CN103087956B - Bacterium for preventing and controlling banana wilt and application thereof - Google Patents
Bacterium for preventing and controlling banana wilt and application thereof Download PDFInfo
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Abstract
The invention discloses a bacterium for preventing and controlling banana wilt and application thereof. The bacterium disclosed by the invention concretely is Bacillus sp. Bsp.6; and the preservation number in the China General Microbiological Culture Collection Center is CGMCC No.6459. The test shows that the Bacillus sp. Bsp.6 CGMCC No.6459 disclosed by the invention can restrain 1# and 4# microspecies of Fusantan oxysporumf. sp. cubense) at the same time; the bacterial agent prepared by taking the Bacillus sp. Bsp.6 CGMCC No.6459 as an active ingredient can prevent and control the banana wilt; the field control effect achieves about 95%; and the Bacillus sp. Bsp.6 CGMCC No.6459 disclosed by the invention and the bacterial agent have significant control effects on the banana wilt. Therefore, rapid development of banana plantation is facilitated to a large extent.
Description
Technical field
The present invention relates to biological technical field, relate in particular to bacterium and the application thereof of a strain control banana blight.
Background technology
Dwarf banana is the hybridization cultivar of banana, because its fruit type is little, external form is beautiful, pericarp is golden yellow, the thin meat of skin is white, the soft sweetness of mouthfeel, delicate fragrance, the feature such as nutritious, liked by consumers in general.Nearly ten years, dwarf banana plantation is rapidly developed.At present, the dwarf banana cultivated area in Guangxi, Guangdong and Hainan is respectively 5000,500 and 200 hectares of left and right.Due to blight harm, the large-scale planting of dwarf banana is not only subject to serious restriction, and needs crop rotation.The banana blight (also claiming Panama disease) being caused by Cuba's point Fusariumsp [Fusarium oxysporumf.sp.cubense (E.F.Simth) Snyder et Hansen] is a kind of destructive silborne fungal diseases, from 1910, Panamanian banana blight great outburst so far, caused heavy loss to the each main banana planting state in South America, Africa and Asia.Cuba point Fusariumsp has 4 physiological strains, wherein, to banana hazardness maximum be No. 1 and No. 4 microspecies, all endanger dwarf banana.
The Prevention Technique of banana blight is made to large quantity research both at home and abroad, related to the many aspects such as chemical prevention, intercropping/crop rotation, disease-resistant variety cultivation and biological control.Because banana blight is soil-borne vascular bundle disease, pathogenic bacteria is from root, basal part of stem infects and spreads after invading in vascular bundle, so Techniques For Chemical Control is almost invalid (Nel B, ViljoenA, Steinberg C, et al.Evaluation of chem ical substances for the mamagement and control ofFusarium wilt of banana[A] .In Claudine Picq, Anne Vezina Eds20d Intem ationalsymposium on Fusarium wilt on banana Brazil[C] .Salvador de Bahia, 2003.).Leek and Brazilian Banana crop rotation can significantly reduce blight incidence (Huang Yonghong, Li Chunyu, Zuo Cunwu, etc. leek to the withered pathogenetic restraining effect of Brazilian Banana. Chinese biological control journal, 2011,27 (3): 344-348).Shop experiment shows, leek leaf extract to the blight control effect of Brazilian Banana and wide No. 1 dwarf banana of powder at 75% left and right (Huang Yonghong, Li Chunyu, Wei Yuerong, Deng. the antagonism of leek to banana blight bacteria and the restraining effect that potted plant banana blight is occurred thereof. Zhejiang Agriculture journal, 2011,23 (6): 1162-1166).Banana fusarium wilt resistance breed of variety mainly adopts tissue cultured seedling variation to select and two kinds of modes of toxin screening.But, the resistant variety selecting often at aspects such as yield and qualities all undesirable (Huang Bingzhi, Tang's moderate seas, Xu Linbing, etc. banana fusarium wilt resistance kind resists withered No. 5 introduction and Experiment. fruit tree, 2009,6:17-19).So far, banana fusarium wilt resistance transgenic breeding not yet starts.Therefore, with the biological control method of environmental protection control banana blight be more and more subject to people's attention (Peng Zengming, etc. banana blight biological control research overview for Zhong Qunyou, Zheng Zhuohui. guangdong agricultural science, 2007,7:64-65).
At present, at the early-stage to the research of banana blight biological control both at home and abroad, mainly concentrate on screening, indoor control or short-term field test aspect to No. 4 microspecies Antagonistic Fungis of banana blight.These Antagonistic Fungis relate to Trichoderma (Thandavelu R, Palaniswami A, Doraiswamy S, et al.The effect of Pseudomonasfluorescens and Fusarium oxysporumf. sp.cubense oninduction of defenceenzymesandphenolics in banana.Biol Plantarum, 2003, 46 (1): 107-110), Penicillium notatum (Wang Yuguang, Lu Juan, Sun Jianbo, Deng. the Isolation and Identification of the Penicillium notatum of a strain antagonism banana blight. Chinese agronomy circular, 2011, 27(04): 178-182), actinomycetes (Ru Xiang, Zeng Tao, Mo Kunlian, Deng. separation and the field control effect test of Luoshan primitive area resisting banana vascular wilt soil actinomycete hung in Hainan. Chinese agronomy circular, 2012, 28 (13): 97-102), genus bacillus (Fu Yeqin, Zhang Keli, Pan Xianxin, Deng. the Molecular Identification of Endophytic antagonistic bacteria BEB2 and the restraining effect to banana blight bacteria thereof. tropical crops journal, 2009, 30(1): 80-85), streptomycete (Lin Mei, Zhang Shaosheng. the restraining effect of streptomycete F-1013 shrimp shell meal fermented liquid to 3 kind of plant pathogenic fungies. University Of Agriculture and Forestry In Fujian's journal (natural science edition), 2010, 39(6): 584-589) and Pseudomonas (Zhong little Yan, Liang Miaofen, Zhen Xizhuan, Deng. the restraining effect of pseudomonas to banana blight bacteria. plant protection, 2009, 35(1) 86-89).
