CN104017748B - Endogeny biocontrol strain as well as preparation method and application of biocontrol bacterial agent prepared from endogeny biocontrol strain - Google Patents
Endogeny biocontrol strain as well as preparation method and application of biocontrol bacterial agent prepared from endogeny biocontrol strain Download PDFInfo
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Abstract
The invention discloses an endogeny biocontrol strain as well as a preparation method and an application of a biocontrol bacterial agent prepared from the endogeny biocontrol strain. The strain is Bacillussubtilis which is preserved in the China General Microbiological Culture Collection Center on January 26th, 2014, the preservation number is CGMCCNO.8812, and the biocontrol bacterial agent is endogeny biocontrol strain cell suspension with the concentration of 1*10<8>cfu/ml. The biocontrol bacterial agent is capable of effectively preventing pomegranate from rot and is free of pollution and high in bacteriostasis rate.
Description
Technical field
The present invention relates to a kind of biological inoculum prevents and treats the field that Punica granatum L. is rotted, more particularly, in one kind raw biocontrol bacterial strain and its
The preparation method of the biocontrol agent made and application.
Background technology
Punica granatum L.（Punica granatum）, call pomegranate, Punicaceae Punica.Originate in the states such as Iran, Afghanistan
Family, in Asia, Africa, Europe along Mediterranean various places, all as cultivation of fruit tree, and especially many with Africa.In China, except east
Outside the extremely cold minority provinces and regions such as north, it is suitable for Punica granatum L. cultivation, China has defined the big producing region of the most influential Punica granatum L. five, point
Wei not Lintong, Zaozhuang, Shandong, Anhui Huaiyuan, Huili, Sichuan and Yunnan Qiaojia County.Punica granatum L. be collect edible, medicinal and view and admire in
In the fruit of one, particularly Punica granatum L. be rich in antioxidant, be slow down aging, prevention of arterial hardening and slow down canceration process
High-level antioxidant.Investigation shows, China Punica granatum L. 20% ~ 30% consumed in the Mid-autumn Festival and National Day, and 10% is used for processing, 50% ~
60% is listing around the Spring Festival through storage Shelf-life.But pomegranate postharvest fruit is in storage, caused a disease by multiple
Bacterium collective effect leads to fruit rot to be gone bad, and usual rotting rate is 30%, up to 70% when serious, has had a strong impact on the economy of Punica granatum L.
It is worth.There are two kinds of viewpoints for storage period pomegranate fruit canker is caused by what pathogenic bacteria：Most of scholar（Wear fragrant billows 1934；
Week and raw 1999；Jin＆Chang 2002, etc.）It is considered by the fresh shell spore (Zythia of Punica granatum L. dry rot pathogenic bacteria Punica granatum L.
Versoniana Sacc.) cause；Pay beautiful girl within 2007 and be considered Fructus Vitis viniferae seat chamber bacterium（Botryosphaeria dothidea）Draw
Rise.The effective measures controlling Pomegranate Fruits Decay Post-harvest at present are that cryopreservation combines chemical preservative, heat, radiation, controlled atmosphere etc..Often
The chemical preservative using has thiophanate methyl（Thiophanate-Methyl）, carbendazim（Carbendazim）, DiMei yan ji
（TBZ）Deng.Chemical prevention is the main path of fruit and vegerable Postharvest diseases preventing and treating, but has carcinogenesis with discovery, and pollutes environment
Chemical bactericide get more and more so as to the application on fruit is restricted.In addition, frequent use of some antibacterial also makes
Pathogenic bacteria produces resistance, and preventive effect is greatly lowered, such as botrytis（Botrytis cinerea）, penicillium（Penicillium
sp.）, pyriform mucormycosiss（Mufcor piriformis Fischer) already to benomyl（Benlate）Resistant, due to
Life-time service benzo miaow series bactericidal agent on mandarin orange（Benzimidazole fungicide）, make originally insensitive to it Citrus chachiensis Hort.
