CN110527647A - A kind of bacillus and its screening technique and application from the inducible system resistant of tomato rhizosphere - Google Patents
A kind of bacillus and its screening technique and application from the inducible system resistant of tomato rhizosphere Download PDFInfo
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Abstract
A kind of bacillus and its screening technique and application from the inducible system resistant of tomato rhizosphere, belongs to microorganism field.In order to which continuous cropping obstacle of tomato is effectively relieved, promote continuous cropping tomato growth, the present invention provides can be with Bei Laisi bacillus velezensis P1, culture presevation number the CCTCC No:M 2019456 of inducible system resistance from tomato rhizosphere.The bacterial strain is the rhizosphere soil by acquiring continuous cropping tomato, is diluted by continuous concentration gradient, is separately cultured using spread plate to the microorganism in the soil supension, is obtained through plate face-off experiment screening.This has the function of good growth-promoting character and field growth-promoting, is able to suppress the generation of verticillium wilt；And when tomato is infected by verticillium wilt opportunistic pathogen, tomato system resistant can be induced, enhancing tomato resists the ability of verticillium wilt evil, which has preferable Utilization prospects in terms of alleviating continuous cropping obstacle of tomato.
Description
Technical field
The invention belongs to microorganism fields, and in particular to a kind of bacillus that can induce system resistant from tomato rhizosphere
And its screening technique and application.
Background technique
Tomato verticillium wilt is the important disease of one of tomato production, is mostly occurred in the middle and later periods of tomato growth, causes to plant
Strain vascular bundle shoal, browning, blade and plant are gradually withered from top to bottom, hinder tomato later period nutriment accumulation and fortune
It is defeated, seriously reduce the yield and quality of tomato.The cause of disease of tomato verticillium wilt is verticillium dahliae (Verticillium
It dahliae), is Deuteromycotina fungi, host range is extensive, is easy to propagate, can be long-term survival in the soil.In recent years, by
In long-term sequential cropping cultivation, tomato verticillium wilt is in the trend gradually aggravated, especially the tomato verticillium wilt of facility cultivation, serious shadow
Tomato production is rung.
It is concentrated mainly on physical method for the control of tomato verticillium wilt at present, such as solarization method, chemical treatment and micro- life
Object prevents and treats three aspects.Physical method is but time-consuming and laborious with safety；Chemical method is in pollution environment, germ drug resistance, food peace
The drawbacks of congruent aspect, increasingly shows.The common microbiological that biological control is carried out to tomato verticillium wilt mainly includes in trichoderma
Some fungies, arbuscular mycorrhizal fungi, some microorganisms in streptomyces etc..But plant rhizosphere growth-promoting bacterium prevents and treats tomato
The case of verticillium wilt is less.
Summary of the invention
In order to which continuous cropping obstacle of tomato is effectively relieved, promote continuous cropping tomato growth, the present invention provides a kind of Bei Laisi gemma
Bacillus Bacillus velezensis P1, culture presevation number CCTCC No:M 2019456.
The present invention also provides the screening techniques of above-mentioned Bei Laisi bacillus velezensis P1: acquisition
The rhizosphere soil of continuous cropping tomato prepares Soil Slurry by the soil of 1g:9mL, water ratio, is diluted, obtained by continuous concentration gradient
10-3Then the soil supension of diluted concentration is separately cultured the microorganism in the soil supension using spread plate,
The plate that verticillium dahliae is carried out after the single colonie of acquisition is purified, which stands facing each other, tests, and screening obtains Bei Laisi bacillus
Bacillus velezensis P1。
The present invention also provides above-mentioned Bei Laisi bacillus velezensis P1 in tomato cultivation
The application of growth-promoting.
It further limits, the growth-promoting refers to fresh weight, plant height or the stem thickness for increasing continuous cropping tomato seedling.
It is raw in tomato cultivation that the present invention also provides above-mentioned Bei Laisi bacillus velezensis P1
The application of object prevention and treatment.
It further limits, the biological control refers to the disease incidence for reducing tomato verticillium wilt.
The present invention also provides above-mentioned Bei Laisi bacillus velezensis P1 to lure in tomato cultivation
Tomato system resistant is led, enhancing tomato resists the application of verticillium wilt evil.
