CN102286412A - Bacillus velezensis and application thereof in tomato blight antagonism - Google Patents
Bacillus velezensis and application thereof in tomato blight antagonism Download PDFInfo
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Abstract
The invention discloses a Bacillus velezensis CGMCC (China General Microbiological Culture Collection Center) No.4992 and application thereof in tomato blight antagonism bacteria. A farmland soil sample on which tomatoes are planted for years in Xinjiang Fukang is subjected to antagonism bacteria screening to obtain a strain with stronger bacteriostatic activity; the strain is further screened and domesticated and bred; and the obtained strain is subjected to morphological specificity and 16S rRNA (Ribose Nucleic Acid) sequencing and system development analysis. The antagonism of the fermentation broth of the strain has outstanding technical effect on solving the tomato blight antagonism bacteria and has wide application field.
Description
Invention field
The present invention relates to the biological control technical field, specifically, the present invention relates to the technical field of a kind of Bei Laisi genus bacillus and the application in the tomato wilt antagonism thereof.
Background technology
Tomato wilt is the soil-borne disease of a kind of serious harm quality of vegetable and output, and the seedling phase of tomato all is subjected to infecting of wilt during becoming the strain phase.Germ survives the winter in soil, propagates with irrigation water and soil, brings serious loss to tomato production when serious.Prophylactico-therapeutic measures based on agricultural chemicals, not only the soil ecology structure is seriously damaged, and to people's healthy and environment structure very big harm, compare with the pesticide residue problem with using the environmental pollution that chemical pesticide brought, biological control method has advantages such as safe, economic, efficient.Originally grind from the biological control aspect of tomato wilt and set about, by antimicrobial screening short of money, application, further reduce usage quantity, the raising Tomato Quality of agricultural chemicals, making me distinguish red tomato industry can develop towards healthy, continuable direction.
Tomato wilt is by the plain disease of the microbial a kind of vascular bundle of blight, pathogenic bacteria is Fusarium oxysporum (Schl) f.sp lycopersici[sacc] snyderet et Hansen, claim tomato point sickle spore bacterium tomato specialized form, belong to the Deuteromycotina fungi, grow 27 ℃～28 ℃, the highest 38 ℃, minimum 5 ℃ of thermophilics of germ, the specialization of tomato wilt bacterium is extremely strong, only endangers tomato, is the tomato wilting germ of Deuteromycotina, fusarium.In recent years, tomato wilt biological control and antimicrobial short of money screening are used and are received publicity, Zhang Qinfang etc. utilize non-virulent sickle-like bacteria ancient unit of weight, equal to 1/24 liang (F047) that tomato wilt is prevented and treated, find that the filtered liquid of F047 liquid culture can effectively suppress the sprouting of tomato wilt bacterium spore, suppressing germination rate is 81.7%.Field adhesion etc. utilizes trichoderma strain T4 that the preventive effect of tomato wilt is reached 93.3%, and it is sturdy that field effect directly perceived shows as the plant that the Trichoderma agent handles, and blade is dark green, well developed root system, bright root is heavy, plant height is handled than derosal increase respectively 2.5,22cm; And all death in 25 days after inserting wilt of contrast tomato seedling.Hao Xiaojuan etc. measure the prevention effect of short genus bacillus JK-2 bacterial strain, and the result shows that JK-2 can reach 83.82% and 74.70% respectively to the potted plant preventive effect and the field efficacy of tomato wilt.
The soil, Xinjiang is vast, and the photo-thermal aboundresources is the area of a few special suitable planting tomato in the world, and Xinjiang processing tomato planting scale enlarges year by year, and ultimate production improves year by year.Xinjiang tomato processing industry utilizes this special resource advantage progressively to develop, and has become China and even the main production area of world's tomato processing industry, and tomato-sauce becomes Xinjiang important export foreign-exchange-earning export products.China becomes third place in the world tomato-sauce production and big export country after the U.S., Italy.But processing tomato still is subjected to the harm of multiple diseases in plantation for many years, wherein tomato wilt is comparatively one of the severe diseases of falling ill, and causes when serious to drop in production over a large area.Carry out antimicrobial short of money screening by the agricultural land soil sample, the bacterium that obtains to have antagonistic action has important practical significance for the solution of tomato wilt bacterium.
