CN102433282A - Bacillus subtilis NB12, as well as culture method and application thereof - Google Patents

Bacillus subtilis NB12, as well as culture method and application thereof Download PDF

Info

Publication number
CN102433282A
CN102433282A CN2011104234353A CN201110423435A CN102433282A CN 102433282 A CN102433282 A CN 102433282A CN 2011104234353 A CN2011104234353 A CN 2011104234353A CN 201110423435 A CN201110423435 A CN 201110423435A CN 102433282 A CN102433282 A CN 102433282A
Authority
CN
China
Prior art keywords
subtilis
bacteria
plant
bacterial strain
strain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN2011104234353A
Other languages
Chinese (zh)
Other versions
CN102433282B (en
Inventor
周而勋
舒灿伟
杨媚
周登博
张德涛
曹琦琦
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China Agricultural University
Original Assignee
South China Agricultural University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South China Agricultural University filed Critical South China Agricultural University
Priority to CN 201110423435 priority Critical patent/CN102433282B/en
Publication of CN102433282A publication Critical patent/CN102433282A/en
Application granted granted Critical
Publication of CN102433282B publication Critical patent/CN102433282B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

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 prodn.Dry thread Pyrenomycetes ( Rhizoctonia solaniK ü hn) is a kind of very important economically plant pathogenic fungi.In China, occupied first of the paddy rice three big 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 hypochnuss such as paddy rice, corn.Control to this disease at present mainly is to use chemical pesticide (sterilant), and mostly sterilant is the interior absorption medicament in single-acting site, and 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, 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 subtilis ( BacillusSpp.) research is more; This bacterium can produce multiple biologically active substance; Like subtilyne, polymyxin, nystatin and linear gramicidins etc., pathogenic bacteria is had the obvious suppression effect, self can also synthesize AMS, lypase and cellulase etc.; Can be used for animal feedstuff additive, in digestive tube, together play a role with the intravital digestive enzymes of animal; 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; Number 200410014484.1 (publication number CN1590535) disclose a kind of subtilis JA like one Chinese patent application, and are all inhibited to various plants pathogenic bacterias such as Rhizoctonia solani Kuhn, fusarium graminearum, withered germ of water-melon; One Chinese patent application number 200910212779.2 (publication number CN101697737A) disclose subtilis and the composite back of tricyclazole have been used to prevent and treat rice blast as sterilant, and control effect is up to 84.67%; One Chinese patent application number 201010175797.0 (publication number CN101857848A) disclose a kind of biocontrol bacillus subtilis WJ-1 for rice sheath blight and microbial inoculum and application; This microbial inoculum is obvious to the indoor live body inoculation of rice sheath blight disease control 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 rice sheath blight disease antagonistic bacterium 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 pathogen bacterium. Zhejiang Agricultural Univ's journal, 1997; Xu Hanhong etc. the preliminary study that Bacillus subtilus BS04 bacterial strain antifungal is plain. agricultural chemicals, 2007; Lixia ZHANG etc. fermentation of bacillus subtilis Optimum of culture medium, Plant Pathology, 2005.
Summary of the invention
The objective of the invention is to, provide a strain to have to suppress the wide spectrum antagonism bacteria strain of the various plants pathogenic bacteria that comprises Rhizoctonia solani Kuhn and fresh kidney beans seedling blight bacterium.
The present invention realizes above-mentioned purpose through following technical scheme:
Subtilis NB12, the classification called after subtilis of this bacterial strain 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 said 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 following: substratum is LB (yeast extract powder 5 g, Tryptones 10 g, NaCl 10 g, agar 15~18 g, water 1000 mL) or BPY substratum (Carnis Bovis seu Bubali cream 5g, yeast extract paste 5g, glucose 5g, peptone 10g, NaCl 5g, agar 20g zero(ppm) water 1000ml; Transfer pH to 7.2), under 30 ℃, rotating speed 180 r/min conditions, cultivate 48h and get final product.Preferred original ph is 7.0,40 mL/250 mL (seed/substratum) liquid amounts.