Further limiting, the induction tomato system resistant refers to after tomato is infected by verticillium wilt opportunistic pathogen,
Bacillus velezensis P1 improves the Defense Enzyme Activities of tomato and the relative expression quantity of defensin gene.
It further limits, the defensive ferment is phenylalanine lyase or β -1,3- dextranase；The defence base
Because of PR gene PR1, PR gene PR2, phenylalanine ammoniacalyase gene PAL or lipoxygenase
Gene LOX.
Bei Laisi bacillus velezensis P1 of the present invention is protected on June 14th, 2019
It is hidden in China typical culture collection center, deposit number is CCTCC No:M 2019456.
Beneficial effect
Verticillium dahliae antagonistic activity with higher is obtained through present invention separation, IAA, siderophore, protease can be produced
With cellulase, there is the Bacillus velezensis P1 bacterial strain for producing antibiotic potentiality, there is good growth-promoting character
With field growth-promoting function.
The present invention also demonstrates above-mentioned Bei Laisi bacillus velezensis P1 and lures in tomato cultivation
The ability for leading systemic resistance of plant, after tomato is infected by verticillium wilt opportunistic pathogen, Bacillus velezensis P1 can drop
Low tomato verticillium wilt disease incidence is embodied in the relative expression for improving tomato correlation Defense Enzyme Activities and related defensin gene
Amount；Mitigate verticillium wilt disease, there are preferable Utilization prospects in terms of alleviating continuous cropping obstacle of tomato.
Detailed description of the invention
Fig. 1 is isolated strains to the plate inhibiting rate of verticillium dahliae, P1, P2 in abscissa, P8, P23, P33, P41,
P44 represents the number for the different strains that screening obtains, and ordinate is bacteriostasis rate (%)；
Fig. 2 is the phylogenetic tree of Bacillus velezensis P1 bacterial strain；
Fig. 3 is the colonial morphology of Bacillus velezensis P1 bacterial strain；
Fig. 4 is that Bacillus velezensis P1 bacterial strain produces siderophore ability；
Fig. 5 is that Bacillus velezensis P1 bacterial strain produces protease ability；
Fig. 6 is Bacillus velezensis P1 bacterial strain cellulase-producing ability；
Fig. 7 is that Bacillus velezensis P1 bacterial strain produces antibiotic potentiality, and wherein B indicates bmyB gene；F is indicated
FenD gene；I indicates ituC gene；S indicates srfA gene；
Fig. 8 is Bacillus velezensis P1 bacterial strain to the growth-promoting and antagonism function of continuous cropping tomato, and wherein CK is represented
The tomato plant of non-inoculating strain, V represent the tomato plant of inoculation verticillium wilt pathogen (Verticillium dahliae), P1
The tomato plant of inoculation Bacillus velezensis P1 bacterial strain is represented, P1&V is represented while being vaccinated with Bacillus
The tomato plant of velezensis P1 bacterial strain and verticillium wilt pathogen (Verticillium dahliae)；
Fig. 9 is influence of the Bacillus velezensis P1 bacterial strain to continuous cropping tomato verticillium wilt disease incidence, in abscissa
CK represents the tomato plant of non-inoculating strain, and V represents the tomato of inoculation verticillium wilt pathogen (Verticillium dahliae)
Plant, P1 represent the tomato plant of inoculation Bacillus velezensis P1 bacterial strain, and P1&V is represented while being vaccinated with
The tomato plant of Bacillus velezensis P1 bacterial strain and verticillium wilt pathogen (Verticillium dahliae)；It is vertical to sit
It is designated as disease incidence (%)；
Figure 10 is influence of the isolated strains to tomato verticillium wilt incidence in point root system system, CK representative pair in abscissa
According to that is, side is only inoculated with sterile water without plant growth-promoting rhizobacteria (PGPR) processing, other represent different strains processing；
Figure 11 is the shadow that Bacillus velezensis P1 bacterial strain defends in dividing root system system Tomato Root System enzyme activity
It rings, the time in abscissa represents pathogen treated the time；H represents control, and only side is inoculated with verticillium wilt opportunistic pathogen, the other side
With sterile water process；After PH represents side progress Bacillus velezensis P1 processing, then in other side inoculation verticillium wilt
Opportunistic pathogen；
Figure 12 is that Bacillus velezensis P1 bacterial strain carries out opposite table to Tomato Root System defence in dividing root system system
The influence reached, the time in abscissa represent pathogen treated the time；H represents control, and only side is inoculated with verticillium wilt opportunistic pathogen,
The sterile water process in the other side；After PH represents side progress Bacillus velezensis P1 processing, then in other side inoculation
Verticillium wilt opportunistic pathogen；
The screening schematic diagram of the PGPR bacterial strain of Figure 13 Induction of Systemic Resistance of Plant.