Summary of the invention
The object of this invention is to provide bacterium and the application thereof of a strain control banana blight.
Bacterium provided by the present invention is specially genus bacillus (Bacillus sp.) Bsp.6.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on August 17th, 2012, and deposit number is CGMCC No.6459.
Another object of the present invention is to provide a kind of microbial inoculum.
The activeconstituents of microbial inoculum provided by the present invention is described genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459.
Concrete, described microbial inoculum is for to be inoculated into described genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 in substratum and to cultivate, and obtaining cell concentration is 3 × 10
4-4.5 × 10
5the bacterium liquid of cfu/mL; In the use procedure of described bacterium liquid, can dilute according to actual needs, adjust working concentration.In one embodiment of the invention, described microbial inoculum is specially and contains 3.8 × 10
5the bacterium liquid of genus bacillus described in cfu/mL (Bacillus sp.) Bsp.6CGMCC No.6459.
Described substratum can be made up of the raw material of following proportioning: analysis for soybean powder (soya bean seed powder), be 5g:10g:20g:1L apart from the proportioning of banana caulo, sucrose and the water of ground 0.5-1.5 rice (as 1.0 meters).
Further, described substratum can be mixed according to the proportioning of 100mL:100mL:20g:800mL by analysis for soybean powder filtrate, Banana Tissue liquid, described sucrose and described water, and pH is that 7.0-7.2(is as pH7.0).
The preparation method of described analysis for soybean powder filtrate specifically can comprise the steps: described analysis for soybean powder to mix according to the ratio of 1g:30mL to 1g:400mL with described water, boils rear filtration, obtains described analysis for soybean powder filtrate; The described time length of boiling is 20-60min.
In one embodiment of the invention, the ratio of described analysis for soybean powder and described water is specially 1g:100mL; The described time length of boiling is specially 20min.
The preparation method of described Banana Tissue liquid specifically can comprise the steps: described banana caulo to mix according to the ratio of 1g:100mL to 1g:25mL with described water, boils rear filtration, obtains described Banana Tissue liquid; The described time length of boiling is 20-40min.
In one embodiment of the invention, the ratio of described banana caulo and described water is specially 1g:50mL; The described time length of boiling is specially 20min.
In one embodiment of the invention, the preparation method of described substratum specifically comprises the steps:
A) preparation of described analysis for soybean powder filtrate: claim described analysis for soybean powder 5g, join described in 500mL in water (distilled water), boil after 20 minutes and filter, obtain described analysis for soybean powder filtrate (about 100mL);
B) preparation of described Banana Tissue liquid: get the described banana caulo 20g apart from 1 meter of left and right, ground, add described water 1000mL, boil after 20 minutes and filter, obtain filtrate and be described Banana Tissue liquid (about 200mL).
C) by 100mL steps A) obtain described analysis for soybean powder filtrate, 100mL step B) obtain described Banana Tissue liquid and 20g sucrose mix after, adjust pH to 7, adding distil water 800mL, obtains described substratum.
Described genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459, or described microbial inoculum following a) or b) application also belong to protection scope of the present invention:
A) suppress wilt;
B) control plant blight.
In above-mentioned application, described wilt specifically can be Cuba's point Fusariumsp (Fusantan oxysporumf.sp.cubense); Described blight can be specifically by the caused blight of Cuba's point Fusariumsp (Fusantan oxysporumf.sp.cubense); Described Cuba's point Fusariumsp (Fusantan oxysporum f.sp.cubense) specifically can be Cuba's point No. 1 microspecies of Fusariumsp (Fusantan oxysporum f.sp.cubense) and/or No. 4 microspecies of Cuba's point Fusariumsp (Fusantan oxysporum f.sp.cubense); Described plant can be banana, concrete as dwarf banana, No. 1, more concrete powder as wide in dwarf banana kind.
Another object of the present invention is to provide a kind of method of utilizing described microbial inoculum control banana blight.
