CN106190917B - The degeneration-resistant production nitrogen pseudomonad of one plant of broad-spectrum disease resistance growth-promoting and its application - Google Patents
The degeneration-resistant production nitrogen pseudomonad of one plant of broad-spectrum disease resistance growth-promoting and its application Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
Abstract
The invention discloses the degeneration-resistant production nitrogen pseudomonad of one plant of broad-spectrum disease resistance growth-promoting and its applications, the degeneration-resistant production nitrogen pseudomonad of the broad-spectrum disease resistance growth-promoting is to produce nitrogen pseudomonad (Pseudomonas azotoformans) WXCDD51, the deposit date is on July 12nd, 2016, deposit number was CGMCC No.12760.Biocontrol bacterial strain of the invention has resistance to ultraviolet drought-resistant characteristic, it is stronger to the control efficiency of graw mold of tomato and leaf mold, it is able to suppress the fungal diseases such as multiple kinds of crops wilt, Causal Organism of Maize Basal Stalk bacterium, rice wilt pathogens, wilsonii pine root fungus, cucumber aphid, cucumber anthracnose, Exserohilum turcicum, spotted wilt of tomato bacterium, sunflower hyphal cluster germ, Botrytis cinerea germ, Fusarium oxysporum, wheel branch sickle-like bacteria, rod method, and there is growth promoting function to tomato seeds, fruit quality can also be improved, reduce rotting rate.
Description
Technical field
The invention belongs to microorganisms technical fields, are related to one plant to graw mold of tomato, leaf mold, Cucumber Target Leaf Spot and thorn five
Add the plurality of plant diseases such as root rot that there is production nitrogen pseudomonad and its application of stronger antagonism.
Background technique
The generation of corps diseases usually destroys the growth of plant, threatens its yield and quality, causes huge loss.Often
The main control method for the plant disease seen has breeding resistant variety, chemical prevention and biological control etc..For some such as tomato
The Common Diseases of gray mold, leaf mold, Cucumber Target Leaf Spot and Radix Acanthopanacis Senticosi root maize ear rot etc., both at home and abroad in terms of screening anti-resource material
The research of long period has been done, has not filtered out ideal anti-source material always, progress of breeding is slow.And chemical pesticide
Long-term a large amount of uses, can be such that germ drug resistance enhances, and pesticide residue endangers human and livestock health, in addition, chemical pesticide is killing germ
While also kill farmland beneficial microbe, destroy Agro-ecological System.And biological control can by Disease management in floor level,
Its advantage is that low toxicity, low-residual, low pollution, environmentally friendly and safe and efficient." 13 " or even from now on longer period agricultural
Work is even more that " will protect supply, and protect income, protect ecology " to be asserted " three protect " target.Green guarantor's ecology is done it is necessary to reduce pesticide, chemical fertilizer
Unreasonable use, therefore the microbial pesticide of Eco-friendly become crop disease control important means.
Summary of the invention
The object of the present invention is to provide one plant of prevention and treatment graw mold of tomato, leaf mold, Cucumber Target Leaf Spot and Radix Acanthopanacis Senticosi root maize ear rots
Etc. plant diseases the degeneration-resistant production nitrogen pseudomonad of broad-spectrum disease resistance growth-promoting and its application, the bacterial strain prevent and treat graw mold of tomato, leaf mold,
The common crops disease effect such as Cucumber Target Leaf Spot and Radix Acanthopanacis Senticosi root maize ear rot is preferable, and, promotion plant growth wide with antimicrobial spectrum,
Improve fruit quality, the resistance to ultraviolet characteristics such as drought-resistant, application value with higher has potential research in biological control
And exploitation advantages.
The purpose of the present invention is what is be achieved through the following technical solutions:
The degeneration-resistant production nitrogen pseudomonad of one plant of broad-spectrum disease resistance growth-promoting, it is production nitrogen pseudomonad (Pseudomonas
Azotoformans) WXCDD51, hereinafter referred to as WXCDD51, it is common to be preserved in China Committee for Culture Collection of Microorganisms
Microorganism center (CGMCC), preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and the deposit date is in July, 2016
12, deposit number was CGMCC No.12760.