Subtilis ( B. subtilis) application of NB12 in the microbial Plant diseases microbial inoculum of preparation control pathogenic.Said 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), the leaf muld of tomato bacterium ( 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 control 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 the obvious suppression effect, and this does not appear in the newspapers in document in the past.Sclerotium is the important source 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 the 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 does not appear in the newspapers in document in the past.
Through experiment confirm, subtilis of the present invention ( B. subtilis) NB12 fermented liquid (microbial inoculum) all shows good control effect to rice sheath blight disease and fresh kidney beans seedling blight; Wherein the preventive effect to the rice leaf hypochnus that exsomatizes reaches 86.2%; This germ sclerotium is sprouted inhibiting rate reach 84.52%; Preventive effect to the pot rice hypochnus 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 great application prospect aspect the control miliary damping-off fungus diseases.The preparation method of its microbial inoculum is ripe; Can be developed into microbial pesticide; Be the substitute of chemical pesticide, can be used for the control of miliary damping-off fungus diseases such as rice sheath blight disease and other Plant diseases, to reduce chemical pesticide residual with pollute, preserve the ecological environment significant; 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, with bringing 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 control 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
Soil sample is gathered in this experiment from orchard, Agricultural University Of South China farm; According to 5 methods samplings of employings such as the landform of gathering ground; Each some 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 -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-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 on culture medium flat plate (each extent of dilution is done 3 repetitions), 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; Choosing to the new substratum purifying to the single bacterium colony that grows on the flat board cultivates; 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 dry thread Pyrenomycetess.Beat the mycelia piece of cut-off footpath 5 mm at the colony edge of pathogenic bacterias such as cultured dry thread Pyrenomycetes 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 dry thread Pyrenomycetes 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 dry thread Pyrenomycetes 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 dry thread Pyrenomycetes are had the bacterial strain of strongly inhibited effect, be numbered NB12, preserve subsequent use.
, the NB12 bacterial strain morphology, molecular biology and Physiology and biochemistry identify
(1) morphology of NB12 bacterial strain is identified
The NB12 bacterial strain is observed after cultivating 3 d on the LB substratum, 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 clone and order-checking, 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 landed 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, and constructing system is grown tree (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 find out by phylogenetic tree (Fig. 1) and similarity factor table (table 1), this bacterial strain with Bacillus amyloliquefaciensFJ641035.1 with 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 and " common bacteria system identification handbook ", the Physiology and biochemistry experimental result that records subtilis NB12 bacterial strain is as shown in table 2.
The Physiology and biochemistry proterties of table 2 bacterial strain NB12
The Physiology and biochemistry proterties Reaction
Catalase +
The 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; – representes 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 sheath blight disease in seedling stage
(1) Rhizoctonia solani Kuhn strains tested
The strong virulence bacterial strain that this experiment supplies the rice sheath blight disease bacteria strain GD-118 of examination to preserve for this laboratory.
(2) seedling stage rice sheath blight disease control test
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 flat board 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, 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 one group to be left intact in addition, also have one group of normal plant group (CK) as negative control (promptly only inoculating the control group of GD-118).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.Disease classification (9 grades) standard adopt (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.) 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 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