Fructus Citri tangerinae black rot（Alternaria citri Ellis et. Pierce）And Geotrichum candidum（Oospora
citriaurantii ex Persoon）Population rises and becomes new problem.With consumer to non-polluted farm product, pollution-free food
With the tight demand of Organic food, exploitation is efficient, low toxicity, the biological pesticide of noresidue have become highly desirable.Biological pesticide is
Refer to by the use of biogenic bioactive substance or biological living as pesticide, and synthetic with native compound structure
Identical pesticide, is environment friendly agricultural free from environmental pollution it is expected to become one of effective way solving chemical pesticide residual contamination.
Plant root, the examples of many successful of leaf diseases Biological control in recent years, significantly excites people that Biological control is expanded to fruit and vegerable and produces
The prevention and control research field of disease afterwards.Especially from nineteen eighty-three, U.S.'s agriculture portion is a collection of beautiful, with, Australia, Fa Deng state based on Wilson
Scientist, has done substantial amounts of research work in this respect, in the U.S., has prevented and treated kernel approaches, pomaceous fruit, mandarin orange and some vegetables
Several Antagonistic Fungis of Postharvest diseases achieve patent or are authorizing.At present, biological prevention is used for pomegranate storage anti-corrosion
Research, there is no relevant report both at home and abroad.In sum, screen effective biocontrol bacteria, develop new, safely and effectively micro- life
Thing antibacterial is the generation controlling Punica granatum L. canker, reduces the loss of storage period Punica granatum L., promotes Punica granatum L. industrial economy sane development
In the urgent need to.
Content of the invention
It is an object of the invention to provide the preparation method of a kind of interior raw biocontrol bacterial strain and its biocontrol agent made and application,
Can effectively prevent rotten, the pollution-free, bacteriostasis rate of Punica granatum L. high.
For achieving the above object, the technical solution used in the present invention is：
Raw biocontrol bacterial strain in one kind, described bacterial strain is bacillus subtilisesBacillus subtilis, oneself was in 2014 1
The moon is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 26th, and its deposit number is CGMCC
NO.8812.Depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is exposed to the sun for Beijing
Area North Star West Road 1 No. 3 Institute of Microorganism, Academia Sinica of institute.
A kind of preparation method according to described bacterial strain, is characterized in that：The first step, the separation of interior raw biocontrol bacterial strain and purification,
The Punica granatum L. blade of collection health weighs 3.0 g, with aseptic water washing 4 ~ 5 times, after sterile scissors are cut into the fritter of 4 ~ 5 mm square,
Successively with 75% alcohol-pickled 5 s, then plus 30 mL sterilized water exist the mercuric chloride of 0.1 % soaks 50 s, aseptic water washing 3 times,
Grind in sterile mortar；Static layering after grinding, supernatant is standby as leachate, and its concentration is 0.1 g/mL, takes supernatant 1
ML carries out gradient dilution to 10-5, from 10-3、10-4、10-5Respectively take 0.2 mL spread plate in dilution suspension, rinsed with last
Liquid 0.2 mL spread plate carries out steriling test, often processes 3 repetitions.It is inverted culture 24 ~ 48 h in 37 DEG C of constant incubators,
Picking single bacterium colony carries out purification using plate streak on beef extract-peptone plating medium；Inoculation after purification in
Beef extract-peptone slant medium, 37 DEG C of culture 24 h, 4 DEG C of Refrigerator stores are standby；Second step, the sieve of interior raw biocontrol bacterial strain
Punica granatum L. rotten pathogenic bacteria is cultivated to producing spore for 28 DEG C on potato dextrose medium flat board, is chosen with inoculating loop by choosing first
A small amount of pathogen spore point is taken to be connected to potato dextrose medium flat board central authorities, two in anomaly plate central authorities 3 cm symmetrical
The separated endophytic bacterial controlled effect of streak inoculation at straight line, only to connect Punica granatum L. rotten pathogenic bacteria for comparison, trains in 28 DEG C of constant incubators
Support, when pathogenic bacteria mycelia will be paved with full ware in compareing, calculate the size of suppression ratio, filter out the preferable bacterial strain of fungistatic effect；
3rd step, the interior raw biocontrol bacterial strain filtering out is preserved, the first store method, will select the endophytic bacterial controlled effect line of culture
It is seeded on beef extract-peptone inclined-plane, after culture 24 h, is placed in 4 DEG C of Refrigerator stores, half a year tube once prevents culture medium from drying up
Strain is dead, uses for interim, second store method preserves for glycerol：By the interior aseptic aqueous suspension of raw biocontrol bacterial strain, fully
Bacteria suspension is made in mixing, takes the sterile glycerol of 20 % fully to mix on vortex oscillator with bacteria suspension and proceeds to 1.5 aseptic mL
In EP pipe, preserve for a long time in 80 DEG C of freezings.