Summary of the invention
Also do not have both at home and abroad at the present situation of Bei Laisi genus bacillus (Bacillus velezensis) at prior art as tomato wilt antimicrobial research short of money, the present invention aims to provide a kind of Bei Laisi genus bacillus (Bacillus velezensis) and as the application in the tomato wilt antagonism bacterium, by verification experimental verification, adopt bacterium that Bei Laisi genus bacillus provided by the invention (Bacillus velezensis) has an antagonistic action to have outstanding technique effect for the solution of tomato wilt bacterium.
Technical scheme of the present invention: the agricultural land soil sample of planting tomato for many years by the Fukang, Xinjiang carries out antimicrobial screening short of money, obtain to have strong bacteriostatic activity bacterial strain, through further screening, domestication breeding, obtain a strain and be numbered LYT-5.By obtaining bacterial strain being carried out morphological specificity and 16S rRNA sequencing and Phylogenetic Analysis, tentatively determined its classification position; Simultaneously characteristics such as the bacteriostasis rate of the fermented liquid of this bacterium LYT-5, stability are furtherd investigate, seen new selection for the former bacterium of biological control tomato wilt provides, its antagonistic action has outstanding technique effect for the solution of tomato wilt bacterium.
A strain provided by the invention is as tomato wilt antimicrobial Bei Laisi short of money genus bacillus (Bacillus velezensis), by separating in the agricultural land soil of planting tomato in outskirts of a town, Fukang, Xinjiang for many years, screen and cultivating, obtain a collection of as tomato wilt antimicrobial microorganism strains short of money, therefrom filter out a strain and be numbered the bacterial strain of LYT-5, have stable and significant antagonistic effect for tomato wilt, through microbiology classification and evaluation, belong to Bei Laisi genus bacillus (Bacillus velezensis).
Concrete, provided by the invention a kind of as tomato wilt antimicrobial Bei Laisi short of money genus bacillus (Bacillus velezensis), called after LYT-5.This bacterial strain was preserved in the international depositary institution of budapest treaty microorganism before the applying date: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC).Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101.Preservation date is on June 27th, 2011, and preserving number is CGMCC No.4992.Be accredited as Bei Laisi genus bacillus (Bacillus velezensis) through microbiology.This bacterial strain optimum growing condition is: 30 ℃ of temperature, substratum (LB) they are peptone 1% (W/V), yeast extract paste 0.5% (W/V), and sodium-chlor 1% (W/V), agar 2% (W/V), pH7.0-7.2 sterilized 20 minutes for 121 ℃; On the LB flat board 30 ℃ constant temperature culture 14-16 hour, bacterium colony median size is creamy white, and is opaque, the edge is irregular, and is flat, surface drying; Its thalline of microscopic examination is rod-short, gives birth in the gemma; According to the 9th edition " uncle Jie Shi systematic bacteriology identification handbook " (" Bergey, s Manual of Systematic Bacterio-logy ") and " commonly used bacterial system identification handbook " the LYT-5 bacterial strain carried out morphology measure, Physiology and biochemistry detects determines that the LYT-5 bacterial strain is the member in the Bei Laisi genus bacillus (Bacillus velezensis).By the comparison of BLAST homology, the similarity of the 16S rRNA sequence of the 16S rRNA sequence of bacterial strain LYT-5 and Bacillus velezensis is the highest, thereby bacterial strain LYT-5 is defined as Bei Laisi genus bacillus (Bacillus velezensis).
Further, a kind of Bei Laisi genus bacillus of the present invention (Bacillus velezensis) is used as tomato wilt antimicrobial short of money.
The present invention further provides the preservation condition of bacterial classification, adopted substratum (LB) composition: peptone 1% (W/V), yeast extract paste 0.5% (W/V), sodium-chlor 1% (W/V), agar 2% (W/V), pH 7.0-7.2 sterilized 20 minutes for 121 ℃.Under 30 ℃ of conditions on its LB medium slant, to cultivate 14-16 hour, it is protective material that aseptic skimmed milk is adopted in the back, cryopreservation after the vacuum lyophilization; Use at ordinary times to be kept on the inclined-plane of LB substratum, 4 ℃ of refrigerators are preserved standby.