Prevent and treat the result and see table 3 and Fig. 2.Visible by table 3, the preventive effect of subtilis NB12 reaches 59.91%.
Table 3 subtilis NB12 is to the control effect of rice sheath blight disease
Handle 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.
, subtilis NB12 do not have the inhibition experiment that fermented liquid is sprouted the Rhizoctonia solani Kuhn sclerotium
(1) the aseptic preparation of fermentation liquid of subtilis NB12
With the activation of subtilis NB12 bacterial strain; Picking list bacterium colony is cultivated on the LB liquid nutrient medium; Under 180 r/min, 30 ℃ and pH 7.0 conditions, cultivate 2 d; With bacterium liquid centrifugal 10 min under 6 000 r/min that fermentation obtains, supernatant filters with biofilter (0.22 μ m), promptly obtains the no fermented liquid (being bacteria-free filtrate) of NB12.
(2) subtilis NB12 does not have the inhibition experiment that fermented liquid is sprouted sclerotium
The GD-118 bacterial strain ripe up to sclerotium about cultivation 14 d on the PDA flat board, collected the sclerotium of size consistent (mean diameter 0.25 cm) and handled (50 sclerotium of every processing) as follows: under the normal temperature sclerotium is soaked 30 min in no fermented liquid, LB substratum and the sterilized water of subtilis NB12; After taking-up is dried,, carry out sclerotium with the PDA flat board and sprout and test by 10/dull and stereotyped, multiple specification that 5 flat boards are made in every processing.When the sclerotium of treating sterilized water contrast was sprouted fully, each sclerotium of handling of investigation was sprouted number and colony diameter.Be regarded as sprouting than big 0.1 cm of sclerotium diameter with colony diameter.Test-results is seen table 4.
The influence that table 4 subtilis NB12 sprouts the Rhizoctonia solani Kuhn sclerotium
Handle 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 to inoculate 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 dry thread Pyrenomycetes mycelia piece (this group echo is NB12) of inoculation diameter 6 mm behind the 1d; BPY liquid nutrient medium, quintozene (PCNB) soup, aqua sterilisa are set simultaneously do above same treatment and contrast (being labeled as NB12 contrast, PCNB and CK1 in order respectively), blank (do not inoculate any bacterium, be labeled as CK2) is set again.Below respectively organize each and handle 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 control effect of fresh kidney beans seedling blight
Visible by 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) soup, but that both compare difference is not remarkable.
Table 5 subtilis NB12 is to the preventive effect of fresh kidney beans seedling blight
Handle Sickness rate (%) Control 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 three multiple MV ± standard errors in the table, adopt the LSR method to carry out variance analysis with the column data SPSS software, have different alphabetical persons and represent 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) supplies examination pathogenic bacteria bacterial strain
Plant pathogenic fungi that this experiment is used and pathogenetic bacteria bacterial strain 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 the leaf muld of tomato bacterium ( C. fulvum) wait pathogenic fungi inoculated by hypha block 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; Petridish places biochemical incubator, cultivates 2~3 d down at 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 yam 200g, sucrose 20g, agar 15 ~ 20g, zero(ppm) water 1000ml) center with subtilis NB12, cultivates bacterium colony under 37 ℃ and is 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) wait (seeing table 6) bacterium liquid, 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 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
Lichee frost epidemic disease mould ( 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 the leaf muld of tomato bacterium ( C. fulvum) wait important cash crop pathogenic fungi all to have had strong inhibitory effects.Simultaneously, this antagonism bacterium to rice leaf spot bacteria ( X. oryzaePv. Oryzae) and xanthomonas oryzae pv. oryzicola ( X. oryzaePv. Oryzicola) wait plant pathogenetic bacteria that very strong antagonistic action is also arranged.Therefore, subtilis NB12 is a wide spectrum antagonism bacterium, can be used for the control of plurality of plant diseases, and is especially better to the control 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 (6)

