CN105838656A - Broad-spectrum disease resisting, growth promotion and stress resisting bacillus capable of preventing and treating tomato gray mold and tomato leaf mold and application of broad-spectrum disease resisting, growth promotion and stress resisting bacillus - Google Patents
Broad-spectrum disease resisting, growth promotion and stress resisting bacillus capable of preventing and treating tomato gray mold and tomato leaf mold and application of broad-spectrum disease resisting, growth promotion and stress resisting bacillus Download PDFInfo
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Abstract
The invention discloses broad-spectrum disease resisting, growth promotion and stress resisting bacillus capable of preventing and treating tomato gray mold and tomato leaf mold and application of the broad-spectrum disease resisting, growth promotion and stress resisting bacillus. The broad-spectrum disease resisting, growth promotion and stress resisting bacillus is bacillus sp. WXCDD105, and is preserved in China General Microbiological Culture Collection Center (CGMCC); the preservation address is 3, No.1 Yard, Western Beichen Road, Chaoyang District, Beijing, the preservation date is May 25, 2016, and the preservation number is CGMCC No.12496. A biocontrol strain provided by the invention has ultraviolet-resisting and drought-resisting properties, has stronger prevention and treatment effects on the tomato gray mold and the tomato leaf mold, and can inhibit fungal diseases including wilt fusarium of a plurality of types of crops, corn foot root bacteria, rice wilt pathogens, acanthopanax senticosus root rot pathogens, corynespora cassiicolai, colletrichum orbiculare, exserohilum turcicum, tomato spot blight fungi, sunflower sclerotinia sclerotiorum, botrytis cinerea, fusarium oxysporum, verticillium fusarium, alternaria alternata and the like; the broad-spectrum disease resisting, growth promotion and stress resisting bacillus has a growth promotion effect on tomato seeds and seedlings and can also improve the quality of fruits and reduce the rotting rate.
Description
Technical field
The invention belongs to microbial technology field, relate to a strain and graw mold of tomato and leaf mold etc. are had bacillus and the application thereof of stronger antagonism.
Background technology
Graw mold of tomato and leaf mold are the plant diseases that a class sickness rate is high, spread speed is fast, particularly even more serious at protecting field, it has also become the Major Diseases during tomato cultivation, have a strong impact on the yield and quality of Fructus Lycopersici esculenti.At present, still based on chemical pesticide during graw mold of tomato and leaf mold are prevented and treated.Although chemical pesticide has played huge effect in terms of plant disease control, but its drawback also becomes increasingly conspicuous.Long-term a large amount of use chemical pesticide can make pathogenic bacteria Drug resistance strengthen.Additionally, beneficial microbe also can be threatened while killing pathogenic bacteria, destroy Agro-ecological System, harm human health.Therefore, possess low stain, low-residual, the Biological control of the human health advantage such as safely is paid close attention to widely by people, and in practical application, plays the most important effect.
Summary of the invention
It is an object of the invention to provide a strain preventing and treating graw mold of tomato and leaf mold works very well, and have that antimicrobial spectrum is wide, biological and ecological methods to prevent plant disease, pests, and erosion lasting period length, promote plant growing, the degeneration-resistant bacillus of broad-spectrum disease resistance growth-promoting improving the characteristics such as fruit quality, resistance to ultraviolet be drought-resistant and application thereof.
It is an object of the invention to be achieved through the following technical solutions:
One strain preventing and treating graw mold of tomato and the degeneration-resistant bacillus of broad-spectrum disease resistance growth-promoting of leaf mold, it is bacillus (Bacillus sp.) WXCDD105, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date is on May 25th, 2016, and deposit number is CGMCC No.12496.
Bacillus (Bacillus sp.) WXCDD105 morphological character: Gram-positive, has brood cell to produce, and raw in brood cell, oval, cyst does not expands.Being seeded on LB solid medium, bacterium colony is creamy white, flat, rough surface, edge are irregular.In liquid medium within during growth, it is commonly formed wrinkle mould.
Bacillus (Bacillus sp.) WXCDD105 physio-biochemical characteristics: aerobic antibacterial, fermented type produces acid, wherein: clark and Lubsreaction, V.P. test is negative, and nitrate reduction reaction, Starch Hydrolysis, catalase reaction, gelatin liquefaction, citrate utilize test to be positive.Growth temperature range 4-37 DEG C, optimum growth temperature is 30 DEG C.