Specific embodiment
The present invention is further described with attached drawing combined with specific embodiments below, the advantages and features of the present invention will be with
Description and it is apparent.But examples are merely exemplary, and it is not intended to limit the scope of the present invention in any way.Art technology
Personnel should be understood that without departing from the spirit and scope of the invention can details and form to technical solution of the present invention
It modifies or replaces, but these modifications and replacement are fallen within the protection scope of the present invention.
The screening and identification of embodiment 1.Bacillus velezensis P1 bacterial strain.
NA culture medium used in the present embodiment is beef-protein medium: beef extract 3.0g, peptone 10.0g,
NaCl 5.0g, agar 20g, distilled water are settled to 1000mL, adjust pH to 7.4-7.6, and agar is not added in fluid nutrient medium.
1) screening of Bacillus velezensis P1:
It acquires in the potted plant experiment of Harbin, Heilongjiang Province Northeast Agricultural University gardening station, kind of simulation field continuous cropping plantation
Eggplant rhizosphere soil collects root system surface soil using root method is trembled, prepares Soil Slurry by the soil of 1g:9mL, water ratio, pass through company
Continuous concentration gradient dilution, obtains 10-3The soil supension of diluted concentration (suspension is diluted to 100 times again).Later, by 200 μ l
Soil supension is coated on 20ml beef extract-peptone agar medium (NA culture medium), is repeated 3 plates, is put in 28 DEG C of insulating boxs
Interior culture 48h, after growing single colonie on each plate, single colonie on each plate of picking is purified.It carries out later big beautiful
The plate of Verticillium dahliae, which stands facing each other, tests, method particularly includes: the 6- of verticillium dahliae (Verticillium dahliae) will be grown
Mm (diameter) agar block accesses potato dextrose agar (PDA), and at agar block about 4cm, use is sterile
Bacterial strain after purification is inoculated with by toothpick, and 4 are inoculated on each PDA.Not to be inoculated with the plate of tested bacteria for control, often
Processing is repeated 3 times.At 28 DEG C after dark culture 7 days, inhibition situation of the observation isolate to hypha,hyphae.
The present invention isolates 7 plants of bacterial strains from tomato rhizosphere, and number is respectively P1, P2, P8, P23, P33, P41, P44,
All there is stronger antagonism (Fig. 1) to verticillium dahliae in plate face-off experiment.
PCR amplification is carried out to isolated antagonistic strain and is sequenced, by sequence in EzBioCloud
(www.ezbiocloud.net) it carries out sequence similarity and compares analysis, and the 16S rDNA gene order of acquisition is submitted to
GenBank database obtains sequence accession number, and selects the building (Fig. 2) of related strain progress phylogenetic tree.With bacterial strain P8
Similarity highest bacterial strain belongs to pseudomonas, is identified by its form, physio-biochemical characteristics, is initially identified as pseudomonas
Bacterium；And the similar strain of remaining bacterial strain belongs to bacillus, through Bacillus subtilis, is initially identified as bacillus
Bacterium (table 1).
The bacterial strain for selecting antagonistic ability strong from the antagonistic strain of screening is as artistic skills object, since P1 and P2 is to big beautiful wheel
The antagonism vigor of branch bacterium is similar, this time only selects P1 as research object.
2) identification of Bacillus velezensis P1:
Physiological and biochemical test is carried out to P1 bacterial strain, display P1 is gram-positive bacteria, in addition to V-P experiment is positive, contact
Enzyme experiment, citrate test negative, combining form observation of characteristics (table 1) using experiment, methyl red, identify P1 bacterial strain
For Bacillus velezensis bacterial strain, it is named as Bacillus velezensis P1.
The observation of 1 isolated strains cultural characteristic of table
Note: Gram's staining, "-" expression are negative, and "+" expression is positive
Embodiment 2.Bacillus velezensis P1 laboratory growth-promoting ability and the measurement of antagonism potentiality.