According to the preparation method of described bacterial strain, it is characterized in that：Described sterile glycerol is 2 with the volume ratio of bacteria suspension：1.
A kind of interior raw biocontrol bacterial strain biocontrol agent described in basis, is characterized in that：Biocontrol agent concentration is 1 × 108cfu/
The interior raw biocontrol bacterial strain cell suspension of mL.
A kind of preparation method according to described biocontrol agent, is characterized in that：The interior raw biocontrol bacterial strain of picking activation is a little, connects
Plant in 10 mL beef extract-peptone fluid mediums, 37 DEG C, 180 r/min shaking table culture 24 h, as seed liquor；To plant
Sub- liquid presses 1%（v/v）Ratio be inoculated in the 1L triangular flask containing 200 mL beef extract-peptone fluid mediums, 37 DEG C,
180 r/min shaking table culture 48 h, obtains fermentation liquid, and 2 500 r/min are centrifuged 6 min, the aseptic water washing of collects thalline, use
It is 1 × 10 that clear water is made into concentration8The interior raw biocontrol bacterial strain cell suspension of cfu/mL, as interior raw biocontrol bacterial strain biocontrol agent.
A kind of biocontrol agent described in basis, in the application of preventing and treating pomegranate storage phase fruit decomposing disease, is characterized in that：By stone
Pomegranate fruit is clean, air-dried with clear water, and pomegranate fruit is immersed 10 min in biocontrol agent, and the residual draining after taking-up in calyx tube is molten
Liquid is simultaneously cooled to 5 DEG C about in advance, then with the thick Polythene Bag individual packing of 0. 025 ~ 0.03 mm, cases and put in storage, and storage temperature is
4 ~ 6 DEG C, relative humidity is 90%.
Advantages of the present invention effect is：Present invention discover that one plant has highly active, stable endogenetic bacteria bacterial strain
Bacillus subtilis NS04.This strain culturing method is simple, has preferable suppression on PDA plate to Punica granatum L. dry rot pathogenic bacteria
Effect processed, suppression ratio is 55.6%, has significant preventive effect, the relative control effect of different storage to pomegranate storage phase fruit decomposing disease
For 100% ~ 54.082%, commodity rate significantly improves 20% ~ 12%, and this bacterial strain is bacillus subtilisesBacillus subtilis,
From Punica granatum L. blade interior tissue, completely not because chemical pesticide is using brought series of problems, green, nontoxic, ring
Protect hence it is evident that the shelf life of pomegranate fruit is significantly extended 90 d, improve the income of orchard worker.Therefore, bacterial strain NS04 makees
For pomegranate postharvest fruit antiseptical bio-control factors, there is wide DEVELOPMENT PROSPECT.
Brief description
Fig. 1 is the inhibition figure of normal control bacterium colony.
Fig. 2 is interior raw biocontrol bacterial strain to Punica granatum L. dry rot pathogenic bacteria inhibitory action figure.