1. subtilis NB12 was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number CGMCC No.5383 on October 24th, 2011.
2. the said subtilis NB12 of claim 1, 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 subtilis NB12 or contains subtilis NB12 fermented liquid bacteria-free filtrate after filtering.
4. the enlarged culturing method of claim 1 or 2 said subtilis NB12, it is characterized in that culture condition is following: with LB or BPY is substratum, under 30 ℃, rotating speed 180 r/min conditions, cultivates 48h;
LB prescription: yeast extract powder 5 g, Tryptones 10 g, NaCl 10 g, agar 15~18 g, water 1000 mL;
The BPY prescription: Carnis Bovis seu Bubali cream 5g, yeast extract paste 5g, glucose 5g, peptone 10g, NaCl 5g, agar 20g zero(ppm) water 1000 mL, transfer pH to 7.2.
5. claim 1 or the 2 said subtilis NB12 application in the Plant diseases microbial inoculum that preparation control plant pathogenic fungi or plant pathogenetic bacteria cause.
6. application according to claim 5 is characterized in that said plant pathogenic fungi is dry thread Pyrenomycetes, 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.
CN 201110423435 2011-12-16 2011-12-16 Bacillus subtilis NB12, as well as culture method and application thereof Expired - Fee Related CN102433282B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 201110423435 CN102433282B (en) 2011-12-16 2011-12-16 Bacillus subtilis NB12, as well as culture method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 201110423435 CN102433282B (en) 2011-12-16 2011-12-16 Bacillus subtilis NB12, as well as culture method and application thereof