WXCDD51 morphological character: Gram-positive does not produce gemma；Bacterium colony is creamy white on LB culture medium, round, prominent
Rise, surface it is smooth, neat in edge, can generate metabolin makes culture medium darken brown.
WXCDD51 physio-biochemical characteristics: WXCDD51 is aerobic bacterium, can utilize glucose and fructose, and oxidized form produces
Acid, wherein nitrate reduction reaction, clark and Lubsreaction, V.P. test be negative, Starch Hydrolysis, contact enzyme reaction, gelatin liquefaction,
Citrate is positive using test.4-37 DEG C of growth temperature range, optimum growth temperature is 25-30 DEG C.
WXCDD51 molecular biology identification result: the 16SrDNA sequence of WXCDD51 bacterial strain is submitted to NCBI's
In GenBank database and Blast comparison is carried out, comparison result discovery belongs to pseudomonad with the higher bacterial strain of its homology
Belong to (Pseudomonas), using 50 plants of bacterium phylogenetic tree constructions of 97% or more similarity, by the phylogenetic tree constructed
It can be seen that, WXCDD51 bacterial strain and Pseudomonas azotoformans (D84009) are in the same branch, this explanation
WXCDD51 bacterial strain and Pseudomonas azotoformans affiliation are nearest, similitude 98.75%.Pass through Appearance View
Examine, physio-biochemical characteristics identification and 16SrDNA sequence analysis, WXCDD51 bacterial strain is accredited as in pseudomonad group
Pseudomonas azotoformans。
WXCDD51 of the invention significantly inhibits effect to botrytis cinerea and the growth of Fulvia fulva Ciferri mycelia, to ash
The inhibiting rate of mildew bacterium and Fulvia fulva Ciferri mycelium morphology factor is respectively 94.9%, 90.7%.
WXCDD51 of the invention shows first to be inoculated with biocontrol bacteria to the greenhouse pot culture effect of graw mold of tomato and leaf mold
It is inoculated with the control efficiency gray mold of pathogen afterwards up to 54.51%, leaf mold is up to 61.46%；It is inoculated with after being first inoculated with pathogen
The control efficiency gray mold of biocontrol microorganisms is up to 45.27%, and leaf mold is up to 50.61%.
WXCDD51 of the invention can effectively inhibit cucumber fusarium axysporum, Melon fusarium Wilt, Causal Organism of Maize Basal Stalk
Bacterium, Fusarium oxysporum, withered germ of water-melon, rice wilt pathogens, wilsonii pine root fungus, tomato wilt bacterium, cucumber foxiness
Germ, Botrytis cinerea germ, cucumber anthracnose, spotted wilt of tomato bacterium, Exserohilum turcicum, sunflower hyphal cluster germ, rod method
The growth of equal disease fungus, antimicrobial spectrum is wider, wherein stronger to the inhibiting effect of grey mould fruit rot of strawberry and Cucumber Target Leaf Spot.
WXCDD51 bacteria suspension of the invention has facilitation to the growth of tomato seeds radicle.Various concentration
The influence of WXCDD51 bacteria suspension and different disposal time to tomato seeds radicle growth amount is up to 1.49 times of CK, minimum
1.06 times of CK, wherein when impregnating 3h in diluting 103 times of bacteria suspensions, radicle longest.
WXCDD51 of the invention does not have a adverse impact to tamato fruit nutritional quality, and having improves fruit quality
Effect.The tamato fruit rotting rate handled through biocontrol bacteria is about 30%, and the rotting rate of CK control group is up to 56%, and biological and ecological methods to prevent plant disease, pests, and erosion is thin
Bacterium has preferable storage antisepsis.Control group is significantly lower than by the processed tamato fruit weight-loss ratio of biocontrol bacteria.Storage
The hardness of 15d, WXCDD51 and CK control group has dropped 28.6% and 42.9% than 0d respectively, biocontrol bacteria processing
Fruit is higher relative to control group hardness.Soluble solid, titratable acid content do not have marked difference at the end of storage.