Fig. 3 is the aspect graph that interior raw biocontrol bacterial strain cultivates 48h.
Fig. 4 is interior raw biocontrol bacterial strain aspect graph under the microscope.
Fig. 5 is the stability diagram that interior raw biocontrol bacterial strain passes on to the suppression of Punica granatum L. dry rot pathogenic bacteria.
Specific embodiment
The invention will be further described with specific embodiment below in conjunction with the accompanying drawings：
Embodiment 1：The screening of interior raw biocontrol bacterial strain NS04：
1. the separation of raw biocontrol bacterial strain and purification in
The Punica granatum L. blade of collection health weighs 3.0 g, and with aseptic water washing 4-5 time, sterile scissors are cut into 4 ~ 5 mm square
Fritter after, successively with 75% alcohol-pickled 5 s, then plus 30 mL the mercuric chloride of 0.1 % soaks 50 s, rinsed with sterile water 3 times,
Sterilized water grinds in sterile mortar；Static layering after grinding, supernatant is standby as leachate, and its concentration is 0.1 g/mL,
Supernatant 1 mL is taken to carry out 10 times of gradient dilutions to 10-5, from 10-3、10-4、10-50.2 mL spread plate is respectively taken in dilution suspension,
Steriling test is carried out with last rinsing liquid 0.2 mL spread plate, often processes 3 repetitions.37 DEG C of constant incubators fall
Put culture 24 ~ 48 h, observe, if comparison does not have bacterium colony to grow, surface sterilizing is described thoroughly, obtained bacterial strain is interior raw biological and ecological methods to prevent plant disease, pests, and erosion
Bacterial strain, picking single bacterium colony is in beef-protein medium（NA）On flat board, purification is carried out using plate streak；After purification
, in NA slant medium, 37 DEG C of culture 24 h, 4 DEG C of Refrigerator stores are standby for inoculation.
2. the screening of raw biocontrol bacterial strain in
The canker being thought with Most scholars pathogenic bacteria Punica granatum L. fresh shell spore (Zythia VersonianaSacc.) it is
Indicator bacteria, measures the inhibitory action to rotten pathogenic bacteria for the endogenetic bacteria using flat board face-off method.First by Punica granatum L. rotten pathogenic bacteria in horse
Bell potato glucose agar medium（PDA）Cultivate to producing spore, with a small amount of pathogen spore of inoculating loop picking for 28 DEG C on flat board
Son point is connected to PDA plate central authorities, the separated endogenetic bacteria of streak inoculation at two symmetrical lines of anomaly plate central authorities 3 cm,
Only to connect Punica granatum L. rotten pathogenic bacteria for comparison, often process 3 repetitions, cultivate in 28 DEG C of constant incubators, pathogenic bacteria mycelia in waiting to compare
When will be paved with full ware, calculate the size of suppression ratio.
Suppression ratio（%）=[（Comparison colony diameter-process colony diameter）/ comparison colony diameter] × 100 %, bacterium colony
Diameter units：Centimetre（cm）.
Isolating and purifying and screening by Punica granatum L. blade endophyte, obtain one plant Punica granatum L. rotten pathogenic bacteria is had preferably antibacterial
The endophyte bacterial strain of activity, suppression ratio is 55.6%, and numbering is NS04（Fig. 1）.
Embodiment 2：The store method of interior raw biocontrol bacterial strain NS04
This strain can be preserved using two methods, the endogenetic bacteria streak inoculation of 24 h will be cultivated on NA inclined-plane, training
Support the rearmounted 4 DEG C of Refrigerator stores of 24 h, half a year tube once prevents the dry strain of culture medium dead, uses for interim.Glycerol is protected
Deposit：By the aseptic aqueous suspension of bacterial strain, fully mix and make bacteria suspension, take sterile glycerol and the bacteria suspension of 20 %（VGlycerol：VBacteria suspension=2：
1）Fully mix on vortex oscillator and proceed in 1.5 aseptic mL EP pipes, preserve for a long time in 80 DEG C of freezings.