By implementing the concrete summary of the invention of the present invention, can reach following beneficial effect: the invention provides and separate Bei Laisi genus bacillus (Bacillus velezensis) the CGMCC No.4992 that obtains, it is a kind of genus bacillus, higher using value is arranged the tomato wilt bacterium aspect preventing and treating, especially can be at the antagonism bacterium of local weather of the isolated adaptation in local habitat and growing environment for improving crop yield, reduce the agricultural chemicals usage quantity, make up good ecotope and have active effect.
Description of drawings
Figure 1 shows that the colonial morphology figure of Bei Laisi genus bacillus (Bacillus velezensis) CGMCC No.4992.
Figure 2 shows that the antagonism bacterium 16S rRNA fragment PCR amplification figure of Bei Laisi genus bacillus (Bacillus velezensis) CGMCC No.4992, among the figure, M is meant standard DNA sequence fragment size.
Figure 3 shows that the 16S rRNA phylogeny tree graph of Bei Laisi genus bacillus (Bacillus velezensis) CGMCC No.4992.
Figure 4 shows that the restraining effect figure of Bei Laisi genus bacillus (Bacillus velezensis) CGMCC No.4992 bacteria suspension to the tomato wilt bacterium.
Below, for embodiment the present invention is described, still, the present invention is not limited to following embodiment.
Embodiment
Below, for embodiment the present invention is described, still, the present invention is not limited to following embodiment.
The main raw and auxiliary material, reagent and the plant and instrument that relate among the present invention: adopt tomato wilt bacterial strain (Fusarium oxysporumf.sp.radicis-lycopersici) for the examination pathogenic bacteria, provide by ACCC (Chinese agriculture microbial strains preservation center); Pedotheque picks up from the agricultural land soil that tomato is planted in outskirts of a town, Fukang, Xinjiang for many years, and sampling back cryopreservation separates immediately to the laboratory; Substratum adopts potato glucose substratum (PDA), LB substratum; The biochemical incubator of MSSPX-250 type, MLS-3020 high-pressure steam sterilizing pan, the single two-sided clean work station of SW-CJ-1F Type B, E360K whizzer, constant temperature shaking table HWY-100.PCR instrument Eppendorf No:5345, electrophoresis apparatus Bio-Rad Mode 200/2.0, gel imaging instrument United-Bio, GK-330C plus, PCR premixed liquid (TaKaRa Biotechnology), all the other reagent are analytical pure.
All raw and auxiliary materials, reagent and the instrument of selecting for use among the present invention all is well known in the art, and other reagent more well known in the art and equipment are all applicable to the enforcement of the following embodiment of the present invention.
Embodiment one: separation, screening and the evaluation of Bei Laisi genus bacillus (Bacillus velezensis) CGMCC No.4992
(1) antimicrobial short of money separates and screening
Gradient dilution partition method: take by weighing tomato rhizosphere soil sample 10g and put into the triangular flask that the 90mL sterilized water is housed and is placed with granulated glass sphere, leave standstill 20-30s behind the vibration 20min, be 10
-1Diluent utilizes gradient method to be diluted to 10 respectively then
-2To 10
-6Diluent.Respectively get the diluent (10 of 0.1mL
-3, 10
-4, 10
-5, 10
- 6) separate application is on the LB substratum, 37 ℃ of cultivations treat that it grows isolate, to the bacterium colony that grows on the flat board separation and purification of ruling, the line of last inclined-plane is preserved standby.
Dull and stereotyped face-off method screening antagonism bacterium: adopt dull and stereotyped face-off method slightly modified, the tomato wilt pathogenic bacteria is activated on the PDA medium slant, after waiting to grow mycelia, punch tool punching bacterium cake places another fresh PDA culture medium flat plate central authorities, and 28 ℃ are continued to cultivate 2d down.Around pathogenic bacteria bacterium sheet, 4 of every ware inoculations are cultivated under 28 ℃ of constant temperature, judge its antagonistic effect according to the size that has or not inhibition zone and antibacterial band with isolated antagonistic strain dibbling to be selected.Simultaneously the bacterial strain with antagonistic action is carried out secondary face-off experiment, filter out the stronger bacterial strain of antagonistic ability in order to subsequent experimental.