1) IAA determination of yield
IAA ability is produced using the P1 bacterial strain that Salkow ski colorimetric method for determining embodiment 1 is screened.In autoclaved 20mL
Pancreas peptone soybean broth fluid nutrient medium (TSB: tryptone 1.5%, soy peptone 0.5%, sodium chloride 0.5%, with steaming
Distilled water is settled to 1000mL, and adjusting pH is 7.2 ± 0.2, uses after 121 DEG C of pressuresteam sterilizations) in filtration sterilization is added
Tryptophan solution (ultimate density 100mg/mL), and it is inoculated with bacterial strain to be determined.
At 28 DEG C, shaking table culture 48h.By fermentation liquid in 8000r.min-1It is centrifuged 10min.2mL supernatant is taken, is added 50
The orthophosphoric acid of μ L 83% (10mM/L) is added 4mL Sackowski ' s color developing agent, is sufficiently mixed, 25 DEG C of colour developings in dark
30min.Light absorption value under 530nm wavelength, and compareed with aseptic culture medium, in triplicate.It is ibid done in aseptic culture medium
Identical processing is ibid done with concentration 0,5,10,20,40,60,80, the heteroauxin titer of 100mg/L as control zeroing
Standard curve calculates the concentration of heteroauxin in fermentation liquid.
2) Soluble phosphorus measures
In National Botanical Research Institute Phosphate (NBRIP) fluid nutrient medium (Portugal
Grape sugar 10g, Ca3(PO4)25g, MgCl2, 6H2O 5g, Mg SO4·7H2O 0.25g, (NH4) 2SO40.1g, KCl 0.2g, fine jade
Rouge 20g, deionized water 1000m L, pH 7.0~8.0) in, the phosphorus measured in ferment product with molybdenum antimony resistance colorimetric method contains
Amount.
3) siderophore generates measurement
Siderophore ability is produced using CAS blue agar medium detection bacterial strain, observes and generates crocus bacterium circle around bacterium,
As shown in figure 4, illustrating that bacterium can produce siderophore.Culture medium the preparation method is as follows:
Solution A: taking 0.079g CAS to be dissolved in 50mL deionized water, and with 10mL 1mmol/L FeCl3Solution mixes；
Solution B: 0.069g HDTMA is taken to be dissolved in 40mL deionized water；A liquid is slowly added in b liquid by dye liquor C (CAS), is mixed well；
High pressure sterilization.The CaCl2 solution of 1mmol/L, the MgSO47H2O solution of 1mmol/L, 10% sour water solution junket egg are first configured
White solution, 10% pipes (sigma) buffer 10mL.Then take CaCl2 solution, the MgSO47H2O of 10mL molten respectively
Liquid, the acid hydrolyzed casein solution of 30mL are added appropriate pipes (sigma) buffer (100 μ l/100mL), are adjusted with NaOH
PH to 6.8-7.0.Add deionized water 450mL, total volume to 500mL.Add 10g agar and CAS solution high pressure sterilization together.Drop
When temperature is to 60 DEG C, with 5mLCAS/100mL culture medium, culture medium (25mL/500mL) is slowly added into along triangle bottle wall.Mixing is equal
It is even, it not generate bubble.Strain to be tested is inoculated on CAS culture medium.
4) cellulase generates measurement
Using 0.1%w/v CMC-Na, 1.5%w/v agar and 0.1M sodium phosphate buffer (pH 6) prepare culture medium, connect
Kind tested bacteria, and be incubated at 30 DEG C 16 hours, it is full of 30 minutes with 0.1%w/v Congo red aqueous solution, is then fallen later
Fall spot, with distilled water flushing plate (10 seconds), then 1M NaCl is used twice (every time 5 minutes), finally, by plate 5%
It is impregnated 5 minutes in w/v acetic acid, to enhance the contrast between enzymatic activity area and unchanged substrate, as a result as shown in figure 5, thoroughly
Bright circle is bigger, and cellulase activity is bigger.