Embodiment 3：The identification of interior raw biocontrol bacterial strain NS04
1. the morphology of NS04 bacterial strain, physio-biochemical characteristics
Morphology and physio-biochemical characteristics adopt conventional method to measure, and measurement result shows that NS04 bacterial strain is cultivated in NA flat board
On base, colony diameter is about 0.76 cm, white, opaque, irregular, protuberance, edge decomposite leaf shape, extends bacterium with incubation time
Fold in the surface that falls（Fig. 2）.NS04 individual cells are in rod-short, 0.63 ~ 0.87 × 1.75 ~ 2.51 μm, single raw or opposite
（Fig. 3）, Gram-positive, there are ellipse or column spore.NS04 bacterial strain and bacillus subtilisesBacillus subtilisPhysio-biochemical characteristics basically identical（Table 1）.
2. the 16S rDNA sequence analysis of NS04 bacterial strain
The extraction of biocontrol bacterial strain NS04 genomic DNA adopts multigelation method, using 16S rDNA universal primer, student on commission
Work biological engineering（Shanghai）Company limited synthesizes：
Forward primer is F27：5 '-AGAGTTTGATCATGGCTCAG-3 '
Downstream primer is R1492：5 '-TACGGTTACCTTGTTACGACTT-3 '
PCR reaction system（Total system is 25 μ L）For：Premix Taq 12 μ L, DNA profiling 1 μ L, each 1 μ L of primer,
Distilled water 10 μ L.PCR reaction condition is：94 DEG C of denaturation 4 min, 94 DEG C of degeneration 30 s, 54 DEG C of annealing 1 min, 72 DEG C
Extend 1.5 min, 30 circulate rear 72 DEG C of extension 10 min.Amplification obtains 16S rDNA 1 500 bp.By product reagent
Raw work biological engineering delivered to after purification by box（Shanghai）Company limited's sequencing obtains 16S rDNA sequence 1449 bp of NS04,
GenBank logs in, and accession number is JX126865.Carry out sequence homology using blast program and compare analysis, use Mega
（5.0）Adjacent method build this bacterial strain phylogenetic tree, combining form, physio-biochemical characteristics simultaneously consult related data, will
NS04 identification of strains is bacillus subtilisesBacillus subtilis.
Embodiment 4：The antimicrobial stability of NS04 bacterial strain
On NA slant medium, NS04 bacterial strain is carried out with continuous passage, 37 DEG C of constant temperature culture 24 h, 4 DEG C of refrigerators are protected
Deposit, continuous transposing 10 generation, after 10 generations passed, the bacterial strain in 10 generations is done with Punica granatum L. rotten pathogenic bacteria respectively using flat board face-off method short of money
Anti- experiment, every group 3 is repeated, and only to connect Punica granatum L. rotten pathogenic bacteria for comparison, when the pathogenic bacteria of matched group covers with whole flat board, records short of money
Anti- situation, and measure the bacterium colony size of Punica granatum L. rotten pathogenic bacteria, in order to calculate bacteriostasis rate.The bacteriostasis rate of measurement result NS04 bacterial strain exists
55.56% ~ 64.20%, not notable to the suppression ratio difference of Punica granatum L. rotten pathogenic bacteria, the suppression to Punica granatum L. rotten pathogenic bacteria for the NS04 bacterial strain is described
Making use can stable heredity（Fig. 4）.
Embodiment 5：The preparation method of NS04 Biocontrol Activity microbial inoculum
By the NS04 bacterial strain transposing preserving on NA inclined-plane, 37 DEG C, culture 24 is activated, the NS04 bacterium of picking activation
Strain is a little, is inoculated into 10 mL beef extract-peptone fluid mediums（NB）In, 37 DEG C, 180 r/min shaking table culture 24 h, makees
For seed liquor；Seed liquor is pressed 1%（v/v）Ratio be inoculated in the 1L triangular flask containing 200 mL NB culture medium, 37 DEG C, 180
R/min shaking table culture 48 h, obtains fermentation liquid, and 2 500 r/min are centrifuged 6 min, the aseptic water washing of collects thalline, use clear water
Being made into concentration is 1 × 108The NS04 strain cell suspension of cfu/mL, as NS04 Biocontrol Activity microbial inoculum.
