CN103952338B

CN103952338B - Pantoea agglomerans strain X M2 and the preparation method of bacteria suspension thereof and the prevention and controls to Pear black spot

Info

Publication number: CN103952338B
Application number: CN201410143195.5A
Authority: CN
Inventors: 张晓宇; 张姝; 王春生; 张泽宇; 张则君; 韩巨才; 刘慧平; 张永杰
Original assignee: Institute Of Storage And Preservation Of Agricultural Products Shanxi Academy Of Agricultural Sciences
Current assignee: Institute Of Storage And Preservation Of Agricultural Products Shanxi Academy Of Agricultural Sciences
Priority date: 2014-04-11
Filing date: 2014-04-11
Publication date: 2016-08-17
Anticipated expiration: 2034-04-11
Also published as: CN103952338A

Abstract

The invention discloses a stain of Pantoea agglomerans (Pantoea agglomeran) strain X M2, it being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 17th, 2014, preserving number is CGMCC No.8822.The present invention describes the morphological characteristic of pantoea agglomerans strain X M2, physiological and biochemical index and 16S rDNA sequencing results etc. in detail.The preparation method of strain X M2 bacteria suspension of the present invention, prepare including seed liquor, fermentation liquid preparation and bacteria suspension prepare three steps and complete.With strain X M2 bacteria suspension of the present invention preventing and treating Pear black spot method include melonidum gather before prevent and treat, melonidum gather after immediately preventing and treating and melonidum pre-cooling after prevent and treat three kinds, prevention effect respectively reaches more than 90%, more than 82% and more than 70%.Preventing and treating chance many, biocontrol effect is good.If it is more preferable that different times is combined prevention effect.

Description

Pantoea agglomerans strain X M2 and the preparation method of bacteria suspension thereof and Pear black spot is prevented Control method

Technical field

The present invention relates to microorganism field, concretely relate to a kind of pantoea agglomerans (Pantoea agglomeran) bacterium Strain XM2 and the prevention and controls to Pear black spot thereof.

Background technology

Pear black spot is a kind of to endanger serious worldwide gas borne fungus diseases, by rod method (Alternaria Alternata) cause, and all have generation after adopting before melonidum is adopted.China is pears plantation and big export country, adopts rear pathogenic microorganism Infect and cause postharvest decay loss serious, directly affect melonidum storing and produce, the effectively preventing and treating of Pear black spot becomes pears industry In problem anxious to be resolved.The postharvest disease using chemical bactericide to control fruit is considered as maximally effective controlling party always Method.But, it is gradually increased along with pathogen is drug-fast, and the adding of people's raising that fruit quality is required and environmental consciousness By force, postharvest fruit and vegetable biological control method, as a kind of new way controlling postharvest disease, is gradually approved by people, and becomes and grind Study carefully focus.In recent years there is the research report much about Pear black spot Biological control.Qi Dongmei etc. have screened a strain bacillus subtilis Bacterium (Bacillus subtilis) H110, pears are adopted the control rate of rear melasma and are reached 86.6% by its fermentation liquid.Xie Li is from soil In be separated to streptomyces aureus (Streptomyces aureus) B-105, its fermentation liquid suppression ratio 67.4% to Pear black spot. Benbow etc. have studied the Cryptococcus informominiatus and the Rhodotorula of Rhodotorula of Cryptococcus The glutinis fungistatic effect to pears disease, Cryptococcus informominiatus and Rhodotorula Pears are processed by glutinis 1 d before gathering, and can efficiently control pears adopts rear melasma.It is withered that Song Cong etc. are separated to a strain Grass bacillus cereus (Bacillus subtilis) J18, melonidum is soaked fruit 30 s in bacteria suspension, sprays inoculation rod method after drying Bacterium, the sickness rate of Pear black spot is only 16.57%, and bacterial strain can be grown on melonidum the most surely.

Summary of the invention

It is an object of the invention to provide a stain of Pantoea agglomerans (Pantoea agglomeran) strain X M2 and to pears black speck Sick prevention and controls.

It is an object of the invention to be achieved through the following technical solutions.

One stain of Pantoea agglomerans (Pantoea agglomeran) strain X M2, is preserved in Chinese micro-life on February 17th, 2014 Thing culture presevation administration committee's common micro-organisms center, preserving number is CGMCC No.8822, address: north, Chaoyang District, Beijing City Occasion West Road 1 No. 3 Institute of Microorganism, Academia Sinica of institute.