The step of utilizing the method for described microbial inoculum control banana blight specifically can comprise following (1) and (2) provided by the present invention:
(1) after tissue culture seedlings of bananas field planting, extract the 1st young leaves, 2-3 sheet young leaves, 4-5 sheet young leaves and 6-7 sheet young leaves out and respectively use 1 time described microbial inoculum these four periods;
The described concentration of using is as follows: the concentration of using described in four times is the 1/5-1/10 of described bacteria suspension concentration, as 1/10(by as described in microbial inoculum carry out 10 times of dilutions);
The described dosage of using is as follows: the dosage of using described in first three time is 60-80g/ strain, and as 60g, the dosage of using described in the 4th time is 100g-200g/ strain (as 150g/ strain);
The described mode of using is as follows: the mode of using described in four times is root filling and executes;
(2) behind banana seedlings field planting land for growing field crops, extract out after young leaves in 1 week until banana seedlings described in 80%-90%, start to use described microbial inoculum, until banana seedlings is taken out after flower bud 1 week described in 80%-90%;
The described frequency of using is for following (a) and (b):
(a), in the time that banana seedlings described in 80%-90% is extracted young leaves out, while reaching 3 months to described banana seedlings field planting land for growing field crops, only, on average monthly use described microbial inoculum 1 time;
(b), in the time of described banana seedlings field planting land for growing field crops 4th month, while taking out after flower bud 1 week to banana seedlings described in 80%-90%, only, on average within every two months, use 1 time described microbial inoculum;
The described concentration of using is as follows: described (a) and (b) in each described in the concentration used be the 1/5-1/10 of described bacteria suspension concentration, as 1/10(by as described in microbial inoculum carry out 10 times of dilutions);
The described dosage of using is as follows: the dosage of using described in each in described (a) is 400g-600g/ strain (as 500g/ strain); The dosage of using described in each in described (b) is 800g-1200g/ strain (as 1000g/ strain).
The described mode of using is as follows: in described (a) for the first time described in the mode used be that root is filled with and executed; Using for the second time and later in described (a), and the mode of at every turn using in described (b) is ring-type charity; Described ring-type charity is to use described microbial inoculum in the vertical corresponding soil part of described banana middle part blade outer rim.
In aforesaid method, while extracting 4-5 sheet young leaves out in step (1) after tissue culture seedlings of bananas field planting, use that described microbial inoculum (using for the third time) is front to be enclosed hindering root 1 apart from glue cup outer rim 1cm place.
In aforesaid method, described blight can be by the caused blight of Cuba's point Fusariumsp (Fusantan oxysporumf.sp.cubense); Described Cuba's point Fusariumsp (Fusantan oxysporum f.sp.cubense) specifically can be Cuba's point No. 1 microspecies of Fusariumsp (Fusantan oxysporum f.sp.cubense) and/or No. 4 microspecies of Cuba's point Fusariumsp (Fusantan oxysporum f.sp.cubense); Described banana specifically can be dwarf banana, more concrete can be No. 1, the wide powder of dwarf banana kind.
In aforesaid method, the 1/5-1/10 of described bacteria suspension concentration doubly dilutes for described microbial inoculum stoste is carried out to 5-10.
Genus bacillus provided by the present invention (Bacillus sp.) Bsp.6CGMCC No.6459 can suppress Cuba's point Fusariumsp (Fusantan oxysporum f.sp.cubense) No. 1 and No. 4 microspecies simultaneously.Microbial inoculum taking described genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 as activeconstituents can prevention and control dwarf banana blight, and its land for growing field crops control effect reaches 95% left and right.Genus bacillus provided by the present invention (Bacillus sp.) Bsp.6CGMCC No.6459 and microbial inoculum thereof have significant control effect for dwarf banana blight, and this will promote the fast development of banana planting industry to a great extent.
Preservation explanation
Strain name: genus bacillus
Latin name: (Bacillus sp.)
Strain number: Bsp.6
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on August 17th, 2012
The preservation center numbering of registering on the books: CGMCC No.6459
Brief description of the drawings
Fig. 1 is the dual test result of bacterial strain Bsp.6.Wherein, A is No. 1 microspecies of Cuba's point Fusariumsp (Fusantan oxysporumf.sp.cubense); B is the face-off of bacterial strain Bsp.6 and No. 1 microspecies of Cuba's point Fusariumsp (Fusantan oxysporumf.sp.cubense); C is No. 4 microspecies of Cuba's point Fusariumsp (Fusantan oxysporum f.sp.cubense); D is the face-off of bacterial strain Bsp.6 and No. 1 microspecies of Cuba's point Fusariumsp (Fusantan oxysporum f.sp.cubense).
Fig. 2 is the detected result of genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 bacteriostatic activity.Wherein, 1-6 expression group respectively 1-group 6.
Fig. 3 is that genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 is to banana blight prevention effect results from pot experiment test (plant survival rate statistics).Wherein, 1-6 expression group respectively 1-group 6.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
Bacterial strain:
Genus bacillus (Bacillus sp.) BEB2, be endogenetic bacteria BEB2 or Endophytic antagonistic bacteria BEB2: be recorded in " Fu Yeqin. the preventive and therapeutic effect research of banana endogenetic bacteria BEB2 bacterial strain to banana blight. University Of Hainan's master thesis; 2008 " literary composition, and " Fu Yeqin; Zhang Keli; Pan Xianxin etc. the Molecular Identification of Endophytic antagonistic bacteria BEB2 and the restraining effect to banana blight thereof. tropical crops journal; 01 phase in 2009 " in a literary composition, the public can obtain from Chinese Academy of Tropical Agricultural Sciences's Haikou experiment centre.