Embodiment 6：Biological and ecological methods to prevent plant disease, pests, and erosion endogenetic bacteria NS04 prevents and treats the application of storage period Punica granatum L. canker
By fresh, healthy, the pomegranate fruit that commodity are ripe plucked（Kind is that Pericarpium Citri Reticulatae Viride is sweet）, cleaned with clear water, natural wind
Dry.2 process of setting：Process 1, pomegranate fruit is immersed 10 min in the cell suspension of the NS04 bacterial strain described in embodiment 5；Place
Reason 2, pomegranate fruit is immersed 1 min in 1 500 times of liquid of 70% thiophanate methyl wettable powder, immerses clear water with pomegranate fruit
10 min are comparison.Drain the residual solution in calyx tube after taking-up and be cooled to 5 DEG C about in advance, the more poly- second with 0. 03 mm thickness
Alkene bag individual packing, vanning warehouse-in.Each of the above processes and sets 3 repetitions, and each repeats 50 fruits.It is housed in temperature after vanning
Spending for (5 ± 1) DEG C, relative humidity is that in 90% mechanical freezer, the 30th d starts observation experiment result, observes 1 every 15d
Secondary, Continuous Observation to 90 d.
Testing index and method：Punica granatum L. canker is divided into 4 grades according to fruit morbidity severity（Table 2）
Result of the test shows：Storage period different time NS04 bacterial strain and 1 500 times of liquid pair of thiophanate methyl wettable powder
The fruit decomposing disease of pomegranate storage phase all has preferable prevention effect, and with the prolongation of storage time, rotten disease index presents
Increased trend, the rotting mechanism of wherein comparison is significantly higher than other process（Table 3）.The relative control effect of NS04 biocontrol bacterial strain is
100% ~ 54.082%, the relative control effect of 1500 times of liquid of thiophanate methyl wettable powder is 100% ~ 81.633% although significantly low
In the preventive effect of 1 500 times of liquid of thiophanate methyl wettable powder, but also effective control the sending out of pomegranate storage phase fruit decomposing disease
Raw（Table 4）.In pomegranate storage 75 d, the fruit commodity rate that NS04 biocontrol bacterial strain is processed is not less than 95.3%, and thiophanate methyl can
The fruit commodity rate that 1 500 times of liquid of WP are processed（98.7%）Compare in 0.05 level no significant difference, but pomegranate storage
During 90 d, the fruit commodity rate of NS04 biocontrol bacterial strain is substantially less than 1 500 times of liquid of thiophanate methyl wettable powder（Table 5）, but
Compare commodity rate with comparison and significantly improve 12% ~ 20%, and this bacterial strain belongs to bacillus subtilisesBacillus subtilis, come
Come from Punica granatum L. blade interior tissue, more green than chemical pesticide, nontoxic, environmental protection is hence it is evident that significantly extend the shelf life of pomegranate fruit
90 d, improve the income of orchard worker.Therefore, bacterial strain NS04 has extensively as pomegranate postharvest fruit antiseptical bio-control factors
Wealthy DEVELOPMENT PROSPECT.
The concrete condition of the present invention is as follows：
The antibacterial that the present invention adopts is the endogenetic bacteria being isolatable from plant Punica granatum L. leaf tissueBacillus subtilis
NS04 bacterial strain, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preservation address is court of Beijing
North Star West Road l institute of positive area 3, preservation date is on January 26th, 2014, and preserving number is CGMCC NO. 8812.