Culture can be dried up to 30 days in WXCDD51 of the invention in 30 DEG C of incubators.Different manual simulations'
Under the conditions of Osmotic treatment, bacterium number amplitude of variation is little, in contrast control strain bacterium number under different artificial artificial drought conditions
Variation is obvious.The bacterium liquid crossed by 10-180min different time sections ultraviolet treatment with irradiation is placed to be cultivated on LB culture medium
48h, can normal growth.Compared with the control group, bacterium number is almost unchanged, and bacteriostatic activity is also several for the processing group of 180min ultraviolet irradiation
It is consistent.
WXCDD51 of the invention has the advantages that
1, compared with common biocontrol bacterial strain, there is excellent characteristic, to averagely imitative in graw mold of tomato and leaf mold ware
Effect can reach 90% or more, and demonstrate biocontrol bacteria by pot experiment and imitate to the prevention and treatment of graw mold of tomato and leaf mold
Fruit.
2, not only stronger to the control efficiency of graw mold of tomato and leaf mold, but also it is able to suppress multiple kinds of crops wilt disease
Bacterium, Causal Organism of Maize Basal Stalk bacterium, rice wilt pathogens, wilsonii pine root fungus, cucumber aphid, cucumber anthracnose, corn
Exserohilum Turcicum, spotted wilt of tomato bacterium, sunflower hyphal cluster germ, Botrytis cinerea germ, Fusarium oxysporum, wheel branch sickle-like bacteria, rod method
Equal fungal diseases, fungistatic effect is preferable, and antimicrobial spectrum is wider, wherein to grey mould fruit rot of strawberry and Cucumber Target Leaf Spot inhibiting effect compared with
By force.
3, there is growth promoting function to tomato seeds, moreover it is possible to improve fruit quality, reduce rotting rate.
4, with resistance to ultraviolet drought-resistant characteristic, can generate metabolin makes culture medium darken brown, and identification is high, can
Basis is done to be prepared into biological pesticide in next step, and preparation method is simple, it is low in cost, it is the biocontrol microorganisms of one plant of function admirable
Strain, with good development and application prospects.
Preservation information:
Classification naming: it produces nitrogen pseudomonad (Pseudomonas azotoformans)；
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 July 12nd, 2016；
Deposit number: CGMCC No.12760.
Detailed description of the invention
Fig. 1 is the selection result of bacterial strain WXCDD51, and a is the inhibiting effect to graw mold of tomato, and b is to leaf muld of tomato
Inhibiting effect；
Fig. 2 is the colonial morphology of bacterial strain WXCDD51；
Fig. 3 is the development tree of bacterial strain WXCDD51；
Fig. 4 is inhibiting effect of the biocontrol bacteria to ash arrhizus bacteria mycelium morphology factor；
Fig. 5 is inhibiting effect of the biocontrol bacteria to Fulvia fulva Ciferri mycelium morphology factor；
Fig. 6 is the influence of the WXCDD51 bacteria suspension and different disposal time of various concentration to tomato seeds radicle growth amount,
Wherein CK, 0-5 respectively indicate water, original bacteria liquid, 10 times of bacteria suspensions of dilution, dilution 102Bacteria suspension, dilution 103Bacteria suspension, dilution 104
Bacteria suspension, dilution WXCDD51 bacteria suspension；
Fig. 7 is influence of the biocontrol bacteria to tamato fruit rotting rate；
Fig. 8 is influence of the biocontrol bacteria processing to tamato fruit weight-loss ratio；
Fig. 9 is influence of the biocontrol bacteria to tamato fruit storage quality；
Figure 10 is that the drought tolerance of biocontrol bacteria measures；
Figure 11 is resistance to ultraviolet Antibacterial Activity；
Figure 12 is the fungistatic effect figure of WXCDD51, and wherein a-p respectively indicates grey mould fruit rot of strawberry, Cucumber Target Leaf Spot, Maize Stem
Basal stem rot, sunflower sclerotiniose, rice seedling blight, Radix Acanthopanacis Senticosi root maize ear rot, rod method, spotted wilt of tomato, cucumber anthracnose, corn are big
Pinta, wheel branch sickle-like bacteria, Fusarium oxysporum, watermelon blight, tomato wilt, cucumber fusarium axysporum, Muskmelon Fusarium wilt；
Figure 13 is the fungistatic effect figure of the plate dual test of WXCDD51 bacteria suspension, and wherein a-l respectively indicates Botrytis cinerea
Disease, tomato wilt, rice seedling blight, rod method, sunflower sclerotiniose, spotted wilt of tomato, the leaf blight of corn, Fusarium equiseti,
Radix Acanthopanacis Senticosi root maize ear rot, Causal Organism of Maize Basal Stalk, watermelon blight, wheel branch sickle-like bacteria.