Cuba's point No. 1 microspecies of Fusariumsp (Fusantan oxysporum f.sp.cubense) and No. 4 microspecies of Cuba's point Fusariumsp (Fusantan oxysporum f.sp.cubense), be No. 1, banana blight bacteria and No. 4 physiological strains: be documented in " Cao Yongjun; Cheng Ping; analogy state brightness etc. No. 1, banana blight bacteria and No. 4 physiological strains growth characteristics on several substratum. tropical crops journal; the 08th phase in 2010 " in a literary composition, the public can obtain from Chinese Academy of Tropical Agricultural Sciences's Haikou experiment centre.
Substratum:
NA substratum: beef extract 3g, yeast extract 1g, peptone 5g, glucose 10g, agar 15g, distilled water 1000mL, pH7.0.
PDA substratum: potato 200g, sucrose 20g, agar 20g, water 1000mL, pH nature (approximately 6.0).
NB liquid nutrient medium: extractum carnis 5g, peptone 10g, sodium-chlor 5g, distilled water 1000mL, pH7.2.
Bacterial strain mother liquor substratum: beef extract 3g, Banana Tissue liquid 100-200mL, peptone 5g, distilled water is settled to 1000mL, pH7.0.
Enlarged culturing bacterium liquid culture medium: analysis for soybean powder 2-10g, adds 200-400mL water, boils after 20-60 minute and filters, and gets its filtrate 100mL, Banana Tissue liquid 100-200mL, and sucrose 20g-40, pH value 7, adding distil water is to 1000mL.
Wherein, the preparation method of described Banana Tissue liquid is as follows: get the banana caulo 10-40g apart from ground 0.5-1.5 rice left and right, the 1000mL that adds water, boils 20-40 minute, filters, and preserves filtrate, is Banana Tissue liquid.
Separation and the qualification of embodiment 1, genus bacillus (Bacillus sp.) Bsp.6
One, the separation of genus bacillus (Bacillus sp.) Bsp.6
1, adopt tissue isolation, obtain the bacterial strain in plant tissue
Gather root (diameter 0.2-0.5cm) 1-2g apart from earth's surface 10-15cm from the land for growing field crops dwarf banana plant body of Chinese Hainan health, rinse 10-30min with tap water, the ethanol that is 70% by volume percent successively again and mass percent are 3.25% clorox sterilization 1min and 3min, then use rinsed with sterile water 3 times.Root is placed in to mortar, adds sterilized water and fully grind.Leave standstill after 15min, suct clear liquid, carry out 10
-1, 10
-2, 10
-3gradient dilution.Respectively get 100 μ L diluents, coat NA culture medium flat plate, the 3rd the rinsed with sterile water liquid of contrast coating 100 μ L.Cultivate after 3-5 days for 28 DEG C, single bacterium colony of picking complete form, obvious difference, streak culture on NA culture medium flat plate, obtain dissimilar bacterial strain.
2, resistant strain separates
(1) Pathogen Causing Banana Fusarium Wilt is streak culture
Get 2 PDA culture medium flat plates, streak inoculation Cuba point No. 1 microspecies of Fusariumsp (Fusantan oxysporumf.sp.cubense) and No. 4 microspecies of Cuba's point Fusariumsp (Fusantan oxysporum f.sp.cubense) respectively, 28 DEG C, cultivate 3-5 days.
(2) dissimilar bacterial strain is streak culture
The dissimilar bacterial strain that step 1 is obtained respectively streak inoculation, in PDA culture medium flat plate, 28 DEG C, is cultivated 1-2 days.
(3) face-off experiment detects fungistatic effect
Picking list bacterium colony in the flat board of each bacterial strain that culture of isolated obtains from step (2), streak inoculation is at a lateral edges of two new PDA culture medium flat plates respectively, (one of two flat boards have been inoculated the PDA substratum blob of viscose after No. 1 microspecies cultivation of Cuba's point Fusariumsp (Fusantanoxysporum f.sp.cubense) to the blob of viscose that contains pathogenic bacteria mycelia at corresponding opposite side placement length of side 3mm in placing and coming from step (1), another placement in two flat boards comes from the PDA substratum blob of viscose of having inoculated in step (1) after No. 4 microspecies of Cuba's point Fusariumsp (Fusantan oxysporum f.sp.cubense) are cultivated), cover lid, seal with preservative film, put under 28 DEG C of conditions and cultivate 5 to 7 days, obtain that wilt-Cuba point No. 1 microspecies of Fusariumsp (Fusantan oxysporum f.sp.cubense) and Cuba's No. 4 microspecies of point Fusariumsp (Fusantan oxysporum f.sp.cubense) are all had to stronger inhibiting bacterial strain, one of them bacterial strain is denoted as to Bsp6.The dual test result of bacterial strain Bsp6 as shown in Figure 1.
Two, the qualification of genus bacillus (Bacillus sp.) Bsp.6
Separate from the following aspects authentication step one the bacterial strain Bsp.6 obtaining:
1, Morphological Identification
For the bacterial strain Bsp.6 in logarithmic phase, after smear staining, adopt observation by light microscope, its Main Morphology is characterized as: size for (0.5～1.0 μ m) × (1.5 ~ 2.3 μ m), ivory buff, opaque, moistening, thalline is surperficial fold, and edge is irregular.
2, analysis of physio biochemical characteristics
Adopt conventional Physiology and biochemistry authentication method to identify bacterial strain Bsp.6, result is as table 1.
The physiological and biochemical property of table 1 bacterial strain Bsp.6
Note: "+" represents to utilize this material, "-" represents to utilize this material.
