, filter with biofilter, obtain bacteria-free filtrate) 100mL, jingganmycin control group spraying concentration is jingganmycin solution 100 mL of 40 μ g/mL, select in addition one group to be left intact as negative control (namely only inoculating the control group of GD-118), also have one group of normal plant group (CK).Spray and with plastics film paddy rice is covered afterwards, wait to contrast morbidity about 10 d to the sickness rate and relative scab height of appropriate level " Invest, Then Investigate " rice seedling, and calculate disease grade and disease index.Disease scale (9 grades) standard adopt (Tang Fang. the Virulence of Rhizoctonia solani Kuhn and genetic diversity Journal of Sex Research. Guangzhou: Agricultural University Of South China's master thesis. 2009:1 – 89.) method, disease index calculate reference literature (Fang Zhongda. plant disease research method (third edition). Beijing: Chinese agriculture press, 1998:11 – 12) method.The calculation formula of sick level and disease index is as follows:

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 the results are shown in Table 3 and Fig. 2.By as seen from Table 3, the preventive effect of subtilis NB12 reaches 59.91%.

Table 3 subtilis NB12 is to the prevention effect of rice sheath blight disease

Process	Sickness rate (%)	Disease index	Preventive effect (%)
				NB12 bacterium liquid group	63.46	19.39 ed	59.91
NB12 bacteria-free filtrate group	71.74	22.69 d	53.09
				The jingganmycin control group	67.39	14.99 e	69.00
Negative control group	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.

, the subtilis NB12 inhibition experiment of the Rhizoctonia solani Kuhn sclerotium being sprouted without fermented liquid

(1) subtilis NB12 is without the preparation of fermented liquid

Subtilis NB12 bacterial strain is activated, picking list bacterium colony is cultivated at the LB liquid nutrient medium, under 180 r/min, 30 ℃ and pH 7.0 conditions, cultivate 2 d, bacterium liquid centrifugal 10 min under 6 000 r/min that fermentation is obtained, supernatant liquor filters with biofilter (0.22 μ m), namely obtain NB12 without fermented liquid (being bacteria-free filtrate).

(2) subtilis NB12 is without the inhibition experiment of fermented liquid to the sclerotium sprouting

The GD-118 bacterial strain is cultivated about 14 d until sclerotium is ripe at the PDA flat board, and the sclerotium of collecting in the same size (mean diameter 0.25 cm) is handled as follows (50 sclerotium of every processing): under the normal temperature with sclerotium subtilis NB12 without fermented liquid, LB substratum and sterilized water in immersion 30 min; After taking-up is dried, by 10/dull and stereotyped, specification that the repetition of 5 flat boards is done in every processing, carry out sclerotium with the PDA flat board and sprout and test.When the sclerotium for the treatment of sterilized water contrast was sprouted fully, each sclerotium of processing of investigation was sprouted number and colony diameter.Be considered as sprouting than large 0.1 cm of sclerotium diameter with colony diameter.Test-results sees Table 4.

The impact that table 4 subtilis NB12 sprouts the Rhizoctonia solani Kuhn sclerotium

Process	Sclerotium germination rate (%)	Colony diameter (cm)	Mycelial growth inhibition rate (%)
				The NB12 bacteria-free filtrate	24	0.22±0.15	71.42
The LB liquid nutrient medium	100	0.83±0.27	–
				Sterilized water	100	0.77±0.27	–

Annotate: – and represent the unrestraint effect

3, subtilis NB12 is to the control experiment of fresh kidney beans seedling blight

(1) strains tested

Be used for the inoculation fresh kidney beans dry thread Pyrenomycetes ( R. solani) bacterial classification system separates from the fresh kidney beans of falling ill by this laboratory and obtain.

(2) subtilis NB12 is to the control of fresh kidney beans seedling blight

In seedling pan, sow fresh kidney beans, when treating that fresh kidney beans differentiate two cotyledons, at first subtilis NB12 ferment filtrate 5mL is sprayed in the plants stems base portion, the Rhizoctonia Solani piece (this group echo is NB12) of inoculation diameter 6 mm behind the 1d, BPY liquid nutrient medium, quintozene (PCNB) liquid, aqua sterilisa are set simultaneously to be done above same treatment and contrasts (being labeled as respectively in order NB12 contrast, PCNB and CK1), blank (do not inoculate any bacterium, be labeled as CK2) is set again.Below respectively organize each and process inoculation 30 strain bacterium, in 28 ℃ of thermostatic chambers, controlling moisture about 70%, 5 d " Invest, Then Investigate " incidences calculate the sickness rate of each processing.