Bacillus (Bacillus sp.) WXCDD105 molecular biology identification result: the 16SrDNA sequence of bacterial strain WXCDD105 is submitted in the GenBanK data base of NCBI and is carried out Blast comparison, comparison result finds that the bacterial strain higher with its homology belongs to Bacillus (Bacillus sp.).Be can be seen that by the phylogenetic tree constructed, belong to bacillus cereus with its several bacterial strains in same branch, be wherein 99.93% with Bacillus subtilis subsp.inaquosorum (AMXN01000021) and Bacllus tequilensis (AYTO01000043) similarity.Identified and 16SrDNA sequence analysis by morphologic observation, physio-biochemical characteristics, tentatively bacterial strain WXCDD105 is accredited as bacillus Bacillus sp..
Bacillus (Bacillus sp.) WXCDD105 of the present invention is obvious to botrytis cinerea and Fulvia fulva Ciferri mycelial growth inhibitory action, and the suppression ratio of ash arrhizus bacteria and Fulvia fulva Ciferri mycelium morphology factor is respectively 98.2%, 95.7%.
The greenhouse pot culture effect of graw mold of tomato and leaf mold is shown by bacillus (Bacillus sp.) WXCDD105 of the present invention, first inoculates the prevention effect gray mold of pathogen up to 60.61% after inoculation biocontrol bacteria, and leaf mold is up to 65.58%；First inoculating the prevention effect gray mold of biocontrol microorganisms up to 47.88% after inoculation pathogen, leaf mold is up to 54.17%.
Bacillus (Bacillus sp.) WXCDD105 of the present invention can suppress cucumber fusarium axysporum effectively, Melon fusarium Wilt, Causal Organism of Maize Basal Stalk bacterium, Fusarium oxysporum, withered germ of water-melon, rice wilt pathogens, Radix Et Caulis Acanthopanacis Senticosi pine root fungus, tomato wilt bacterium, cucumber aphid, Botrytis cinerea pathogenic bacteria, cucumber anthracnose, spotted wilt of tomato bacterium, Exserohilum turcicum, sunflower hyphal cluster germ, the growth of the pathogenic fungi such as rod method, most pathogen fungistatic effects reach more than 85%, antimicrobial spectrum is wider, bacteriostasis is relatively strong and has bacteriolysis, lasting period is longer.
Bacillus (Bacillus sp.) the WXCDD105 bacteria suspension of the present invention has facilitation to the growth of tomato seeds radicle.Bacteria suspension and the different disposal time of variable concentrations are up to 3.35 times of CK to the impact of tomato seeds radicle growth amount, 2 times of minimum CK, and when wherein soaking 3h in diluting 10 times of bacteria suspensions, radicle is the longest.The bacteria suspension diluting 10 times has obvious growth-promoting functions to growth of seedling.Process group adds mean fresh and the dry weight of tomato seedling, and the average plant height of tomato seedling, stem are thick, root length adds 25.33%, 17.5%, 10.7% the most respectively.
Bacillus subtillis bacillus (Bacillus sp.) WXCDD105 of the present invention has the effect improving fruit quality.The tamato fruit rotting rate processed through biocontrol bacteria is about 30%, and the rotting rate of CK matched group reaches 56%, and biocontrol bacteria has preferably preserves antisepsis.The tamato fruit weight-loss ratio processed by biocontrol bacteria is significantly lower than matched group.Preserve 15d, WXCDD105, CKX and CK matched group hardness have dropped 18.5%, 28.6% and 42.9% than 0d respectively, the fruit that biocontrol bacteria processes is higher relative to matched group hardness.At the end of storage, soluble solid, titratable acid content do not have marked difference.
Bacillus (Bacillus sp.) WXCDD105 of the present invention can be dried cultivation up to 90 days in 30 DEG C of incubators.Under the conditions of the Osmotic treatment of different manual simulations, bacterium number amplitude of variation is little, and control strain bacterium number change under the artificial artificial drought conditions of difference is obvious by contrast.The antibacterial liquid crossed through 10-180min different time sections ultraviolet treatment with irradiation places cultivation 48h in LB culture medium, all can normal growth.The process group of 180min ultra-vioket radiation is compared with matched group, and bacterium number is almost unchanged, and bacteriostatic activity the most almost keeps consistent.