3,16s rDNA sequence homology analysis
Ordinary method is cultivated above-mentioned steps one and is separated the bacterial strain Bsp.6 obtaining, extract total DNA of bacterial strain as gene amplification template, with bacterium 16s rDNA universal primer, 27f:5 '-AGAGTTTGATCCTGGCTCAG-3 ', 1492r:5 '-TACGGTTACCTTGTTACGACTT-3 ' carries out PCR reaction.The program of answering is: 95 DEG C of sex change 30s, 55 DEG C of annealing 1min, 72 DEG C of extensions 2min, totally 30 circulations.After finishing, reaction carries out DNA sequencing, sequence assembly and similarity analysis, and sequence alignment completes online by American National biotechnology information center ncbi database (http://www.ncbi.nlm.nih.gov).
The sequence of the 16s rDNA of bacterial strain Bsp.6 refers to sequence 1 in sequence table.
4, determining of bacterial strain Bsp.6 growth optimum temperuture
By 12 part of 100 μ L OD
600value is that the bacterium liquid of 0.5 bacterial strain Bsp.6 is inoculated in respectively in 12 500mL triangular flasks that 200mL NB liquid nutrient medium is housed, and under differing temps (10～45 DEG C, concrete value is as shown in table 2) condition, 160rpm constant temperature culture is measured its OD after 24 hours
600value.
Result is as shown in table 2, and result shows in the time cultivating for 26～33 DEG C, the bacterium liquid OD of bacterial strain Bsp.6
600value is 2.385～2.701, shows that 26～33 DEG C of this strain culturing temperature are advisable, and optimum temperuture is 30 DEG C, OD
600value is 2.701.
Table 2 bacterial strain BSP.6 bacterial strain upgrowth situation in the time that condition of different temperatures is cultivated
| Temperature | 10℃ | 15 |
20℃ | 24℃ |
| OD 600Value | 0 | 1.908±0.030 | 2.013±0.154 | 2.275±0.014 |
| Temperature | 26℃ | 28℃ | 30℃ | 33℃ |
| OD 600Value | 2.423±0.037 | 2.561±0.008 | 2.701±0.022 | 2.385±0.017 |
| Temperature | 36℃ | 39℃ | 42℃ | 45℃ |
| OD 600Value | 2.212±0.056 | 1.839±0.036 | 0.679±0.012 | 0 |
5, determining of bacterial strain Bsp.6 growth optimum pH
By 13 part of 100 μ L OD
600value is that the bacterium liquid of 0.5 bacterial strain Bsp.6 is inoculated in respectively in 13 500mL triangular flasks that 200mL NB liquid nutrient medium is housed, and respectively nutrient solution is adjusted under Initial pH (4～12, as shown in table 3) condition, cultivates after 24 hours for 160rpm28 DEG C and measures its OD
600value.
Result is as shown in table 3, and result shows in the time that Medium's PH Value 6.5～8.5 is cultivated, bacterium liquid OD
600value is 2.516～2.701, shows that this strain culturing pH value is advisable with 6.5～8.5, and optimum pH is 7, OD
600value reaches 2.701.
Table 3 bacterial strain BSP.6 bacterial strain upgrowth situation in the time of different pH value
In view of results such as above-mentioned form, analysis of physio biochemical characteristics and 16s rDNA sequence homology analysis, step 1 is separated to the bacterial strain Bsp.6 obtaining and be accredited as genus bacillus (Bacillus sp.).This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on August 17th, 2012, and deposit number is CGMCC No.6459.
The detection of embodiment 2, genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 bacteriostatic activity
Genus bacillus (Bacillus sp.) the Bsp.6CGMCC No.6459 that the present embodiment obtains embodiment 1 detects the inhibition of Cuba's point No. 1 microspecies of Fusariumsp (Fusantan oxysporum f.sp.cubense) and No. 4 microspecies.Simultaneously taking genus bacillus (Bacillus sp.) BEB2 as contrast.Divide into groups as shown in table 4.
Table 4 genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 bacteriostatic activity experiment grouping
| Group | Antagonistic Fungi | |
| 1 | Genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 | No. 1 microspecies of Cuba's |
| 2 | Genus bacillus (Bacillus sp.) BEB2 | No. 1 microspecies of Cuba's point Fusariumsp |
| 3 | - | No. 1 microspecies of Cuba's |
| 4 | Genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 | No. 4 microspecies of Cuba's |
| 5 | Genus bacillus (Bacillus sp.) BEB2 | No. 4 microspecies of Cuba's point Fusariumsp |
| 6 | - | No. 4 microspecies of Cuba's point Fusariumsp |
Note: "-" represents not add corresponding bacterial strain.
Side streak inoculation Antagonistic Fungi along a straight line on PDA culture medium flat plate, and access respectively the pathogenic bacteria bacterium piece of diameter 5mm at opposite side, both are at a distance of 3cm left and right, and between guarantee group and group, experimental bacteria and pathogenic bacteria inoculum size equate.After inoculation, be placed at 28 DEG C and cultivate 6 ~ 7d, antibacterial band occurs, observes pathogenic bacteria colony growth situation in each group, measures antibacterial distance (distance between Antagonistic Fungi and pathogenic bacteria).In experiment, establish 3 repetitions for every group, test repeats 2 times, results averaged.