5) protease generates measurement
In order to measure casein hydrolysis, after the NA culture medium high pressure sterilization of 1L, addition 15.0mL high pressure sterilization is crossed de-
Rouge milk prepares plate after mixing with the volume of each culture dish (diameter 9cm) 20ml culture medium, solidifies to culture medium
Afterwards, the HCl for being inoculated with the addition 2.0mL 0.1mol/L into culture dish (plate) after Bacillus velezensis P1,48h is molten
Liquid observes transparent haloing existing for periphery of bacterial colonies, as shown in Figure 6.
6) antagonism potentiality measure
Using the total DNA of Bacillus velezensis P1 as template, with primers F NDF1/FNDR1, BMBF2/BMBR2,
SRFAF1/SRFAR1, ITUCF1/ITUCR3 be primer, detection bacillus synthesis fengycin, bacillomycin,
The case where surfactin, iturin.PCR uses 25 μ l systems, including 1 μ l of template, each 1 μ l, dd water of upstream and downstream primer
14.7 μ l, PCR mix, 7.3 μ l.SRFAF1/SRFAR1 (srfA273bp), ITUCF1/ITUCR3 (ituC 575bp),
The condition of BMBF2/BMBR2 (bmyB 395bp) three genes: 95 DEG C of initial denaturation 4min；94 DEG C of denaturation 1min, 62 DEG C of annealing
1min, 70 DEG C of extension 1min, 30 circulations, 70 DEG C extend lasting 5min eventually.The annealing temperature of FNDF1/FNDR1 (fenD 293bp)
Degree is 52 DEG C, and other conditions are consistent with the deposition condition of the other three antibiotic, as shown in Figure 7.
As a result as follows: the IAA yield of Bacillus velezensis P1 is 23.70 μ g/mL, to the dissolution energy of aluminum phosphate
Power highest is 77.65mg/L, is inferior to the dissolution to aluminum phosphate to the solubility of ferric phosphate, is 29.46mg/L, is secondly phosphoric acid
Calcium is 21.48mg/L, and measures through PCR, display Bacillus velezensis P1 have synthesis fengycin,
The potentiality of tetra- kinds of antibiotic of bacillomycin, surfactin, iturin.
The application of the field growth-promoting and antagonism function of embodiment 3.Bacillus velezensis P1 bacterial strain.
1) seed seedling-raising and processing
It will be seeded in after kind of tomato seeds agricultural university 708 sterilizes in tomato continuous cropping soil, and simulate the field under normal growth state
Between condition plant, carry out following processing (table 2), " CK " represents the tomato of non-inoculating strain, and " Huang withers ", which represents, to be inoculated with Huang and wither
The tomato of sick pathogen (Verticillium dahliae), P1 represent kind of inoculation Bacillus velezensis P1 bacterial strain
Eggplant, " P1& Huang withers " represent while being vaccinated with Bacillus velezensis P1 bacterial strain and verticillium wilt pathogen
The tomato of (Verticillium dahliae).
2 RANDOMIZED BLOCK DESIGN of table
2) processing of Bacillus velezensis P1 and pathogen
By PDA culture medium at 121 DEG C, high pressure sterilization 15min.By verticillium wilt pathogen (Verticillium dahliae)
It is seeded in culture medium.It cultivates 7 days at 28 DEG C in the dark.Culture is filtered by double gauze.Conidium is included in
Blood red tally, concentration are adjusted to 107Conidium mL-1.Above-mentioned isolate is seeded in the LB culture medium of sterilizing, in dark
48h is cultivated at 28 DEG C, is adjusted with microplate reader, its light absorption value (OD is made600) it is 1.In order to observe bacterium isolate to plant growth
It influences, after field planting, pours light absorption value (OD every plant of tomato root600) be 1 10mL bacterial inoculum, be inoculated with same volume without
The processing of bacterium water is control；After 7 days, it is inoculated with pathogen spore suspension 10mL.3 repetitions of each processing repeat 20 basins every time.
3) morbidity survey
From (after inoculation 20 days) when there is verticillium wilt illness, institutes an inquiry disease incidence and calculate disease index.Each repetition
10 plants of random searching, 15 leaves of every plant of random searching.Disease incidence=(the morbidity number of sheets/investigation total number of sheets) × 100%
4) data are analyzed
Original experiment data is arranged using the groupware (word, Excel) of 2016 version of Office；Data point
Analysis uses SAS 9.1 editions, and variance analysis uses Tukey ' s HSD, is analyzed under the level of p < 0.05；Chart production uses
The word software and GraphPad Prism 7 of 2016 version of Office.