Bacillus (Bacillus sp.) WXCDD105 of the present invention can produce multiple antibiotic substance such as biting ferrum element, protease.
The bacillus WXCDD105 of the present invention has the advantage that
1, compared with common biocontrol bacterial strain, having excellent characteristic, in ware, average imitation all can reach more than 95%, and demonstrates biocontrol bacteria to graw mold of tomato and the prevention effect of leaf mold by pot experiment.
2, stronger to the prevention effect of graw mold of tomato and leaf mold, and the fungal diseases such as multiple kinds of crops wilt, Causal Organism of Maize Basal Stalk bacterium, rice wilt pathogens, Radix Et Caulis Acanthopanacis Senticosi pine root fungus, cucumber aphid, cucumber anthracnose, Exserohilum turcicum, spotted wilt of tomato bacterium, sunflower hyphal cluster germ, Botrytis cinerea pathogenic bacteria, Fusarium oxysporum, wheel branch Fusarium spp., rod method can be suppressed, and multiple diseases therein is had bacteriolysis, antimicrobial spectrum is relatively wide, and lasting effect is stronger.
3, tomato seeds and seedling are had growth promoting function, moreover it is possible to improve fruit quality, reduce rotting rate.
4, there is the characteristic that resistance to ultraviolet is drought-resistant, and preparation method is simple, with low cost, it is the biocontrol bacterial strain of a strain function admirable, there is good development prospect.
Preservation information:
Classification And Nomenclature: bacillus (Bacillus sp.)；
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)；
Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3；
Preservation date: on May 25th, 2016；
Deposit number: CGMCC No.12496.
Accompanying drawing explanation
Fig. 1 is the selection result of bacterial strain WXCDD105, and a is the inhibitory action to graw mold of tomato, and b is the inhibitory action to leaf muld of tomato；
Fig. 2 is the colonial morphology of bacterial strain WXCDD105；
Fig. 3 is the growth tree of bacterial strain WXCDD105；
Fig. 4 is the biocontrol bacteria inhibitory action to ash arrhizus bacteria mycelium morphology factor；
Fig. 5 is the biocontrol bacteria inhibitory action to Fulvia fulva Ciferri mycelium morphology factor；
Fig. 6 is WXCDD105 bacteria suspension and the impact on tomato seeds radicle growth amount of the different disposal time of variable concentrations, and wherein CK, 0-5 represent water, original bacteria liquid, 10 times of bacteria suspensions of dilution, dilution 10 respectively2Bacteria suspension, dilution 103Bacteria suspension, dilution 104Bacteria suspension, dilution 105Bacteria suspension；
Fig. 7 is the biocontrol bacteria impact on tamato fruit rotting rate；
Fig. 8 is the impact that biocontrol bacteria processes on tamato fruit weight-loss ratio；
Fig. 9 is the biocontrol bacteria impact on tamato fruit storage quality；
Figure 10 is that the drought tolerance of biocontrol bacteria measures；
Figure 11 is (the original bacteria liquid dilution 10 of drought tolerance Counting alive microbial7)；
Figure 12 is drought tolerance Antibacterial Activity；
Figure 13 is the detection of antibacterial substance；
Figure 14 is the fungistatic effect figure of WXCDD105, and wherein a-p represents grey mould fruit rot of strawberry, Cucumber Target Leaf Spot Causal Organism of Maize Basal Stalk, sunflower sclerotiniose, rice seedling blight, Radix Acanthopanacis Senticosi root maize ear rot, rod method, spotted wilt of tomato, cucumber anthracnose, the leaf blight of corn, wheel branch Fusarium spp., Fusarium oxysporum, watermelon blight, tomato wilt, cucumber fusarium axysporum, Muskmelon Fusarium wilt respectively；
Figure 15 is the fungistatic effect figure of WXCDD105 bacteria suspension, and wherein a-h represents watermelon blight, Fusarium oxysporum, spotted wilt of tomato, tomato wilt, sunflower sclerotiniose, rice seedling blight, wheel branch Fusarium spp., Semen Maydis brown foot rot respectively.