Antibacterial range measurements as shown in Figure 2, result shows, genus bacillus (Bacillus sp.) Bsp.6CGMCCNo.6459 all has good inhibition to Cuba's point No. 1 microspecies of Fusariumsp (Fusantan oxysporum f.sp.cubense) and No. 4 microspecies, particularly the inhibition of No. 1 microspecies of Cuba's point Fusariumsp (Fusantan oxysporum f.sp.cubense) is significantly higher than to (p<0.05) control strain genus bacillus (Bacillus sp.) BEB2.
Genus bacillus (Bacillus sp.) the Bsp.6CGMCC No.6459 that the present embodiment obtains embodiment 1 has carried out pot experiment detection to the prevention effect of the banana blight being caused by Cuba point No. 1 microspecies of Fusariumsp (Fusantan oxysporum f.sp.cubense) and/or No. 4 microspecies.Simultaneously with genus bacillus (Bacillus sp.) BEB2 bacterial strain in contrast.Experiment grouping and inoculation mode are as shown in table 5.
Table 5 genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 is to banana blight prevention effect pot experiment experiment grouping and vaccination ways
| Group | Inoculation (0d) first | Inoculation (30d) for the second time |
| 1 | - | No. 1 microspecies of Cuba's point Fusariumsp |
| 2 | - | No. 4 microspecies of Cuba's |
| 3 | Genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 | No. 1 microspecies of Cuba's |
| 4 | Genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 | No. 4 microspecies of Cuba's |
| 5 | Genus bacillus (Bacillus sp.) BEB2 | No. 1 microspecies of Cuba's |
| 6 | Genus bacillus (Bacillus sp.) BEB2 | No. 4 microspecies of Cuba's point Fusariumsp |
Note: "-" represents not add corresponding bacterial strain.
By hindering root inoculation method to potted plant dwarf banana (No. 1, the wide powder of kind) plant inoculation Antagonistic Fungi (two kinds of genus bacillus in table 5) and pathogenic bacteria (two kinds of pathogenic bacterias in table 5).The inoculum size of two kinds of Antagonistic Fungis is the bacterium liquid (3 × 10 of 50mL
4cfu/mL), the spore suspension (1 × 10 of 50mL for pathogenic bacteria inoculation
4cfu/mL).Last is inoculated the plant forms of observing dwarf banana for latter 1 month, incidence, and add up survival rate.
In experiment, process dwarf banana 30 strains for every group, test repeats 3 times, results averaged
Result demonstration, the 1st group and the 2nd group starts to occur disease symptom on the 7th day after last inoculation, all extremely complete in one month; Inoculate morbidity 4 strains in latter 1 month at last for the 3rd group, inoculate morbidity 6 strains in latter 1 month at last for the 4th group, inoculate morbidity 30 strains in latter 1 month at last for the 5th group, inoculate morbidity 12 strains in latter 1 month at last for the 6th group.To the statistics of survival rate as shown in Figure 3, inoculate latter 1 month at last, the plant survival rate of the 3rd group is that the plant survival rate of 95.6%, the 4 group is 93.3%; The plant survival rate of the 6th group is that the plant survival rate of 86.7%, the 5 group is 66.7%.
The present embodiment has been studied the prevention effect of large Tanaka genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 microbial inoculum to dwarf banana blight.
One, the preparation of genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 microbial inoculum
1, preparation substratum
(1) Banana Tissue liquid preparation
Get the banana caulo 20g apart from 1 meter of left and right, ground, the 1000mL that adds water, boils 20 minutes, and the filtrate (200mL altogether) after filtration is Banana Tissue liquid.
(2) bacterial strain mother liquor substratum preparation
Bacterial strain mother liquor substratum preparation: 3g beef extract, 10g peptone and 15g agar-agar are joined in 800mL distilled water, after heating for dissolving, adjust pH7.0, then add described Banana Tissue liquid 100mL, be settled to 1000mL with distilled water.
(3) analysis for soybean powder filtrate preparation
Claim analysis for soybean powder (powder that soya bean seed is worn into) 5g, join in 500mL distilled water, boil after 20 minutes and filter, the filtrate (100mL altogether) after filtration is analysis for soybean powder filtrate.
(4) enlarged culturing bacterium liquid culture medium preparation
Get described analysis for soybean powder filtrate 100mL, add described Banana Tissue liquid 100mL and sucrose 20g, after dissolving, adjust pH to 7.0, adding distil water 800mL, obtains described enlarged culturing bacterium liquid culture medium.
Calculate described in this 1000mL in enlarged culturing bacterium liquid culture medium the service condition of each raw material as follows:
Analysis for soybean powder: (100mL ÷ 100mL) × 5g=5g;
Banana caulo: (100mL ÷ 200mL) × 20g=10g;
Sucrose 20g;
Water 1000mL.
The proportioning for preparing the required each raw material of described enlarged culturing bacterium liquid culture medium is: analysis for soybean powder: banana caulo: sucrose: water=5g:10g:20g:1000mL.