(3) subtilis NB12 is to the prevention effect of fresh kidney beans seedling blight

By as seen from Table 5, subtilis NB12 ferment filtrate can obviously suppress the generation of fresh kidney beans seedling blight, and preventive effect reaches 65.38%, and is slightly higher than the preventive effect (61.54%) of quintozene (PCNB) liquid, but that both compare difference is not remarkable.

Table 5 subtilis NB12 is to the preventive effect of fresh kidney beans seedling blight

Process	Sickness rate (%)	Prevention effect (%)
			NB12	30± 0.00	65.38± 0.00a
PCNB	33.33± 5.77	61.54± 6.66a
			The NB12 contrast	86.67± 11.55
CK1	86.67± 5.77
			CK2	0.00± 0.00

Annotate: data are mean value ± standard errors of three repetitions in the table, and same column market demand SPSS software adopts the LSR method to carry out variance analysis, has different alphabetical persons and represents significant difference (P=0.05).CK1 is the aqua sterilisa contrast, and CK2 is not for inoculating the blank of any bacterium.

, subtilis NB12 is to the inhibition of other various plants pathogenic fungi and bacterium experiment

(1) for examination pathogenic bacteria bacterial strain

The plant pathogenic fungi that this experiment is used and pathogenic bacteria are the bacterial strain that preserve in this laboratory.

(2) to the restraining effect of pathogenic fungi

Inoculating needle with sterilization is inoculated in the PDA culture medium flat plate on one side with subtilis NB12, simultaneously with Pyricularia oryzae ( P. oryzae), fusarium graminearum ( G. zeae), banana blight bacteria ( F. oxysporumF. sp. Cubense), the banana anthrax-bacilus ( C. musae), peronophythora litchi ( P. litchii), the sugarcane black rot ( C. adiposum) and Fulvia fulva ( C. fulvum) etc. the inoculated by hypha block of pathogenic fungi in PDA culture medium flat plate the other side, cultivation (a kind of pathogenic bacteria of each plating and subtilis NB12 stand facing each other) stands facing each other, culture dish places biochemical cultivation case, cultivates 2～3 d under 28 ℃, observes antibacterial situation and inhibition zone size (table 6).

(3) to the restraining effect of pathogenetic bacteria

Inoculating needle with sterilization is inoculated in PSA flat board (peeling potato 200g, sucrose 20g, agar 15 ~ 20g, distilled water 1000ml) center with subtilis NB12, and 37 ℃ of lower bacterium colonies of cultivating are about 5 mm big or small (24～48 h).Cover at culture dish and to add 3.0 mL chloroforms, left-hand thread culture dish 2 h are with killing bacteria.Ventilating kitchen take out dechlorination in culture dish, pour into after imitative about 200 μ L rice leaf spot bacterias ( X. oryzaePv. Oryzae) or xanthomonas oryzae pv. oryzicola ( X. oryzaePv. Oryzicola) etc. (seeing Table 6) bacterium liquid, evenly be laid on media surface, dry up at super clean bench, then 28 ℃ of lower cultivations about 24 h are observed antibacterial situation and inhibition zone size (table 6).

Table 6 subtilis NB12 is to the inhibition result of plant pathogenic fungi and bacterium

Pathogenic bacteria	Subtilis NB12 inhibition zone radius (cm)
		Glorosprium musarum Cookeet Mass ( Cm)	1.68±0.15
Pyricularia oryzae ( Po)	2.05±0.19
		Fusarium graminearum ( Gz)	1.21±0.06
Banana blight ( Foc)	1.40±0.07
		Peronophythora Litchii ( Pl)	1.27±0.06
The sugarcane black rot ( Ca)	0.89±0.10
		Leaf muld of tomato ( Cf)	1.28±0.06
Paddy rice bacterial leaf spot bacterium ( Xooe)	1.33±0.12
		Xanthomonas oryzae pv. oryzicola ( Xooa)	1.39±0.12