Detailed description of the invention
Below in conjunction with the accompanying drawings technical scheme is further described; but it is not limited thereto; every technical solution of the present invention is modified or equivalent, without deviating from the spirit and scope of technical solution of the present invention, all should contain in protection scope of the present invention.
The degeneration-resistant bacillus of broad-spectrum disease resistance growth-promoting that the present invention provides is bacillus (Bacillus sp.) WXCDD105, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date is on May 25th, 2016, and deposit number is CGMCC No.12496.
One, the Screening and Identification of biocontrol bacteria
Use microscope slide point sample dilution method, the plate streaking partition method isolated and purified WXCDD105 bacterial strain that obtains from the Rhizosphere Soil of plant, then with graw mold of tomato and leaf mold as indicator bacteria, use that duplex paper is around-France to be screened, biocontrol bacterial strain can be obtained, see Fig. 1.
Bacterial strain carries out form, cultural character and physio-biochemical characteristics identify, specifically comprise the following steps that biocontrol microorganisms WXCDD105 respectively at flat lining out, observe single colony characteristics in 30 DEG C of constant temperature culture 48h.Examine under a microscope trophosome and the shape of brood cell, size and growing state, see Fig. 2.From the test of glucose oxidative fermentation, Starch Hydrolysis test, catalase test, aerobic or anaerobic test, nitrate reduction test, gelatin liquefaction test, citrate utilization test, methylred reaction test, the physio-biochemical characteristics of V.P. test for identification bacterial strain WXCDD105.
Bacterial strain is identified: take 1mL bacterium solution centrifugal collection thalline, extract test kit (TIANGEN BIOTECH) by bacterial genomes DNA and extract genomic DNA, with genomic DNA as template, use universal primer to carry out 16SrDNA and carry out PCR amplification, 16SrDNA pcr amplification product is identified, utilize glue to reclaim test kit and the agarose gel containing target DNA is carried out glue recovery, PCR reclaims afterproduct transfers to Hua Da science and technology to check order, the 16SrDNA gene order that order-checking is obtained is analyzed with Blast in ncbi database, and carry out sequence analysis with the sequence reported.Be can be seen that by the phylogenetic tree (Fig. 3) constructed, belong to bacillus cereus with its several bacterial strains in same branch, be wherein 99.93% with Bacillus subtilis subsp.inaquosorum (AMXN01000021) and Bacllus tequilensis (AYTO01000043) similarity.Identified and 16SrDNA sequence analysis by morphologic observation, physio-biochemical characteristics, be tentatively bacillus Bacillus sp. by WXCDD105 identification of strains.
Two, biocontrol bacteria is to botrytis cinerea and the inhibitory action of Fulvia fulva Ciferri mycelial growth
According to 2% (V/V) inoculum concentration, WXCDD105 biocontrol bacteria is accessed in 50mL PDA liquid medium, inoculate ash arrhizus bacteria and the Fulvia fulva Ciferri of one piece of diameter 7mm respectively, matched group does not connect pathogenic bacteria, 28 DEG C, 120r/min cultivate 7d, mycelia is collected by filtered through gauze, drying is weighed, and each process repeats for 3 times.Result shows: biocontrol microorganisms WXCDD105 all significantly inhibits effect to botrytis cinerea and Fulvia fulva Ciferri mycelial growth, and the suppression ratio of ash arrhizus bacteria and Fulvia fulva Ciferri mycelium morphology factor is respectively 98.2%, 95.7%, is shown in Table 1 and Fig. 4, Fig. 5.
Three, the biocontrol bacteria greenhouse control effect testing to graw mold of tomato and leaf mold
Tomato variety is east agriculture 713, and the tomato seeds of accelerating germination is sowed in flowerpot, 5, every basin, each process 5 basin by accelerating germination after soaking with clear water.The spore suspension that the new ash arrhizus bacteria cultivated of sprinkling and Fulvia fulva Ciferri are made respectively is on tomato leaf.First deliver a child the fungi-proofing pathogen process group that is followed by: 500 times of diluents of biological and ecological methods to prevent plant disease, pests, and erosion bacterium solution are sprayed tomato leaf, after 1d, spray ash arrhizus bacteria and Fulvia fulva Ciferri spore the most respectively.First connect pathogen and be followed by biocontrol microorganisms process group: after spraying pathogen 1d, spray 500 times of diluents of biocontrol microorganisms again.After each process inoculation, 25 DEG C of moisturizings are cultivated, and 15d observes incidence after inoculation.According to following standard survey blade mined grade, calculate disease index and prevention effect by formula (1), (2).Stage division is as follows:
0 grade: without scab；
1 grade: lesion area accounts for whole leaf area less than 5%；
3 grades: lesion area accounts for whole leaf area 6%-10%；
5 grades: lesion area accounts for whole leaf area 11%-20%；
7 grades: lesion area accounts for whole leaf area 21%-40%；
9 grades: lesion area accounts for whole leaf area more than 40%.