2, cultivate bacterial strain, obtain enlarged culturing bacterium liquid (being microbial inoculum)
(1) bacterial strain mother liquor is cultivated
Under 28 DEG C of temperature condition, with the mono-bacterium colony of the streak culture genus bacillus of NA culture medium flat plate (Bacillus sp.) Bsp.6CGMCC No.6459, treat single colony diameter 3mm left and right, picking list bacterium colony, be inoculated in the above-mentioned bacterial strain mother liquor substratum of 1000mL, under 28 DEG C, 150rpm condition, shaking culture 24 hours, makes bacterial strain mother liquor.
(2) bacterium liquid enlarged culturing
The bacterial strain mother liquor (3.8 × 10 that 50mL step (1) is obtained
5cfu/mL) join in the described enlarged culturing bacterium liquid culture medium of 2000L, under 28 DEG C, 130rpm condition, shaking culture 24 hours, obtains enlarged culturing bacterium liquid, is the microbial inoculum of described genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459.The content of described genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 in described enlarged culturing bacterium liquid is about 3.8 × 10
5cfu/mL.
Two, utilize step 1 gained microbial inoculum to carry out control in field to dwarf banana blight
Test arranges two groups altogether, genus bacillus (Bacillus sp.) the Bsp.6CGMCC No.6459 microbial inoculum treatment group that group 1 obtains for step 1; Group 2 is not for carrying out the control group of any drug treating.The experimental plot area of each group is 50 mu, plants 120 strains of dwarf banana plant on every mu of experimental plot, amounts to for the dwarf banana plant of every group of test and is 6000 strains.Described dwarf banana is No. 1, the wide powder of dwarf banana kind.
1, cool canopy nursery stage is used microbial inoculum
Cool canopy nursery stage, the field planting of individual plant dwarf banana tissue cultured seedling is at 10cm(round mouth diameter) × 12cm(cup is high) glue cup in.Group 1 is used described enlarged culturing bacterium liquid (being microbial inoculum) 4 times altogether, is and fills with root inoculation.When growing the 1st young leaves the 1st time after the field planting of dwarf banana tissue cultured seedling, come into effect, by 10 times of distilled water dilutings for described enlarged culturing bacterium liquid, 60g diluent is used in every strain; While growing 2-3 sheet young leaves for any of several broadleaf plants seedling the 2nd time, weaker concn and consumption are with the 1st time; While growing 4-5 sheet young leaves for any of several broadleaf plants seedling the 3rd time, and hindering root one circle apart from glue cup outer rim 1cm place with the long fruit knife insertion soil of 10cm, then use described enlarged culturing bacterium liquid, weaker concn and consumption are with the 1st time; The 4th is that any of several broadleaf plants seedling is used while growing 6th～7 young leaves, and weaker concn is with the 1st time, and 150g diluent is used in every strain, uses field planting land for growing field crops after two weeks.And group 2(control group) do not carry out any drug treating, general planting is cultivated, with any of several broadleaf plants seedling in group 1 in same time definite value in land for growing field crops.
2, the field production phase use microbial inoculum
(1) growth early stage (transplant～the 3 month in land for growing field crops):
After 80-90% any of several broadleaf plants seedling is extracted young leaves out, in 1 week, any of several broadleaf plants seedling of group 1 starts to use described enlarged culturing bacterium liquid (being microbial inoculum), and site of administration is the root (root is filled with and executed) of any of several broadleaf plants seedling, and by 10 times of described enlarged culturing bacterium liquid dilutions, 500g diluent is used in every strain.Subsequently, on average within every 1 month, use once, use in the form of a ring, charity is in the vertical corresponding soil position of dwarf banana middle part blade outer rim, and weaker concn and consumption using first after transplanting with land for growing field crops is identical, until stop when full 3 months of any of several broadleaf plants seedling field planting land for growing field crops.And group 2(control group) not carrying out any drug treating, general planting is cultivated.
(2) grow mid-term to taking out the flower bud phase: (4th month～take out the flower bud phase)
, group 1 any of several broadleaf plants seedling every strain amount of application is 1000g diluent when from transplant in land for growing field crops the 4th month, and in the same step of application concentration and method of application (1), site of administration is depending on the position of dwarf banana middle part blade outer rim from for the second time and later using growth early stage.Within every about two months, use once, until the dwarf banana of 80-90% while taking out after flower bud 1 week, stops using.And group 2(control group) not carrying out any drug treating, general planting is cultivated.
3, the statistics of step 1 gained microbial inoculum to dwarf banana blight control in field effect
The number of banana blight disease plant in statistics group 1 and group 2 respectively, and be step 1 gained microbial inoculum according to following formula (1) calculating group 1() control in field effect to dwarf banana blight, according to following formula (2) calculating group 2(control group) in the sickness rate of dwarf banana blight.
Formula (1):
Prevention effect=(group 1 is preserved plant number-group 1 disease plant number) ÷ group 1 is preserved plant number × 100%
Formula (2):
Sickness rate=group 2 disease plant number ÷ groups 2 are preserved plant number × 100%
Result shows, through a growth season, in group 1, preserves plant 5960, wherein disease plant 297 strains; Group 2 is preserved plant 5943, wherein disease plant 1682 strains.To organize after the above-mentioned formula of 1 substitution, calculate step 1 gained microbial inoculum to dwarf banana blight land for growing field crops control effect in 95.0% left and right, group 2(control group) sickness rate of dwarf banana blight is 28.3% left and right.In addition, the plant falling ill in two groups is carried out to Race Identification, find that the dwarf banana blight of disease plant is mainly caused by Cuba's No. 1 microspecies of point Fusariumsp (Fusantan oxysporum f.sp.cubense).