Interpretation of result is as follows:
1) influence of the Bacillus velezensis P1 bacterial strain to continuous cropping tomato growth of seedling
After connecing bacterium processing 20 days, the influence to tomato growth is measured, inoculation Bacillus velezensis P1 bacterial strain
Processing, as shown in table 2, increasing maximum is overground part fresh weight, increases 51%, secondly increases 33% for overground part dry weight,
Plant height increases 26%, and stem thickness increases by 15%, and underground part fresh weight also increases 10%, and underground part dry weight is compared with the control without aobvious
Write difference.It can be seen that the processing of Bacillus velezensis P1 bacterial strain promotes the growth of continuous cropping tomato, have significant
The ability of tomato overground part growth is promoted, as shown in Figure 8.
Influence (P < 0.05) of the 3 Bacillus velezensis P1 bacterial strain of table to tomato growth
2) influence of the Bacillus velezensis P1 bacterial strain to continuous cropping tomato verticillium wilt disease incidence
Being inoculated with processing of the Bacillus velezensis P1 bacterial strain from object all reduces the generation of verticillium wilt.Fig. 8 shows, with
Control is compared, and P1 processing reduces 16% disease incidence, compared with inoculation verticillium wilt processing, while being inoculated with P1 and verticillium wilt cause of disease
The disease incidence of bacterium processing reduces 23.6%.
The above result shows that the Bacillus velezensis P1 bacterial strain tool that the present invention is isolated from plant rhizosphere soil
There is higher growth-promoting ability, and being capable of producing IAA, dissolving phosphoric acid salt, generation siderophore, cellulase-producing, production protease.Continuous cropping
After tomato seedling applies this bacterium, overground part dry weight and fresh weight of plant seedlings, plant height, stem thickness and the underground part fresh weight of plant are significantly improved, is dropped simultaneously
The low disease incidence of verticillium wilt.Therefore, the present invention screens the continuous cropping that tomato can be effectively relieved in the Bei Laisi bacillus obtained
Obstacle, in tomato sequential cropping cultivation with good application prospect.
The ability of embodiment 4.Bacillus velezensis P1 induction tomato system resistant.
1) screening of tomato system resistant bacterial strain is induced
In order to compare Bacillus velezensis P1 induction tomato system resistant ability, selected while having separated its
He sieves bacterial strain together.It is selected as avoiding the direct interaction between PGPR and V.dahliae, using Split-Root Nutrient Solution Method (split-
Root), setting divides root device, to be inoculated with PGPR and V.dahliae respectively on the different root systems of same strain tomato.To promote
Tomato seedling lateral root growth carries out vernalization to carry out a point root, by tomato seeds, after seed sprouting shows money or valuables one carries unintentionally, by the seed embryo of exposing
Root cuts off top part with the scalpel sterilized, obtains tomato and divides root seedling, then plants seed in equipped with sterile soil
Nutritive cube in.When tomato length to 4 true leaves, tomato seedling is colonized in point root implant system: by tomato seedling root system point
For two parts of equivalent, planted in two plastic tubs equipped with 700g sterile soil respectively.
After being colonized 7d, pouring 20ml concentration in the tomato root for the wherein side for dividing root device is 107CFU/ml(OD600
The PGPR suspension of value about 1), to be inoculated with 20ml sterile water as control；After 5d, pouring 20ml concentration in the root other side is
107The V.dahliae spore suspension of CFU/ml.2 processing are set altogether: a) being compareed, and side is inoculated with sterile water (H2O), the other side is
Pathogen handles (V.d), b) processing, side inoculation PGPR suspension, the other side is that pathogen handles the processing of (V.d) pathogen.
3 repetitions of every processing, every 20 basin of repetition.According to 3 the method for embodiment, the disease incidence of tomato is measured, calculates disease index.Show
It is intended to as shown in figure 13.