Result shows: first inoculating the prevention effect gray mold of pathogen up to 60.61% after inoculation biocontrol bacteria, leaf mold is up to 65.58%.First inoculating the prevention effect gray mold of biocontrol microorganisms up to 47.88 after inoculation pathogen, leaf mold, up to 54.17%, is shown in Table 2, table 3.The prevention effect first inoculating pathogen after inoculation biocontrol bacteria inoculates biocontrol microorganisms higher than after first inoculation pathogen.
Four, the antimicrobial spectrum of biocontrol bacteria measures
Mycelial growth rate method is used to measure four kinds of biocontrol microorganisms bacteriostatic activity to 16 kinds of plant pathogenic fungis, experimental group takes 100 μ L bacterium solution and coats on PDA plate, matched group takes 100 μ L sterilized water and coats on PDA plate, at plate center inoculation cause of disease truffle (diameter 8mm), each pathogenic bacteria is repeated for 3 times, cultivate 7d for 28 DEG C, measure colony diameter, calculate bacteriostasis rate.Bacteriostasis rate=(matched group pathogenic bacteria bacterium colony net growth-experimental group pathogenic bacteria bacterium colony net growth)/matched group pathogenic bacteria bacterium colony net growth × 100%；Colony growth diameter-former bacterium cake diameter after bacterium colony net growth=cultivation 7d.Result shows that this bacterial strain is respectively provided with good inhibition to 16 kinds of pathogenic fungi, and most pathogenic bacterias all can be reached more than 85%, illustrates that this bacterial strain antimicrobial spectrum is wide, is shown in Table 4, Figure 14.
Five, the mensuration of biocontrol bacteria bacteriostasis
Flat board opposite culture method is used to measure bacteriostasis, the pathogen cake of cut-off footpath 8mm is placed in the middle of flat board, two-layer hollow filter paper ring is placed in the position (2cm) of bacterium cake both sides equidistance, experimental group drips the original bacteria liquid of 50 μ L WXCDD105, matched group is sterilized water, each pathogenic bacteria is repeated for 3 times, 28 DEG C of cultivations, pathogen colony diameter (the pathogen diameters in i.e. two filter paper ring air line distances) is measured after opposite culture 7d (now matched group all covers with plate) and 30d, calculate both differences, i.e. bacteriolyze distance.The pathogen diameter of the pathogen diameter-opposite culture 30d of bacteriolyze distance=opposite culture 7d.Result shows that the bacteriostasis of WXCDD105 bacterial strain is relatively strong, and multiple diseases is had bacteriolysis.Illustrating that this bacterial strain has good biological and ecological methods to prevent plant disease, pests, and erosion specially good effect, lasting effect is strong, is shown in Table 5, Figure 15.
Six, the growth-promoting of tomato seeds is tested by biocontrol bacteria
After tomato seeds is disinfected, equipped with the undiluted bacteria suspension of 10ml, 10 times of bacteria suspensions of dilution, dilution 102Times bacteria suspension, dilution 103Times bacteria suspension, dilution 104Times bacteria suspension, dilution 105In the test tube of times bacteria suspension and sterilized water, it is respectively put into 250 tomato seeds, take out after soaking 3h, 10h, 24h under above-mentioned variable concentrations, the tomato seeds of each process group is neatly put in the sterile petri dish being covered with filter paper, 25 tomato seeds put at random by each culture dish, it is then respectively adding 3ml sterilized water, is placed in 28 DEG C of incubators cultivation, supplements sterilized water at regular time and quantity.Each process group repeats for three times, and the radicle of record germination tomato seeds is long.By what the growth-promoting result of the test of tomato seeds was drawn, when seed being soaked 3h with the bacteria suspension of dilution 10 times, there is preferable growth-promoting functions.WXCDD105 bacteria suspension and sterilized water with dilution 10 times soak tomato seeds 3h, are then seeded in sterile soil flowerpot, and every basin sows 15, often process and are repeated 3 times.After planting 30d measure that its plant height, stem be thick respectively, root length and the fresh dry weight of plant, dry weight, plant weights weighs after drying in 80 DEG C.