Isolate many strains bacterium from tomato rhizosphere soil sample, adopt dull and stereotyped face-off method, according to having or not inhibition zone to judge whether it has antagonistic action, preliminary screening goes out the bacterial strain that the former bacterium of tomato wilt is had certain antagonistic ability.Bacterial strain with antagonistic action is carried out secondary face-off experiment, filter out the stronger bacterial strain of antagonistic ability in order to subsequent experimental.
Multiple sieve obtains to have the bacterial isolates of good antagonism tomato wilt bacterium effect, called after LYT-5, cultivate 72 hours its can the former bacterium mycelia of obvious suppression tomato wilt the growth (see figure 1), through repeatedly its fungistatic effect of experimental verification is comparatively stable.
Referring to accompanying drawing 1, the LYT-5 bacterium of acquisition was cultivated 14-16 hour under 30 ℃ of conditions on the LB culture medium flat plate, and bacterium colony median size is creamy white, and is opaque, and the edge is irregular, and is flat, surface drying; Its thalline of microscopic examination is rod-short, gives birth in the gemma; According to the 9th edition " uncle Jie Shi systematic bacteriology identification handbook " (" Bergey, s Manual of Systematic Bacterio-logy ") and " commonly used bacterial system identification handbook " the LYT-5 bacterial strain carried out morphology measure, and shape, size, the biochemical reactions of bacterial strain LYT-5 detected, LYT-5 bacterial strain biological characteristics of the present invention is as shown in table 1.
(2) pcr amplification antagonism bacterium 16S rRNA sequence and order-checking thereof
The a small amount of single bacterium colony of picking is put into the EP pipe that fills 25 μ L sterilized waters, and 100 ℃ are boiled 8-10min, after put into mixture of ice and water 5min rapidly.Centrifugal 10000r/min, 5min, 4 ℃ of preservations, the time spent gets
Table 1: the physio-biochemical characteristics of bacterial strain LYT-5
Supernatant.Pcr amplification 16S rRNA sequence.
Primer 1 (27F): 5 '-TCCTCCGCTTATTGATATGC-3 ';
Primer 2 (1492R): 5 '-CAAACTTGGTCATTAGAGGA-3 '.
The pcr amplification reaction system is 50 μ L, contains 24 μ L premix Taq, primer 11 μ L, primer 21 μ L, template 2 μ L, sterilized water 22 μ L.Amplification condition: 94 ℃ of 4min, 94 ℃ of 55s, 53 ℃ of 30s, 72 ℃ of 90s, 30 circulations; 72 ℃ of 7min.Amplified production (about 1500bp) is through 1% agarose gel electrophoresis isolation identification, and the PCR product directly carries out two-way order-checking.
(3) 16S rRNA sequence alignment and Phylogenetic Analysis
16S rRNA sequence and the nucleotide sequence in the GenBank database that order-checking is obtained carry out the BLAST analysis, therefrom obtain close 16S rRNA sequence, with Neighbor-joining method constructing system evolutionary tree among Clustal X software and the MEGA 4.1.
With the 16S rRNA sequence of bacterial 16 S rRNA sequence universal primer 27F and 1492R pcr amplification LYT-5, the PCR product is through 1% agarose gel electrophoresis, and gel imaging is referring to shown in the accompanying drawing 2.Be about through the 16S rRNA sequence of sequencing analysis LYT-5 amplification and be 1500bp.The 16S rRNA sequence of LYT-5 is carried out BLAST analyze, the result shows that the similarity of LYT-5 and bacterial strain Bacillus velezensis LMG 22478 is the highest, has reached 99%.
The 16S rRNA sequence of LYT-5 is carried out after BLAST analyzes the constructing system evolutionary tree in ncbi database.Shown in accompanying drawing 3, evolutionary distance is the shortest between bacterial strain LYT-5 and the Bacillus velezensis LMG 22478, is the allied species of Bacillus velezensis.In conjunction with morphological structure feature and the physio-biochemical characteristics of LYT-5, determine that it is Bei Laisi genus bacillus (Bacillus velezensis).