Specific embodiment
Further description of the technical solution of the present invention with reference to the accompanying drawing, and however, it is not limited to this, all to this
Inventive technique scheme is modified or replaced equivalently, and without departing from the spirit and scope of the technical solution of the present invention, should all be covered
Within the protection scope of the present invention.
The degeneration-resistant pseudomonad of broad-spectrum disease resistance growth-promoting provided by the invention is to produce nitrogen pseudomonad (Pseudomonas
Azotoformans) WXCDD51 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, and the deposit date is on July 12nd, 2016, deposit number was
CGMCC No.12760。
One, the screening and identification of biocontrol bacteria
It isolates and purifies to obtain from the Rhizosphere Soil of plant using glass slide point sample dilution method, plate streaking partition method
WXCDD51 bacterial strain, then using graw mold of tomato and leaf mold as indicator bacteria, screened using duplex paper is around-France, can be obtained life
Anti- bacterial strain, is shown in Fig. 1.
Form, cultural character and physio-biochemical characteristics identification are carried out to bacterial strain, the specific steps are as follows: by biocontrol microorganisms
WXCDD51 observes single colonie feature in 30 DEG C of constant temperature incubation 48h respectively in flat lining out.Trophosome is observed under the microscope
With the shape, size and growing state of brood cell, Fig. 2 is seen.From the test of glucose oxidative fermentation, Starch Hydrolysis test, catalase examination
It tests, the test of aerobic or anaerobic, nitrate reduction test, gelatin liquefaction test, citrate utilization test, clark and Lubsreaction examination
It tests, the physio-biochemical characteristics of V.P. test for identification bacterial strain WXCDD51.
Identify bacterial strain: thalline were collected by centrifugation for the bacterium solution for taking 2mL to cultivate to logarithmic phase, extracts bacterium base with CTAB method
Because of a group DNA, using genomic DNA as template, 16SrDNA is carried out using universal primer and carries out PCR amplification, 16SrDNA PCR amplification
Ago-Gel containing target DNA is carried out glue recycling using plastic recovery kit by product identification, and product is handed over after PCR is recycled
It is sequenced by Hua Da science and technology, the 16SrDNA gene order that sequencing obtains is analyzed in ncbi database with Blast,
And sequence analysis is carried out with reported sequence.Similarity is constructed in 97% or more 50 plants of bacterium using MEGA5.0 software
Phylogenetic tree.See Fig. 3.As a result, it has been found that pseudomonas (pseudomonas) is belonged to the higher bacterial strain of its homology, by
The phylogenetic tree constructed can be seen that WXCDD51 bacterial strain with Pseudomonas azotoformans (D84009) same
In one branch, this illustrates that WXCDD51 bacterial strain and pseudomonas azotoformans affiliation are nearest.Pass through Appearance View
Examine, physio-biochemical characteristics identification and 16SrDNA sequence analysis, WXCDD51 bacterial strain is accredited as in pseudomonad group
pseudomonas azotoformans。
Two, the inhibiting effect that biocontrol bacteria grows botrytis cinerea and Fulvia fulva Ciferri mycelia
According to 2% (V/V) inoculum concentration respectively by WXCDD51, control bacterium X biocontrol bacteria access in 50mL PDA Liquid Culture
In liquid, it is inoculated with the ash arrhizus bacteria and Fulvia fulva Ciferri of one piece of diameter 7mm respectively, control group does not connect germ, 28 DEG C, 120r/min culture
7d collects mycelia, drying weighing, 3 repetitions of each processing with filtered through gauze.The result shows that: biocontrol microorganisms WXCDD51 is to tomato ash
Mildew bacterium and the growth of Fulvia fulva Ciferri mycelia significantly inhibit effect, the inhibition to ash arrhizus bacteria and Fulvia fulva Ciferri mycelium morphology factor
Rate is respectively 94.9%, 90.7%.It is shown in Table 1 and Fig. 4, Fig. 5.