Publications (2)

Publication Number Publication Date
CN102433282A true CN102433282A (en) 2012-05-02
CN102433282B CN102433282B (en) 2013-05-01

Family

ID=45981629

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 201110423435 Expired - Fee Related CN102433282B (en) 2011-12-16 2011-12-16 Bacillus subtilis NB12, as well as culture method and application thereof

Country Status (1)

Country Link
CN (1) CN102433282B (en)

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103421722A (en) * 2013-09-05 2013-12-04 华南农业大学 Litchi endogenous bacillus subtilis strain and biological agent and application thereof
CN103667099A (en) * 2013-08-13 2014-03-26 浙江大学 Bacillus subtilis for prevention and control of Fusarium diseases of crops and application thereof
CN103952328A (en) * 2013-11-13 2014-07-30 陕西省微生物研究所 Bacillus subtilis strain A33, microbial inoculum and preparation method and application thereof
CN104109643A (en) * 2014-04-23 2014-10-22 中国海洋大学 Bacillus spp with broad-spectrum antibacterial activity
CN104195071A (en) * 2014-08-06 2014-12-10 江苏农林职业技术学院 Bacillus subtilis B33 and application thereof
CN104232511A (en) * 2014-08-06 2014-12-24 长江大学 Bacillus subtilis for preventing and treating phytophthora blight of pepper
CN104351258A (en) * 2014-10-20 2015-02-18 辽宁科技大学 Compound biopesticide for preventing and curing rice sheath blight disease and preparation method thereof
CN105145639A (en) * 2015-10-13 2015-12-16 苏州科大微龙农业科技有限公司 Cellulase organic copper mixture for controlling root rot of tomato phytophthora
CN106472370A (en) * 2016-09-30 2017-03-08 贵州尚品源农业科技有限公司 Oryza sativa L. Ctenopharyngodon idellus symbiosis deinsectization cultural method
CN107502570A (en) * 2017-08-18 2017-12-22 华中农业大学 One plant of Biocontrol Bacillus subtilis BJ 1 and its application
WO2017186543A3 (en) * 2016-04-24 2018-03-22 Bayer Cropscience Aktiengesellschaft Use of fluopyram and/or bacillus subtilis for controlling fusarium wilt in plants of the musaceae family
CN108048357A (en) * 2017-12-26 2018-05-18 武汉科诺生物科技股份有限公司 A kind of bacillus subtilis and its cultural method
CN108192851A (en) * 2018-03-13 2018-06-22 长沙艾格里生物肥料技术开发有限公司 Dongfanghong bacteria strain, Dongfanghong bacteria agent and its application
CN108192852A (en) * 2018-03-20 2018-06-22 吉林省农业科学院 One plant of Bacillus subtillis GB519 for promoting paddy growth and antagonism Pyricularia oryzae and application
CN109156477A (en) * 2018-09-14 2019-01-08 河北省农林科学院植物保护研究所 Application of the bacillus subtilis BAB-1 in prevention and treatment leaf muld of tomato
CN109355233A (en) * 2018-12-04 2019-02-19 沈阳化工研究院有限公司 A kind of bacillus amyloliquefaciens and its application
CN109554317A (en) * 2018-12-29 2019-04-02 陕西省微生物研究所 Bei Laisi bacillus and its application in wheat sharp eyespot disease prevention growth-promoting
CN110172428A (en) * 2019-06-11 2019-08-27 山东碧蓝生物科技有限公司 It is a kind of with it is disease-resistant and improve fruit quality bacillus subtilis and its application
CN112625957A (en) * 2020-12-28 2021-04-09 上海交通大学 Bacillus subtilis LJBS06 and application thereof
CN113201478A (en) * 2021-05-27 2021-08-03 广西民族大学 Siamese bacillus Gxun-36 secreting iron carrier and application thereof
CN114015592A (en) * 2021-09-30 2022-02-08 海南大学 Endophytic biocontrol strain HN075 for preventing and treating litchi frost blight and application thereof
CN114134064A (en) * 2021-09-16 2022-03-04 华南农业大学 Bacillus subtilis XX and application thereof in prevention and treatment of soil-borne fungal diseases
CN115011505A (en) * 2022-04-24 2022-09-06 山东省农业科学院 Bacillus subtilis and application thereof
CN115141785A (en) * 2022-08-26 2022-10-04 甘肃省科学院生物研究所 Bacillus subtilis and application thereof in cabbage planting
CN115927084A (en) * 2022-10-17 2023-04-07 三峡大学 Multifunctional bacillus subtilis and application thereof in preventing bacterial leaf blight of rice
CN116836856A (en) * 2023-06-26 2023-10-03 上海市农业科学院 Bacillus subtilis, pathogenic bacteria antagonist and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101760438A (en) * 2009-12-23 2010-06-30 珠海市农业科学研究中心 Broad-spectrum antifungal plant endophytic bacillus subtillis and application thereof
CN101935628A (en) * 2010-06-13 2011-01-05 浙江大学 Bacillus subtilis and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101760438A (en) * 2009-12-23 2010-06-30 珠海市农业科学研究中心 Broad-spectrum antifungal plant endophytic bacillus subtillis and application thereof
CN101935628A (en) * 2010-06-13 2011-01-05 浙江大学 Bacillus subtilis and application thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KUMAR,K.VIJAY KRISHNA ET AL.: "Screening and Selection of Elite Plant Growth Promoting Rhizobacteria (PGPR) For Suppression of Rhizoctonia solani and Enhancement of Rice Seedling Vigor", 《JOURNAL OF PURE AND APPLIED MICROBIOLOGY》 *
刘永锋: "枯草芽孢杆菌(Bacillus subtilis)Bs-916胞外抗菌蛋白质的纯化及其鉴定", 《中国博士学位论文全文数据库 农业科技辑》 *
彭灯水: "枯草芽孢杆菌12-041和12-059对玉米纹枯病生物防治的研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 *
黎起秦 等: "芽孢杆菌对水稻纹枯病的防治效果", 《中国生物防治》 *