2) tomato Defense Enzyme Activities
Plant early period is handled such as this example 1) as described in, set 2 processing altogether: control, side are inoculated with sterile water (H2O), the other side
(V.d) is handled for pathogen；Processing, side are inoculated with Bacillus velezensis P1 suspension (PGPR), and the other side is disease
Opportunistic pathogen handles the processing of (V.d) pathogen.3 repetitions of every processing, every 20 basin of repetition.0 after being inoculated with V.dahliae, 12,
24,48,96h takes Tomato Root System sample.Tomato two sides root system is cleaned rapidly, and with distilled water flushing 3 times.The same processing
3 plants mix, 3 repetitions of every processing.A part of fresh sample is for measuring plant root Defense Enzyme Activities.A part tinfoil packet
It is good, be put in it is quick-frozen in liquid nitrogen after, -80 DEG C of preservations extract (Trizol method) for RNA.
The measurement of tomato Defense Enzyme Activities is respectively according to (Wang M, Wu C, Cheng Z, the et al.Growth such as Wang
and physiological changes in continuously cropped eggplant(Solanum melongena
L.)upon relay intercropping with garlic(Allium sativum L.)[J].Frontiers in
Plant Science, 2015,6 (262)) method measure phenylalanine lyase；By Lever (Lever M.A new
reaction for colorimetric determination of carbohydrates[J].Analytical
Biochemistry, 1972,47 (1): 273-9.) method carry out β -1 with DNS coloration method, 3- dextranase enzymatic activity is surveyed
It is fixed.
3) measurement of root system defensin gene
Tomato Root System total serum IgE is extracted with Trizol, RNA carries out reverse transcription after the completion of extracting.After reverse transcription, by reverse transcription
Product be placed in -20 DEG C of preservations, the fluorescent quantitative measurement for root system defensin gene.In conjunction with described previously, this experiment is with Actin
Gene is internal reference, carries out the expression of quantitative PCR analysis defense-related gene PR1, PR2, PAL, LOX etc..Quantitative PCR reaction
It is carried out on IQ5 real-time PCR system (Bio-Rad).Specific primer and condition are shown in Table 4.PCR cycle reaction is completed
Melting curve analysis is carried out later, and PCR final product carries out agarose gel electrophoresis detection.Each PCR amplification is all provided with without template
Negative control.
4 design of primers of table
Interpretation of result is as follows:
Figure 10 is shown, in split-root experiment, compared with the control, 6 plants of bacterial strains can reduce the disease incidence of verticillium wilt,
The tomato disease incidence of Bacillus velezensis P1 processing is 47.67%, is only second to P2, illustrates Bacillus
Tomato can be improved to the resistance of verticillium wilt pathogen in velezensis P1 bacterial strain.After the pathogen that withers is infected, warp
The Tomato Root System beta-1,3-glucanase (β -1,3-glucanase) of Bacillus velezensis P1 processing is after connecing bacterium
48hpi is significantly higher than the processing for being only inoculated with verticillium wilt, and there were significant differences for other times processing.Since connecing bacterium for 24 hours pi,
Phenylalanine lyase (PAL) activity of P1 processing is all remarkably higher than the processing of inoculation verticillium wilt pathogen.
Figure 11 is shown, before connecing bacterium, Bacillus velezensis P1 processing (PH) Tomato Root System with compare between phase
Defensin gene is closed without significant difference.From 12hpi to 96hpi, the expression of two genes of PR1 and PR2 in the Tomato Root System of P1 processing
It is all remarkably higher than control, and reaches maximum respectively at pi for 24 hours and 96hpi.Tomato Root System is handled from pi to 96hpi for 24 hours, P1
The expression quantity of PAL gene is significantly higher than always control, and peaks in pi for 24 hours.Bacillus velezensis P1 processing
The expression of LOX gene is significantly higher than control in 12hpi, pi for 24 hours, and other times are interior without significant difference.
These results suggest that Bacillus velezensis P1 bacterial strain can when tomato is infected by extraneous pathogen,
Tomato correlation defensive ferment and gene relative expression is induced to increase, this is conducive to tomato and resists disease.