Three, greenhouse control effect testing of the biocontrol bacteria to graw mold of tomato and leaf mold
Tomato variety is eastern agriculture 713, and the tomato seeds of vernalization are sowed every basin 5 in flowerpot by vernalization after being impregnated with clear water
Grain, 5 basins of each processing.Spore suspension made of the ash arrhizus bacteria and Fulvia fulva Ciferri newly cultivated is sprayed respectively on tomato leaf.
It first delivers a child and fungi-proofing is followed by pathogen processing group: 500 times of dilutions of biological and ecological methods to prevent plant disease, pests, and erosion bacterium solution are sprayed into tomato leaf, sprinkling ash respectively again after 1d
Mildew bacterium and Fulvia fulva Ciferri spore.It first connects pathogen and is followed by biocontrol microorganisms processing group: spraying biocontrol microorganisms 500 again after spraying pathogen 1d
Times dilution.After each processing inoculation, 25 DEG C of moisturizing cultures, 15d observes incidence after inoculation.According to following standard survey leaf
Piece mined grade calculates disease index and control efficiency.It is calculated by formula (1), (2).Stage division:
0 grade: disease-free spot；
1 grade: lesion area accounts for entire 5% or less leaf area；
3 grades: lesion area accounts for entire leaf area 6-10%；
5 grades: lesion area accounts for entire leaf area 11-20%；
7 grades: lesion area accounts for entire leaf area 21-40%；
9 grades: lesion area accounts for entire 40% or more leaf area.
The result shows that: first it is inoculated with the control efficiency gray mold that pathogen is inoculated with after biocontrol bacteria up to 54.51%, leaf mold
Up to 61.46%.The control efficiency gray mold of biocontrol microorganisms is first inoculated with after inoculation pathogen up to 45.27%, leaf mold is reachable
50.61%, it is shown in Table 2, table 3.First the control efficiency of inoculation pathogen is inoculated with after being higher than first inoculation pathogen after inoculation biocontrol bacteria
Biocontrol microorganisms.
Four, the antimicrobial spectrum measurement of biocontrol bacteria
Bacteriostatic activity of four kinds of biocontrol microorganisms to 16 kinds of plant pathogenic fungis, experimental group are measured using mycelial growth rate method
100 μ L bacterium solutions are taken to be coated on PDA plate, control group takes 100 μ L sterile waters to be coated on PDA plate, is inoculated in plate center
Cause of disease fungus block (diameter 8mm), 3 repetitions of each germ, 28 DEG C of culture 7d measure colony diameter, calculate bacteriostasis rate.Bacteriostasis rate=
(control group germ bacterium colony net growth-experimental group germ bacterium colony net growth)/control group germ bacterium colony net growth ×
100%；Colony growth diameter-original bacteria cake diameter after bacterium colony net growth=culture 7d.The result shows that the bacterial strain is to 16 kinds of cause of diseases
Fungi has good inhibitory effect, wherein it is stronger to the inhibiting effect of grey mould fruit rot of strawberry and Cucumber Target Leaf Spot, illustrate the bacterial strain
Antimicrobial spectrum is wide, and bacteriostasis is strong, is shown in Table 4, Figure 12.
Five, the plate dual test of biocontrol bacteria
Four kinds of biocontrol microorganisms are measured to the bacteriostatic activity of 12 kinds of plant pathogenic fungis using flat-plate bacterial colony face-off method, are advised unified
The filter paper of lattice (overall diameter 13mm, interior diameter 8mm) is placed at distance center equidistant (2cm), takes 50 μ L experimental group bacterium solutions
It is added dropwise on filter paper, control group adds 50 μ L of sterile water, and plate center is inoculated with cause of disease fungus block (diameter 8mm), 3 weights of each germ
Multiple, 28 DEG C of culture 7d measure pathogen colony diameter, calculate bacteriostasis rate.Bacteriostasis rate=(control group germ bacterium colony net growth-
Experimental group germ bacterium colony net growth)/control group germ bacterium colony net growth × 100%.The result shows that using tablet face-off method
The germ is measured to keep the inhibitory effect of 12 kinds of disease fungus substantially with the inhibitory effect using mycelial growth rate method measurement
Unanimously, illustrate the germ inhibiting effect preferably and stablize, be shown in Table 5, Figure 13.