Result shows: biocontrol bacteria WXCDD105 bacteria suspension has facilitation to tomato seeds growth.WXCDD105 bacteria suspension and the different disposal time of variable concentrations are up to 3.35 times of CK to the impact of tomato seeds radicle growth amount, 2 times of minimum CK, and when wherein soaking 3h in diluting 10 times of bacteria suspensions, radicle is the longest.To in the growth-promoting functions of tomato seedling, WXCDD105 bacterial strain process group adds mean fresh and the dry weight of tomato seedling, and the average plant height of tomato seedling, stem are thick, root length adds 25.33%, 17.5%, 10.7% the most respectively, see Fig. 6 and Biao 6.
Seven, the biocontrol bacteria impact on adopting rear tamato fruit physiology quality measures
Selecting that fresh full, bright in colour, size Maturity is consistent, have no mechanical damage and the commercially available little tamato fruit of disease is for testing, dry after fruit sterile water wash, line label of going forward side by side is weighed, then to be respectively put into concentration be 1 × 108The WXCDD105 Bacteria suspension of cfu/mL soaks 4min, naturally dries after taking-up, put in freshness protection package and preserve at room temperature, with 15d for the storage cycle, every 3d, the Decay of tamato fruit is observed, and the weight-loss ratio of calculating tamato fruit of weighing.Sample the hardness to fruit at the end of storage period, soluble solid, titratable acid content are measured.Test using sterilized water immersion as negative control, this laboratory preserve biocontrol bacteria X as positive control, often group fruit 30, test in triplicate.
Tamato fruit rotting rate is investigated: the investigation of rotting rate is calculated by formula (3):
Weight-loss ratio: tamato fruit weight-loss ratio calculates according to formula (4):
Hardness: take the position at tamato fruit maximum gauge and prune peel, and slowly insert sarcocarp with the GY-4 fruit hardness meter after zeroing, record reading.
Soluble solid: returned to zero by hand refractometer deionized water, takes l0g filtered through gauze extrusion juice 2-3 and drips in refractive power plane, record reading after being pulled an oar by tamato fruit.
Titratable acid content: weigh 10g tamato fruit and be placed in mortar, transfer to after grinding in 100mL volumetric flask, then by rinsing the distilled water of mortar, proceed in volumetric flask in the lump, then be settled to graduation mark with distilled water, shake up.Filter after static 30min.Draw 20mL filtrate and proceed in triangular flask, add 2-3 and drip acid phthalein solution, titrate by the NaOH solution demarcated, to pink colour aobvious at the beginning of solution, and colour-fast for titration end-point, the consumption of record NaOH solution in 30s.Test is with distilled water as blank titration, in triplicate.
Computing formula:
In formula: Vc sample extracting solution cumulative volume, mL；
VsThe volume of taken filtrate, mL during titration；
The concentration of c NaOH volumetric solution, mol/L；
V1The NaOH solution volume that titration filtrate consumes, mL；
V0The volume of consumed NaOH solution, mL during titration distilled water；
M sample quality, g；
F conversion factor, g/mmol (conversion factor of tamato fruit is calculated by malic acid, i.e. 0.067).
Result shows: tamato fruit nutritional quality is not exerted an adverse impact by biocontrol microorganisms WXCDD105.The tamato fruit rotting rate processed through biocontrol bacteria is about 30%, and the rotting rate of CK matched group reaches 56%, and biocontrol bacteria has preferably preserves antisepsis, sees Fig. 7.The tamato fruit weight-loss ratio processed by biocontrol bacteria is significantly lower than matched group, sees Fig. 8.Preserving 15d, the hardness of WXCDD105, X and CK matched group have dropped 18.5%, 28.6% and 42.9% than 0d respectively, and the fruit that biocontrol bacteria processes is higher relative to matched group hardness.At the end of storage, soluble solid, titratable acid content do not have marked difference, see Fig. 9.