Cited By (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103667099B (en) * 2013-08-13 2015-08-26 浙江大学 A kind of subtilis for preventing and treating crop fusarium disease and application thereof
CN103667099A (en) * 2013-08-13 2014-03-26 浙江大学 Bacillus subtilis for prevention and control of Fusarium diseases of crops and application thereof
CN103421722A (en) * 2013-09-05 2013-12-04 华南农业大学 Litchi endogenous bacillus subtilis strain and biological agent and application thereof
CN103421722B (en) * 2013-09-05 2015-08-19 华南农业大学 One strain lichee endophytic Bacillus subtilis and biotechnological formulation thereof and application
CN103952328A (en) * 2013-11-13 2014-07-30 陕西省微生物研究所 Bacillus subtilis strain A33, microbial inoculum and preparation method and application thereof
CN103952328B (en) * 2013-11-13 2016-08-17 陕西省微生物研究所 A kind of bacillus subtilis strain A33, microbial inoculum and preparation method and application
CN104109643A (en) * 2014-04-23 2014-10-22 中国海洋大学 Bacillus spp with broad-spectrum antibacterial activity
CN104109643B (en) * 2014-04-23 2017-07-21 中国海洋大学 A kind of bacillus with broad spectrum antibiotic activity
CN104232511A (en) * 2014-08-06 2014-12-24 长江大学 Bacillus subtilis for preventing and treating phytophthora blight of pepper
CN104195071A (en) * 2014-08-06 2014-12-10 江苏农林职业技术学院 Bacillus subtilis B33 and application thereof
CN104351258A (en) * 2014-10-20 2015-02-18 辽宁科技大学 Compound biopesticide for preventing and curing rice sheath blight disease and preparation method thereof
CN105145639A (en) * 2015-10-13 2015-12-16 苏州科大微龙农业科技有限公司 Cellulase organic copper mixture for controlling root rot of tomato phytophthora
CN109068649A (en) * 2016-04-24 2018-12-21 拜耳农作物科学股份公司 Fluopyram and/or bacillus subtilis are used to prevent and treat the purposes of the wilt disease of Musaceae plant
WO2017186543A3 (en) * 2016-04-24 2018-03-22 Bayer Cropscience Aktiengesellschaft Use of fluopyram and/or bacillus subtilis for controlling fusarium wilt in plants of the musaceae family
CN106472370A (en) * 2016-09-30 2017-03-08 贵州尚品源农业科技有限公司 Oryza sativa L. Ctenopharyngodon idellus symbiosis deinsectization cultural method
CN107502570A (en) * 2017-08-18 2017-12-22 华中农业大学 One plant of Biocontrol Bacillus subtilis BJ 1 and its application
CN107502570B (en) * 2017-08-18 2019-07-16 华中农业大学 One plant of Biocontrol Bacillus subtilis BJ-1 and its application
CN108048357B (en) * 2017-12-26 2021-01-05 武汉科诺生物科技股份有限公司 Bacillus subtilis and culture method thereof
CN108048357A (en) * 2017-12-26 2018-05-18 武汉科诺生物科技股份有限公司 A kind of bacillus subtilis and its cultural method
CN108192851A (en) * 2018-03-13 2018-06-22 长沙艾格里生物肥料技术开发有限公司 Dongfanghong bacteria strain, Dongfanghong bacteria agent and its application
CN108192851B (en) * 2018-03-13 2021-03-19 长沙艾格里生物肥料技术开发有限公司 Oriental rhodobacter strain, oriental rhodobacter agent and application thereof
CN108192852A (en) * 2018-03-20 2018-06-22 吉林省农业科学院 One plant of Bacillus subtillis GB519 for promoting paddy growth and antagonism Pyricularia oryzae and application
CN109156477A (en) * 2018-09-14 2019-01-08 河北省农林科学院植物保护研究所 Application of the bacillus subtilis BAB-1 in prevention and treatment leaf muld of tomato
CN109355233A (en) * 2018-12-04 2019-02-19 沈阳化工研究院有限公司 A kind of bacillus amyloliquefaciens and its application
CN109355233B (en) * 2018-12-04 2021-04-09 沈阳化工研究院有限公司 Bacillus amyloliquefaciens and application thereof
CN109554317B (en) * 2018-12-29 2022-04-01 陕西省微生物研究所 Bacillus belgii and application thereof in preventing diseases and promoting growth of wheat sheath blight
CN109554317A (en) * 2018-12-29 2019-04-02 陕西省微生物研究所 Bei Laisi bacillus and its application in wheat sharp eyespot disease prevention growth-promoting
CN110172428A (en) * 2019-06-11 2019-08-27 山东碧蓝生物科技有限公司 It is a kind of with it is disease-resistant and improve fruit quality bacillus subtilis and its application
CN112625957A (en) * 2020-12-28 2021-04-09 上海交通大学 Bacillus subtilis LJBS06 and application thereof
CN113201478A (en) * 2021-05-27 2021-08-03 广西民族大学 Siamese bacillus Gxun-36 secreting iron carrier and application thereof
CN114134064B (en) * 2021-09-16 2023-08-29 华南农业大学 Bacillus subtilis XX and application thereof in preventing and controlling soil-borne fungus diseases
CN114134064A (en) * 2021-09-16 2022-03-04 华南农业大学 Bacillus subtilis XX and application thereof in prevention and treatment of soil-borne fungal diseases
CN114015592A (en) * 2021-09-30 2022-02-08 海南大学 Endophytic biocontrol strain HN075 for preventing and treating litchi frost blight and application thereof
CN115011505A (en) * 2022-04-24 2022-09-06 山东省农业科学院 Bacillus subtilis and application thereof
CN115011505B (en) * 2022-04-24 2023-04-18 山东省农业科学院 Bacillus subtilis and application thereof
CN115141785A (en) * 2022-08-26 2022-10-04 甘肃省科学院生物研究所 Bacillus subtilis and application thereof in cabbage planting
CN115927084A (en) * 2022-10-17 2023-04-07 三峡大学 Multifunctional bacillus subtilis and application thereof in preventing bacterial leaf blight of rice
CN115927084B (en) * 2022-10-17 2024-09-10 三峡大学 Multifunctional bacillus subtilis and application thereof in preventing bacterial leaf blight of rice
CN116836856A (en) * 2023-06-26 2023-10-03 上海市农业科学院 Bacillus subtilis, pathogenic bacteria antagonist and application thereof
CN116836856B (en) * 2023-06-26 2024-06-04 上海市农业科学院 Bacillus subtilis, pathogenic bacteria antagonist and application thereof