Six, biocontrol bacteria tests the growth-promoting of tomato seeds
After tomato seeds disinfection treatment, equipped with 102 times of undiluted bacteria suspension of 10ml, 10 times of bacteria suspensions of dilution, dilution bacterium
Suspension, 104 times of bacteria suspensions of dilution, dilutes in the test tube of 51 times of bacteria suspensions and sterile water 103 times of bacteria suspensions of dilution, is respectively put into
250 tomato seeds impregnate 3h, 10h under above-mentioned various concentration, take out afterwards for 24 hours, and the tomato seeds of each processing group are neat
For pendulum in the sterile petri dish for being covered with filter paper, each culture dish is put into 25 tomato seeds at random, and it is sterile to be then respectively adding 3ml
Water is placed in 28 DEG C of incubators and cultivates, supplements sterile water at regular time and quantity.Each processing group repeats three times, record germination tomato species
The radicle of son is long.The result shows that: biocontrol bacteria WXCDD51 bacteria suspension has facilitation to tomato seeds growth.Various concentration
The influence to tomato seeds radicle growth amount of WXCDD51 bacteria suspension and different disposal time be up to 1.49 times of CK, it is minimum
It is 1.06 times of CK, wherein in dilution 103When impregnating 3h in times bacteria suspension, radicle longest is shown in Fig. 6.
Seven, biocontrol bacteria measures the influence for adopting rear tamato fruit physiology quality
Select the commercially available small tamato fruit that fresh full, bright in colour, size maturity is consistent, has no mechanical damage with disease
It for testing, will be dried after fruit sterile water wash, line label of going forward side by side weighing, then being respectively put into concentration is 1 × 108cfu/mL
WXCDD51 bacteria suspension in impregnate 4min, naturally dry after taking-up is put into freshness protection package and stores at room temperature, with 15d be storage
The period is hidden, is observed every Decay of the 3d to tamato fruit, and weigh and calculate the weight-loss ratio of tamato fruit.Storage period knot
Sampling is measured the hardness of fruit, soluble solid, titratable acid content when beam.Test is control with sterile water immersion
Group, every group fruit 30, test is in triplicate.
The investigation of tamato fruit rotting rate: the investigation of rotting rate is calculated by formula (3):
Weight-loss ratio: tamato fruit weight-loss ratio is calculated according to formula (4):
Hardness: the position at tamato fruit maximum gauge is taken to prune pericarp, and slow with the GY-4 fruit hardness meter after zeroing
It is slowly inserted pulp, record reading.
Soluble solid: hand refractometer is returned to zero with deionized water, and 10g gauze mistake is taken after tamato fruit is beaten
Filter squeezes out juice 2-3 drop in refractive power plane, record reading.
Titratable acid content: weighing 10g tamato fruit and be placed in mortar, be transferred in 100mL volumetric flask after grinding, then will
The distilled water for rinsing mortar, is transferred in volumetric flask, then be settled to graduation mark with distilled water together, shakes up.Mistake after static 30min
Filter.It draws 20mL filtrate to be transferred in triangular flask, the sour phthalein solution of 2-3 drop is added, is titrated with the NaOH solution demarcated, until molten
Liquid just shows pink colour, and colour-fast in 30s is titration end-point, records the dosage of NaOH solution.Test is dripped by blank of distilled water
It is fixed, in triplicate.
Calculation formula:
In formula: Vc-sample extracting solution total volume, mL；
The volume of taken filtrate, mL when Vs-titration；
The concentration of c-NaOH titrating solution, mol/L
V1-titration filtrate consumption NaOH solution volume, mL；
The volume of consumed NaOH solution, mL when V0-titration distilled water；
M-sample quality, g；
(conversion factor of tamato fruit is calculated by malic acid, i.e., 0.067) by f-conversion factor, g/mmol；
The result shows that: biocontrol microorganisms WXCDD51 does not have a adverse impact to tamato fruit nutritional quality.Through biocontrol bacteria
The tamato fruit rotting rate of processing is about 30%, and the rotting rate of CK control group is up to 56%, and biocontrol bacteria has preferable storage
Antisepsis is shown in Fig. 7.Control group is significantly lower than by the processed tamato fruit weight-loss ratio of biocontrol bacteria, sees Fig. 8.Storage the
The hardness of 15d, WXCDD51 and CK control group has dropped 28.6% and 42.9%, the fruit of biocontrol bacteria processing than 0d respectively
It is higher relative to control group hardness.Soluble solid, titratable acid content do not have marked difference at the end of storage, see Fig. 9.