Eight, the research of the drought-resistant and resistance to ultraviolet of biocontrol bacteria
Drought-resistant: to take the mono-bacterium colony of WXCDD105 and transfer in 10mL LB fluid medium, 30 DEG C, 24h is cultivated in 180r/min concussion, and culture fluid dilutes 10 respectively-1, then take 50 μ L dilution bacterium solution in the test tube gone out, be stoppered with cotton plug.Placing in 30 DEG C of incubators and cultivate, whether each have survival every bacterium colony of detection in 10 days.It is repeated 3 times.Result shows: WXCDD105 bacterial strain arid is cultivated still has survival in 90 days, is shown in Table 7.
Utilize polyethylene glycol 6000 (PEG6000) manual simulation drought condition.Appropriate PEG is added in LB fluid medium, makes ultimate density (w/v) be respectively 0%, 5%, 10%, 15%, 20%, 25% and 30%, be sub-packed in test tube, often pipe 10ml, seal with defat tampon.After inoculum concentration according to 2% inoculates strains tested, as 37 DEG C, the shaking table concussion of 200r/min is cultivated 48h and is measured viable count and measure OD600.It is repeated 3 times.Result shows: bacterium number not the biggest difference under different manual simulation drought conditions.But difference is obvious under the conditions of matched group bacterial strain different disposal.Illustrate that WXCDD105 bacterial strain has drought-resistant characteristic.See Figure 10.
Resistance to ultra-vioket radiation: take WXCDD105 seed liquor 2% and be inoculated in 10mL LB fluid medium, 30 DEG C, 24h is cultivated in 180r/min concussion, and culture fluid dilutes 10 respectively-1Then 50 μ L dilution bacterium solution are taken to the microscope slide gone out (every strain bacterium 18 pieces of microscope slides of preparation), with spreading rod, bacterium solution is spread out, ultra-vioket radiation is started after drying up, add 50 μ L sterilized water to microscope slide every 10min, stir evenly, take on the flat board of 10 μ L to the best LB with pipettor, change next microscope slide every 10min again to operate ibid, until 180min.In triplicate.Result shows: the antibacterial liquid crossed through 10-180min different time sections ultraviolet treatment with irradiation is placed and cultivated 48h in LB culture medium, all can normal growth, be shown in Table 8.
Take WXCDD105 seed liquor 2% to be inoculated in 10mL LB fluid medium, 30 DEG C, 24h is cultivated in 180r/min concussion, plate dilution assay method is used to calculate viable count after culture fluid is placed under uviol lamp irradiation 180min, and with cucumber fusarium axysporum as indicator bacteria, use flat board face-off method to measure bacteriostatic activity.With under natural light, the culture fluid that other condition of culture are the most identical compares.Being repeated 3 times, result shows: ultra-vioket radiation process group bacteria concentration is 1.35 × 1010Cfu/mL, matched group bacteria concentration is 1.47 × 1010Cfu/mL, process group and matched group bacterium number are almost unchanged；The antibacterial distance of process group is 4.62mm, and the antibacterial distance of matched group is 4.36mm, the most consistent before and after bacteriostatic activity, is shown in Table 9 and Figure 11,12.
Nine, the detection of antibacterial substance:
Protease detects: by inoculation on protease detection culture medium flat plate (defatted milk powder 1%, Carnis Bovis seu Bubali cream 0.3%, peptone 1%, sodium chloride 0.5%), cultivates 3d-7d for 10 DEG C, observes with or without enzymolysis circle.
Bite ferrum element detection: by inoculation CAS-flat board (bite ferrum element detection culture medium: A liquid: a, 60.5mg CAS (the reddish black S of cheese) is dissolved in 50mL deionized water；B, 10mL ferric iron solution；C, 72.9mg HDTMA is dissolved in 40mL deionized water.Tri-solution mixing of above-mentioned a, b, c are settled to 100mL, pH 7.0.B:1000mL NA culture medium, is adjusted to 6.8 by the NaOH solution of 12g50% (W/V) by medium pH.During use, A liquid and the mixing of B liquid.), cultivate 3-7d for 10 DEG C, observe with or without enzymolysis circle.Result shows: WXCDD105 bacterial strain produces protease and bites ferrum element.See Figure 13.