Embodiment two: the somatomedin of Bei Laisi genus bacillus (Bacillus velezensis) CGMCC No.4992
Referring to following mode, but should determine the somatomedin that it is concrete according to Bei Laisi genus bacillus provided by the invention (Bacillus velezensis) CGMCC No.4992 characteristic.
Bacterial strain LYT-5 is received on the LB substratum that contains different salt (NaCl) concentration, microbiotic (penbritin) concentration, pH, insert 30 ℃ of incubators and cultivated 14 hours.Again bacterial strain LYT-5 is received on the LB substratum, insert respectively in 4 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ the incubator and cultivate.The results are shown in Table 2.
Draw the most suitable somatomedin of bacterial strain LYT-5 by table 3.
Table 2: the influence that temperature, pH, salt, microbiotic are grown to bacterial strain LYT-5
| Temperature (℃) | 4 | 10 | 15 | 20 | 25 | 30 |
| Growing state | + | + | + | + | + | ++++ |
| Temperature (℃) | 35 | 40 | 45 | 50 | 55 | 60 |
| Growing state | +++ | ++ | + | - | - | - |
| pH | 1 | 2 | 3 | 4 | 5 | 6 |
| Growing state | - | - | - | - | - | + |
| pH | 7 | 8 | 9 | 10 | ||
| Growing state | ++ | + | - | - | ||
| NaCl concentration | 0.5％ | 1％ | 2％ | 3％ | 4％ | 5％ |
| Growing state | + | ++ | ++ | ++ | ++ | + |
| NaCl concentration | 6％ | 7％ | 8％ | 9％ | 10％ | |
| Growing state | - | - | - | - | - | |
| Penbritin μ g/ml | 50 | 60 | 70 | 80 | 90 | 100 |
| Growing state | + | - | - | - | - | - |
Embodiment three: Bei Laisi genus bacillus (Bacillus velezensis) CGMCC No.4992 fermented liquid is to the antagonistic ability of tomato wilt bacterium
After LYT-5 antagonism bacterium obtained purifying, single colony inoculation was cultivated 48h for 32 ℃ on the LB inclined-plane; Each picking one transfering loop inserts in the triangular flask of the 250mL that 100mL LB liquid nutrient medium is housed, in 32 ℃, and 200r/min shaking culture 24h; Mixing is drawn the 0.1mL bacteria suspension and is uniformly coated on the fresh PDA solid plate, after treating that bacterium liquid absorbs fully, to activate good pathogenic bacteria on the PDA flat board places with punch tool punching bacterium cake and contains antimicrobial culture medium flat plate short of money central authorities, every strain antagonism bacterium is provided with three repetitions, is control group with the pathogenic bacteria PDA flat board that adds sterilized water; Place 28 ℃ of cultivations, measured pathogenic bacteria bacterium colony size respectively at the 3rd day, the 5th day, the 7th day, colony diameter adopts the right-angled intersection method to measure.
The bacteriostasis rate calculation formula:
Bacterial strain LYT-5 all has antagonistic action preferably for tomato wilt and cotton wilt, and referring to accompanying drawing 4, from upgrowth situation, the tomato wilt bacterium grows on brassboard and almost completely is suppressed.The result shows that antagonism bacterium LYT-5 nutrient solution all keeps higher level to the inhibiting rate of tomato wilt bacterium in a week, and the inhibiting rate in five days reaches 100%, still can enough remain on more than 98% to ten day time.See Table 3:
Table 3 antagonism bacteria culture fluid is to the restraining effect of tomato wilt bacterium
Annotate: CK represents not add the control group of antagonism bacterium bacteria suspension;
LYT-5 has represented to add the experimental group of antagonism bacterium bacteria suspension;
Claims (2)
1. one kind as tomato wilt antimicrobial Bei Laisi short of money genus bacillus (Bacillus velezensis), and its deposit number is CGMCC No.4992.
2. Bei Laisi genus bacillus as claimed in claim 1 (Bacillus velezensis) CGMCC No.4992 is using as tomato wilt antimicrobial short of money.
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