Eight, the drought-resistant and resistance to ultraviolet research of biocontrol bacteria
It is drought-resistant: take the switching of WXCDD51 single colonie in 10mL LB liquid medium, 30 DEG C, 180r/min shake culture
For 24 hours, culture solution is diluted 10 respectively-1, then take 50 μ L dilution bacterium solution into the test tube to have gone out, be stoppered with cotton plug.It places
It is cultivated in 30 DEG C of incubators, each whether has survival every the bacterium colony of detection in 5 days.It is repeated 3 times.The result shows that: WXCDD51 bacterial strain
Arid culture still has survival in 30 days, is shown in Table 6.
Utilize Macrogol 6000 (PEG6000) manual simulation's drought condition.LB liquid medium is added in suitable PEG
In, making ultimate density (w/v) is respectively 0%, 5%, 10%, 15%, 20%, 25% and 30%, is sub-packed in test tube, every pipe
10ml is sealed with degreasing tampon.After being inoculated with strains tested according to 2% inoculum concentration, as 37 DEG C, the shaking table of 200r/min shakes
Culture 48h measurement viable count simultaneously measures OD600.It is repeated 3 times.The result shows that: bacterium number does not have under different manual simulation's drought conditions
There is too big difference.But difference is obvious under the conditions of control group bacterial strain different disposal.Illustrate that WXCDD51 bacterial strain has drought-resistant spy
Property.See Figure 10.
Resistance to ultraviolet irradiation: taking WXCDD51 seed liquor 2% to be inoculated in 10mL LB liquid medium, and 30 DEG C, 180r/min
Shake culture dilutes 10 for 24 hours, by culture solution respectively-1, then take (every plant of bacterium standard in 50 μ L dilution bacterium solution to the glass slide to have gone out
Standby 18 pieces of glass slides), bacterium solution is spread out with spreading rod, starts ultraviolet irradiation after drying, every 10min plus 50 μ L sterile waters to carrying glass
On piece stirs evenly, and is taken on 10 μ L to the plate of good LB with pipettor, then changes next glass slide operation every 10min and be same as above, directly
To 180min.In triplicate.The result shows that: it is put by the bacterium liquid that 10-180min different time sections ultraviolet treatment with irradiation is crossed
Set and cultivate 48h on LB culture medium, can normal growth, be shown in Table 7.
Take WXCDD51 seed liquor 2% to be inoculated in 10mL LB liquid medium, 30 DEG C, 180r/min shake culture for 24 hours,
Culture solution is placed to irradiate in the UV lamp after 180min, viable count is calculated using plate dilution assay method, and with grey mould fruit rot of strawberry
For indicator bacteria, bacteriostatic activity is measured using tablet face-off method.With under natural light, other condition of culture culture solution all the same is done
Control.It is repeated 3 times, the results showed that ultraviolet irradiation processing group bacteria concentration is 1.27 × 1010Cfu/mL, control group bacteria concentration are 1.3
×1010Cfu/mL, processing group and control group bacterium number are almost unchanged；It is also almost consistent before and after bacteriostatic activity, it is shown in Table 8 and Figure 11.