Also Published As

Publication number Publication date
CN102433282B (en) 2013-05-01

Similar Documents

Publication Publication Date Title
CN102433282B (en) Bacillus subtilis NB12, as well as culture method and application thereof
CN100540656C (en) A kind of subtilis and microbial inoculum thereof and application
CN102533593B (en) Burkholderia cepacia SD7 and culturing method and application thereof
CN100334201C (en) Bacillus subtilis and its uses
CN105219681B (en) A kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM and preparation method and application
CN105557756B (en) Plant bacteriostasis method adopting bacillus methylotrophicus strain NKG-1
CN102433281B (en) Streptomyces katrae NB20, as well as culture method and application thereof
CN109022315A (en) The biological and ecological methods to prevent plant disease, pests, and erosion bacillus of one plant of broad-spectrum disease resistance and its application
CN101967455B (en) Bacillus amyloliquefaciens EA19 for controlling wheat root diseases and preparation thereof
CN105886428A (en) Streptomyces albidoflavus and applications thereof in microbial fertilizers
CN104164394A (en) Antagonistic phytopathogen strain and application thereof
CN103540542A (en) Bidirectional burkholderia as well as culture method and application thereof
CN108148794A (en) A kind of the bacillus subtilis DYr3.3 and preparation method and application of broad-spectrum antibacterial activity
CN104480046A (en) Brevibacillus laterosporus strain and application thereof
CN103436457A (en) Burkholderia cepacia, and cultivation method and application thereof
CN113699065B (en) Bacillus vallismortis and application thereof
CN104017749B (en) Endogenous bacillus subtilis positive mutant strain, preparation method thereof, prepared biocontrol agent and application of biocontrol agent in preventing and controlling pomegranate dry rot
CN105543132A (en) Bacillus methylotrophicus YB-F7 and application thereof in preventing plant diseases
CN104789509B (en) Raw bacillus pumilus and its application in one plant of bark of eucommia
CN110317747A (en) A kind of bacillus amyloliquefaciens JT68 and its application in prevention and treatment tea anthracnose
CN106701623A (en) Bacillus atrophaeus antagonistic to lycium chinensis root rot and application of bacillus atrophaeus
CN108641989A (en) One plant of Methylotrophic bacillus and its application
CN105907662A (en) Bacillus subtillis and application thereof
CN107699526A (en) One plant of actinomycetes strain for preventing and treating gray mold and its application
CN113604376A (en) Sugarcane endophytic bacillus subtilis and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20130501

Termination date: 20151216

EXPY Termination of patent right or utility model