Table 1 biocontrol bacteria inhibitory action to mycelium morphology factor
Table 2 biocontrol bacteria is to graw mold of tomato greenhouse preventive effect
Table 3 biocontrol microorganisms is to leaf muld of tomato greenhouse preventive effect
The antimicrobial spectrum (8.4 × 10 of table 4 WXCDD1058cfu/mL)
Note: pure increment=bacterium colony average diameter-bacterium cake diameter
Suppression ratio (%)=(the pure increment of matched group bacterium colony-pure increment of experimental group bacterium colony) pure increment × 100% of/matched group
The bacteriostasis of table 5 WXCDD105
Note: former bacterium cake diameter 8mm
Table 6 biocontrol bacteria growth-promoting functions to tomato seedling
The drought-resistant result of study of table 7 bacterial strain WXCDD105
Note: "+" indicate bacterial growth；"-" indicates without bacterial growth
Table 8 bacterial strain WXCDD105 resistance to ultraviolet result of study
| Process time/min | WXCDD105 | Process time/min | 105 |
| 10 | + | 100 | + |
| 20 | + | 110 | + |
| 30 | + | 120 | + |
| 40 | + | 130 | + |
| 50 | + | 140 | + |
| 60 | + | 150 | + |
| 70 | + | 160 | + |
| 80 | + | 170 | + |
| 90 | + | 180 | + |
Note: "+" indicate bacterial growth；"-" indicates without bacterial growth
Table 9 bacterial strain WXCDD105 resistance to ultraviolet count results
| Bacterial strain | Matched group bacteria concentration (cfu/ml) | Process group bacteria concentration (cfu/ml) |
| WXCDD105 | 1.47×1010 | 1.35×1010 |
Claims (8)
1. strain preventing and treating graw mold of tomato and the degeneration-resistant bacillus of broad-spectrum disease resistance growth-promoting of leaf mold,
Its Classification And Nomenclature is bacillus (Bacillus sp.), and deposit number is CGMCC No.12496.
2. the broad-spectrum disease resistance of preventing and treating graw mold of tomato as claimed in claim 1 and leaf mold promotees
Raw degeneration-resistant bacillus is used for preventing and treating botrytis cinerea and Fulvia fulva Ciferri.
3. the broad-spectrum disease resistance of preventing and treating graw mold of tomato as claimed in claim 1 and leaf mold promotees
Raw degeneration-resistant bacillus is for suppressing the growth of pathogenic fungi.
4. the broad-spectrum disease resistance of preventing and treating graw mold of tomato as claimed in claim 3 and leaf mold promotees
Raw degeneration-resistant bacillus is for suppressing the growth of pathogenic fungi, it is characterised in that described pathogenic fungi
For cucumber fusarium axysporum, Melon fusarium Wilt, Causal Organism of Maize Basal Stalk bacterium, Fusarium oxysporum, west
Cucurbit wilt bacterium, rice wilt pathogens, Radix Et Caulis Acanthopanacis Senticosi pine root fungus, tomato wilt bacterium, Fructus Cucumidis sativi
Brown patch germ, Botrytis cinerea pathogenic bacteria, cucumber anthracnose, spotted wilt of tomato bacterium, the big speckle of Semen Maydis
Pathogenic bacteria, sunflower hyphal cluster germ or rod method.
5. the broad-spectrum disease resistance of preventing and treating graw mold of tomato as claimed in claim 1 and leaf mold promotees
Raw degeneration-resistant bacillus is used for promoting tomato seeds radicle growth.
6. the broad-spectrum disease resistance of preventing and treating graw mold of tomato as claimed in claim 1 and leaf mold promotees
Raw degeneration-resistant bacillus is used for promoting Growth of Tomato Seedling.
7. the broad-spectrum disease resistance of preventing and treating graw mold of tomato as claimed in claim 1 and leaf mold promotees
Raw degeneration-resistant bacillus is used for improving Quality of Tomato Fruit.
8. the broad-spectrum disease resistance of preventing and treating graw mold of tomato as claimed in claim 1 and leaf mold promotees
Raw degeneration-resistant bacillus is for drought-resistant and resistance to ultraviolet.
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