In sum, subtilis NB12 to dry thread Pyrenomycetes ( R. solani), the banana anthrax-bacilus ( C. musae), Pyricularia oryzae ( P. oryzae), fusarium graminearum ( G. zeae), banana blight bacteria ( F. oxysporumF. sp. Cubense), peronophythora litchi ( P. litchii), the sugarcane black rot ( C. adiposum) and Fulvia fulva ( C. fulvum) etc. Important Economic crop pathogenic fungi all have strong restraining effect.Simultaneously, this Antagonistic Fungi to rice leaf spot bacteria ( X. oryzaePv. Oryzae) and xanthomonas oryzae pv. oryzicola ( X. oryzaePv. Oryzicola) etc. plant pathogenetic bacteria very strong antagonistic action is also arranged.Therefore, subtilis NB12 is the wide spectrum Antagonistic Fungi, can be used for the control of plurality of plant diseases, and is especially better to the prevention effect of rice sheath blight disease and fresh kidney beans seedling blight.

SEQUENCE LISTING

＜110〉Agricultural University Of South China

＜120〉subtilis NB12 and cultural method thereof and application

<130>

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1044

<212> DNA

<213> 16S rDNA

<400> 1

tgcctaatac atgcaagtcg agcggacaga tgggagcttg ctccctgatg ttagcggcgg 60

acgggtgagt aacacgtggg taacctgcct gtaagactgg gataactccg ggaaaccggg 120

gctaataccg gatggttgtt tgaaccgcat ggttcagaca taaaaggtgg cttcggctac 180

cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct caccaaggcg 240

acgatgcgta gccgacctga gagggagatc gctcttactg ggactgagac acggcccaga 300

ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac 360

gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga agaacaagtg 420

ccgttcaaat agggcggcac cttgacggta cctaaccaga aagccacggc taactacgtg 480

ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg 540

ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg gagggtcatt 600

ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta gcggtgaaat 660

gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt aactgacgct 720

gaggagcgaa agcgtgggga gcgaacacga ttagataccc tggtagtcca cgccgtatac 780

gatgagtgct aagtgttagg gggtttccgc cccttagtgc tgcagctaac gcattaagaa 840

ctccgcctgg ggagtacggg cgcaagactg aaactctaag gaattgactg gggcccgcac 900

aagcggagga gcatgtggtt aattcgaagc aacgcgaaga accatacagg tctggcatct 960

ctgacatcta gagatagacg tgcatcggcg gcagagtgac aagtggagca tggttgtcgt 1020

cagctcgatc gtgcgagttg gtag 1044

Claims (5)

1. Subtilis ( Bacillus subtilis) NB12, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number CGMCC No.5383 on October 24th, 2011.
2. 2. the described subtilis NB12 of claim 1 is characterized in that the 16S rDNA sequence of this bacterial strain is shown in SEQ ID NO:1.
3. 3. a bacteria agent is characterized in that containing claim 1 or 2 described subtilis NB12 or contains subtilis NB12 fermented liquid bacteria-free filtrate after filtering.
4. 4. the enlarged culturing method of claim 1 or 2 described subtilis NB12 is characterized in that culture condition is as follows: take LB substratum or BPY substratum as substratum, cultivate 48h under 30 ℃, rotating speed 180 r/min conditions;

   LB culture medium prescription: yeast extract powder 5 g, Tryptones 10 g, NaCl 10 g, agar 15～18 g, water 1000 mL;

   The BPY culture medium prescription: extractum carnis 5g, yeast extract paste 5g, glucose 5g, peptone 10g, NaCl 5g, agar 20g, distilled water 1000 mL, regulate pH to 7.2.
5. 5. claim 1 or the 2 described subtilis NB12 application in the Plant diseases microbial inoculum that preparation control plant pathogenic fungi or plant pathogenetic bacteria cause;

   Described plant pathogenic fungi is dry thread Pyrenomycetes, Glorosprium musarum Cookeet Mass, Pyricularia oryzae, fusarium graminearum, banana blight bacteria, peronophythora litchi, sugarcane black rot or Fulvia fulva;

   Described plant pathogenetic bacteria is rice leaf spot bacteria or xanthomonas oryzae pv. oryzicola.

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