The present inventor is according to as above method, the prevention effect that step 1 gained genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 microbial inoculum has been carried out 4 years is continuously measured, set genus bacillus (Bacillussp.) BEB2 simultaneously and contrasted as bacterial strain, its time of application, method of application and consumption are all identical with step 1 gained genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 microbial inoculum.The prevention effect statistics of 4 years is as shown in table 6: genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 at control in field dwarf banana blight effect stability between 93.2% ~ 95.3%; Genus bacillus (Bacillus sp.) BEB2 to the prevention effect of dwarf banana blight between 79.3% ~ 83.9%, both significant differences (p<0.05).
Table 6 genus bacillus (Bacillus sp.) Bsp.6CGMCC No.6459 microbial inoculum was to 4 of dwarf banana blight years control effects
Fruit statistics
| ? | The 1st year | The 2nd year | The 3rd year | The 4th year |
| Bsp.6 microbial inoculum | 95.3% | 94.2% | 93.5% | 93.2% |
| BEB2 microbial inoculum | 83.9% | 80.7% | 79.6% | 79.3% |
Claims (6)
1. genus bacillus (Bacillus sp.) Bsp.6, it is CGMCC No.6459 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. microbial inoculum, is characterized in that: genus bacillus described in claim 1 (Bacillussp.) Bsp.6 CGMCC No.6459 is inoculated in substratum and is cultivated, and obtaining cell concentration is 3 × 10
4-4.5 × 10
5the bacterium liquid of cfu/mL;
Described substratum is made up of following raw material: the proportioning of analysis for soybean powder, banana caulo, sucrose and water apart from ground 0.5-1.5 rice is 5g:10g:20g:1L.
3. microbial inoculum according to claim 2, is characterized in that: described substratum is mixed according to the proportioning of 100mL:100mL:20g:800mL by analysis for soybean powder filtrate, Banana Tissue liquid, described sucrose and described water, and pH is 7.0 ~ 7.2.
4. microbial inoculum according to claim 3, is characterized in that: the preparation method of described analysis for soybean powder filtrate comprises the following steps: analysis for soybean powder and water are mixed according to the ratio of 1g:30mL to 1g:400mL, boil rear filtration, obtain described analysis for soybean powder filtrate;
The preparation method of described Banana Tissue liquid comprises the following steps: banana caulo is mixed according to the ratio of 1g:100mL to 1g:25mL with water, boil rear filtration, obtain described Banana Tissue liquid.
5. genus bacillus claimed in claim 1 (Bacillus sp.) Bsp.6CGMCC No.6459, or in claim 2-4, arbitrary described microbial inoculum a) or b) is applied following:
A) suppress wilt;
B) control plant blight;
Described blight is by Cuba's point No. 1 microspecies of Fusariumsp (Fusantan oxysporumf.sp.cubense) and/or No. 4 caused blights of microspecies of Cuba's point Fusariumsp (Fusantan oxysporum f.sp.cubense); Described plant is dwarf banana.
6. the method for utilizing arbitrary described microbial inoculum control banana blight in claim 2-4, comprises the steps:
(1) after tissue culture seedlings of bananas field planting, extract the 1st young leaves, 2-3 sheet young leaves, 4-5 sheet young leaves and 6-7 sheet young leaves out and respectively use 1 time described microbial inoculum these four periods;
The described concentration of using is as follows: the concentration of using described in four times is the 1/5-1/10 of described bacteria suspension concentration;
The described dosage of using is as follows: the dosage of using described in first three time is 60-80g/ strain, and the dosage of using described in the 4th time is 100g ~ 200g/ strain;
The described mode of using is as follows: the mode of using described in four times is root filling and executes;
(2) behind banana seedlings field planting land for growing field crops, extract out after young leaves in 1 week until banana seedlings described in 80%-90%, start to use described microbial inoculum, until banana seedlings is taken out after flower bud 1 week described in 80%-90%;
The described frequency of using is for following (a) and (b):
(a) from banana seedlings described in 80%-90% is extracted out when young leaves, to described banana seedlings field planting land for growing field crops 3 months time only, on average monthly use described microbial inoculum 1 time;
(b), in the time of described banana seedlings field planting land for growing field crops 4 months, while taking out after flower bud 1 week to banana seedlings described in 80%-90%, only, on average within every two months, use 1 time described microbial inoculum;
The described concentration of using is as follows: described (a) and (b) in each described in the concentration used be the 1/5-1/10 of described bacteria suspension concentration;
The described dosage of using is as follows: the dosage of using described in each in described (a) is 400g ~ 600g/ strain; The dosage of using described in each in described (b) is 1000g/ strain;
The described mode of using is as follows: in described (a) for the first time described in the mode used be that root is filled with and executed; Using for the second time and later in described (a), and the mode of using described in each in described (b) is ring-type charity; Described ring-type charity is to use described microbial inoculum in the vertical corresponding soil part of described banana middle part blade outer rim;
Described blight is by Cuba's point No. 1 microspecies of Fusariumsp (Fusantan oxysporumf.sp.cubense) and/or No. 4 caused blights of microspecies of Cuba's point Fusariumsp (Fusantan oxysporum f.sp.cubense); Described banana is dwarf banana.
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