Inhibiting effect of 1 biocontrol bacteria of table to mycelium morphology factor
2 biocontrol bacteria of table is to graw mold of tomato greenhouse preventive effect
3 biocontrol microorganisms of table are to leaf muld of tomato greenhouse preventive effect
The antimicrobial spectrum (1.3 × 10 of table 4WXCDD519cfu/mL)
Note: pure increment=bacterium colony average diameter-bacteria cake diameter
Inhibiting rate (%)=(the pure increment of the control group bacterium colony-pure increment of experimental group bacterium colony) pure increment of/control group ×
100%
The plate dual test of table 5WXCDD51
Note: former bacteria cake diameter 8mm
The 6 drought-resistant result of study of bacterial strain WXCDD51 of table
Note: "+" indicates bacterial growth；"-" indicates no bacterial growth
The resistance to ultraviolet result of study of 7 bacterial strain WXCDD51 of table
| Handle time/min | WXCDD51 | Handle time/min | 51 |
| 10 | + | 100 | + |
| 20 | + | 110 | + |
| 30 | + | 120 | + |
| 40 | + | 130 | + |
| 50 | + | 140 | + |
| 60 | + | 150 | + |
| 70 | + | 160 | + |
| 80 | + | 170 | + |
| 90 | + | 180 | + |
Note: "+" indicates bacterial growth；"-" indicates no bacterial growth
The resistance to ultraviolet count results of 8 bacterial strain WXCDD51 of table
| Bacterial strain | Control group bacteria concentration (cfu/ml) | Processing group bacteria concentration (cfu/ml) |
| WXCDD51 | 1.3×1010 | 1.27×1010 |
Claims (5)
1. the degeneration-resistant production nitrogen pseudomonad of one plant of broad-spectrum disease resistance growth-promoting, classification naming be produce nitrogen pseudomonad (Pseudomonas azotoformans), deposit number is CGMCC No.12760, which is characterized in that drought-resistant and resistance to ultraviolet.
2. the degeneration-resistant production nitrogen pseudomonad of broad-spectrum disease resistance growth-promoting as described in claim 1 is for preventing and treating botrytis cinerea and leaf mold
The purposes of germ.
3. the degeneration-resistant use for producing nitrogen pseudomonad and being used to inhibit the growth of disease fungus of broad-spectrum disease resistance growth-promoting as described in claim 1
On the way, it is characterised in that the disease fungus is cucumber fusarium axysporum, Melon fusarium Wilt, Causal Organism of Maize Basal Stalk bacterium, sharp spore reaping hook
Bacterium, withered germ of water-melon, rice wilt pathogens, wilsonii pine root fungus, tomato wilt bacterium, cucumber aphid, strawberry ash
Mildew bacterium, cucumber anthracnose, spotted wilt of tomato bacterium, Exserohilum turcicum, sunflower hyphal cluster germ or rod method.
4. the degeneration-resistant production nitrogen pseudomonad of broad-spectrum disease resistance growth-promoting as described in claim 1 is for promoting tomato seeds radicle growth
Purposes.
5. the degeneration-resistant use for producing nitrogen pseudomonad and being used to improve Quality of Tomato Fruit of broad-spectrum disease resistance growth-promoting as described in claim 1
On the way.
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| CN111778183B (en) * | 2020-06-30 | 2021-11-30 | 中国农业科学院烟草研究所 | Acidophilic nitrogen-producing pseudomonas strain and application thereof |
| CN112143672B (en) * | 2020-09-18 | 2021-08-10 | 北京农学院 | Pseudomonas aurantiaca CM-7 and application thereof |
| CN112111426B (en) * | 2020-09-22 | 2022-10-21 | 河南科技学院 | Pseudomonas flaxseed and application thereof in biocontrol of green Chinese onion purple spot |
| CN112358994B (en) * | 2020-11-17 | 2021-07-27 | 甘肃省科学院生物研究所 | Pseudomonas and application thereof |
| CN112940964B (en) * | 2021-01-29 | 2023-05-09 | 西南大学 | Karst trough area stony desertification soil improvement microbial agent, and preparation and application thereof |
| CN113249243B (en) * | 2021-03-18 | 2022-04-26 | 中国中医科学院中药研究所 | Pseudomonas, microbial biocontrol microbial inoculum containing pseudomonas and application of microbial biocontrol microbial inoculum |
| CN113337422A (en) * | 2021-05-14 | 2021-09-03 | 领先生物农业股份有限公司 | Broad-spectrum disease-resistant growth-promoting pseudomonas defense microbial inoculum and preparation method and application thereof |
| CN114196663B (en) * | 2021-12-14 | 2024-01-23 | 中国科学院合肥物质科学研究院 | Soil-activated microbial inoculum immobilized microsphere and preparation method thereof |
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