Nucleotides sequence list
<110>Northeast Agricultural University
<120>a kind of bacillus and its screening technique and application from the inducible system resistant of tomato rhizosphere
<130>
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<170> PatentIn version 3.5
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<213> PR-1a-F
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cgttgcttgt ttcattacct tc 22
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ctgcccgaaa ttgactgctg 20
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aatgcatcag gcacctctca agtat 25
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tgctcatcgt cttccgtttc 20
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cccaatattg ccgcaatgac 20
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Claims (9)
1. a kind of Bei Laisi bacillus velezensis P1, culture presevation number CCTCC No:M
2019456。
2. the screening technique of Bei Laisi bacillus velezensis P1 described in claim 1, feature exist
In, the rhizosphere soil of continuous cropping tomato is acquired, prepares Soil Slurry by the soil of 1g:9mL, water ratio, it is dilute by continuous concentration gradient
It releases, obtains 10-3Then the soil supension of diluted concentration divides the microorganism in the soil supension using spread plate
From culture, the plate that verticillium dahliae is carried out after the single colonie of acquisition is purified, which stands facing each other, is tested, and screening obtains Bei Laisi gemma bar
Bacterium Bacillus velezensis P1.
3. the Bei Laisi bacillus velezensis P1 described in claim 1 growth-promoting in tomato cultivation is answered
With.
4. Bei Laisi bacillus velezensis P1 biological control in tomato cultivation described in claim 1
Application.
5. Bei Laisi bacillus velezensis P1 described in claim 1 induces tomato in tomato cultivation
System resistant, enhancing tomato resist the application of verticillium wilt evil.
6. application according to claim 3, which is characterized in that the growth-promoting refer to increase continuous cropping tomato seedling fresh weight,
Plant height or stem thickness.
7. application according to claim 4, which is characterized in that the biological control refers to the morbidity for reducing tomato verticillium wilt
Rate.
8. application according to claim 5, which is characterized in that the induction tomato system resistant refers in tomato by Huang
It withers after pathogen infection, Bacillus velezensis P1 improves the Defense Enzyme Activities of tomato and the relative expression of defensin gene
Amount.
9. application according to claim 8, which is characterized in that the defensive ferment is phenylalanine lyase or β -1, the Portugal 3-
Dextranase；The defensin gene is PR gene PR1, PR gene PR2, phenylalanine ammoniacalyase
Gene PAL or lipoxygenase gene LOX.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112899201A (en) * | 2021-03-16 | 2021-06-04 | 云南省农业科学院农业环境资源研究所 | Bacillus belgii, application thereof and method for preventing and treating banana wilt |
| CN115316252A (en) * | 2022-10-10 | 2022-11-11 | 中国科学院昆明植物研究所 | Method for establishing root division system in herbaceous root hemiparasitic plant vessel |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107254427A (en) * | 2017-08-15 | 2017-10-17 | 云南农业大学 | A kind of Bei Laisi Bacillus strains JN5 and its application |
| CN107964514A (en) * | 2017-09-08 | 2018-04-27 | 华夏春秋科技发展（北京）有限责任公司 | A kind of Bei Laisi bacillus and its application on plant |
| CN109055281A (en) * | 2018-09-19 | 2018-12-21 | 北京化工大学 | Bei Laisi bacillus ZF2 and its application in control of plant disease |
-
2019
- 2019-08-21 CN CN201910774544.6A patent/CN110527647A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107254427A (en) * | 2017-08-15 | 2017-10-17 | 云南农业大学 | A kind of Bei Laisi Bacillus strains JN5 and its application |
| CN107964514A (en) * | 2017-09-08 | 2018-04-27 | 华夏春秋科技发展（北京）有限责任公司 | A kind of Bei Laisi bacillus and its application on plant |
| CN109055281A (en) * | 2018-09-19 | 2018-12-21 | 北京化工大学 | Bei Laisi bacillus ZF2 and its application in control of plant disease |
Non-Patent Citations (1)
| Title |
|---|
| CRISTINA RUIZ-GARCÍA等: "Bacillus velezensis sp. nov., a surfactant-producing bacterium isolated from the river Vélez in Málaga, southern Spain", 《INT J SYST EVOL MICROBIOL》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112899201A (en) * | 2021-03-16 | 2021-06-04 | 云南省农业科学院农业环境资源研究所 | Bacillus belgii, application thereof and method for preventing and treating banana wilt |
| CN115316252A (en) * | 2022-10-10 | 2022-11-11 | 中国科学院昆明植物研究所 | Method for establishing root division system in herbaceous root hemiparasitic plant vessel |
| CN115316252B (en) * | 2022-10-10 | 2023-01-31 | 中国科学院昆明植物研究所 | Method for establishing root division system in herbaceous root hemiparasitic plant vessel |
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