The interior raw biocontrol bacterial strain of the present invention through morphological observation, customary physiological biochemical test and 16S rDNA sequence analysis,
It is accredited asBacillus subtilisNew strains, there are following characteristics：24 h bacterium colonies are cultivated on NA plating medium
Diameter is about 0.76 cm, white, opaque, irregular, protuberance, edge decomposite leaf shape, and after 48 h, fold in bacterium colony surface.
Individual cells are in rod-short, 0.63 ~ 0.87 × 1.75 ~ 2.51 μm, single raw or opposite, Gram-positive, have ellipse or
Column spore.Produce catalase；V-P reacting positive；Indole test is negative；Using citrate；Do not utilize propionate；Reduction
Nitrate；Hydrolysis starch, gelatin, tyrosine, casein；Produce acid using glucose, L-arabinose, PEARLITOL 25C, D- xylose；Energy
The nutrient broth of pH 5.7 grows；10%NaCl remains to grow；10 DEG C ~ 50 DEG C of growth temperature range.
The present invention is conventionally prepared, within raw biocontrol bacterial strain as producing bacterial strain, by liquid fermentation, in preparation
Raw biocontrol bacterial strain biocontrol agent；Tested by indoor flat plate, evaluate its inhibitory action to Punica granatum L. dry rot pathogenic bacteria mycelial growth；Logical
Cross Punica granatum L. corrosion-resistanting test in freezer, evaluate its prevention effect to storage period Punica granatum L. canker.
Embodiment described above is only that the preferred embodiment of the present invention is described, the not structure to the present invention
Think and protection domain is defined, on the premise of without departing from design concept of the present invention, ordinary skill technical staff in this area
The various modifications that technical scheme is made and improvement, all should fall into protection scope of the present invention.
Claims (4)
1. raw biocontrol bacterial strain in one kind, described bacterial strain is bacillus subtilises（Bacillus subtilis）, oneself was in 2014 1
The moon is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 26th, and its deposit number is CGMCC
NO.8812.
2. a kind of interior raw biocontrol bacterial strain biocontrol agent according to claim 1, is characterized in that：Biocontrol agent concentration be 1 ×
108The interior raw biocontrol bacterial strain cell suspension of cfu/mL.
3. a kind of preparation method of biocontrol agent according to claim 2, is characterized in that：The interior raw biocontrol microorganisms of picking activation
Strain is a little, is inoculated in 10 mL beef extract-peptone fluid mediums, 37 DEG C, 180 r/min shaking table culture 24 h, as kind
Sub- liquid；Seed liquor is pressed 1%（v/v）Ratio be inoculated in the 1L triangular flask containing 200 mL beef extract-peptone fluid mediums
In, 37 DEG C, 180 r/min shaking table culture 48 h, obtain fermentation liquid, 2 500 r/min are centrifuged 6 min, collects thalline is with aseptic
Water washing, being made into concentration with clear water is 1 × 108The interior raw biocontrol bacterial strain cell suspension of cfu/mL, as interior raw biocontrol bacterial strain life
Anti- microbial inoculum.
4. a kind of biocontrol agent according to claim 2 is in the application of preventing and treating pomegranate storage phase fruit decomposing disease, its feature
It is：Pomegranate fruit is clean, air-dried with clear water, pomegranate fruit is immersed 10 min in biocontrol agent, drains in calyx tube after taking-up
Residual solution and be cooled to 5 DEG C about in advance, then with the thick Polythene Bag individual packing of 0. 025 ~ 0.03 mm, vanning warehouse-in, protect
Deposit temperature and be 4 ~ 6 DEG C, relative humidity is 90%.
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| CN108300678B (en) * | 2018-03-12 | 2021-06-15 | 桂林理工大学 | Preparation method and application of Leersia hexandra endophytic bacteria capable of reducing hexavalent chromium |
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| CN110468060B (en) * | 2018-05-15 | 2021-05-07 | 北京市园林科学研究院 | Pantoea strain and application thereof in biological control |
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