Described pantoea agglomerans (Pantoea agglomeran) strain X M2 is from Jin Tan village, Shanxi province Taiyuan city Prunus mume Sieb. Et Zucc. fruit Real surface separates and purification obtains.At bacteriolyze meat soup (LB) culture medium culturing 48 h ~ 72 h.Pantoea agglomerans (Pantoea Agglomeran) strain X M2 is gram negative bacteria, rod-short, and size is (0.5 ~ 0.9) μ m (1.0 ~ 2.5) μ M, amphitrichous, there is mobility's (see figure 1).On LB culture medium flat plate after aerobic cultivation 24 h ~ 48 h, produce surface light Sliding, protruding, translucent, neat in edge, the yellow color colonies of a diameter of 2.0 mm ~ 3.0 mm.Strain X M2 is facultative anaerobe, Growth pH scope is 5.7 ~ 7.5, and growth temperature range is 4 DEG C ~ 40 DEG C, NaCl concentration scope 0% ~ 7%.Other physiology Biochemical indicator is as shown in table 1:

Table 1 strain X M2 physiological and biochemical property

Obtain the 16S rDNA fragment of strain X M2 through 16S rDNA sequencing: PCR amplification, check order a length of 1386 Bp, the sequence accession number in GenBank is KF150143.16S rDNA sequence is entered with the sequence in GenBank data base Row BLAST comparison, result shows, the homology of the pantoea agglomerans (P. agglomerans) in strain X M2 and general Pseudomonas Pantoea Property reaches 99.8%, and combining form feature and physiological and biochemical index determine that strain X M2 is pantoea agglomerans (P. Agglomerans).

Described bacteriolyze meat soup (LB) culture medium is made up of the raw material of following mass/volume part: yeast powder 5, peptone 10 , NaCl 5, agar powder 18, distilled water 1000；pH 7.4-7.6.The preparation method of pantoea agglomerans strain X M2 bacteria suspension, including Following steps:

(1) seed liquor preparation is with the Inoculating needle picking one ring fresh pantoea agglomerans mono-bacterium colony of XM2 from test tube slant, inoculates In LB culture fluid, 28 DEG C, 160 rpm/min concussion constant temperature culture 24 h, prepare the seed liquor of pantoea agglomerans XM2；

(2) seed liquor of pantoea agglomerans XM2 is accessed in LB culture fluid according to the ratio of 1% by fermentation liquid preparation, 28 DEG C 160 rpm/min shake constant temperature culture 72 h, prepare the fermentation liquid of pantoea agglomerans XM2；

(3) fermentation liquor 10000 rpm/min of pantoea agglomerans XM2 is centrifuged 10 min by bacteria suspension preparation, abandons supernatant Liquid；In precipitation, add aquesterilisa, be again centrifuged 10 min through 10000 rpm/min, abandon supernatant；Again add in precipitation Sterilized water, mixing, it is thus achieved that the bacteria suspension of pantoea agglomerans XM2.

The pantoea agglomerans strain X M2 controlling experiment to Pear black spot

1. the different disposal bacterium solution of the pantoea agglomerans strain X M2 controlling experiment to Pear black spot

(1) pantoea agglomerans strain X M2 is configured to following 4 kinds of bacterium solution by test method:

A: being configured to concentration is 1 × 10⁶The fermenation raw liquid of cfu/mL；

B: fermenation raw liquid filtrate after 0.22 μm membrane filtration；

After C: fermenation raw liquid is centrifugal, abandoning supernatant, precipitate sterilized water and clean 2 times, being prepared as concentration is 1 × 10⁶cfu/ The bacteria suspension of mL；

D: fermenation raw liquid is at 121 DEG C of 100KPa autoclaving 20 min, and being prepared as concentration is 1 × 10⁶The heat of cfu/mL is killed Dead liquid；

Pear black spot bacterium spore suspension is prepared:

Fresh Pear black spot bacterium flat board adds 10 mL sterilized water, scrapes bacterium cake surface gently with sterilizing spreader, wash Under the conidium of fresh pathogen, collect washing liquid.Spore suspension is made by the four of sterilizing layers of filtered through gauze.Optical microscope Microscopy suspension miospore situation, is configured to desired concn.

Above-mentioned 4 kinds of pantoea agglomerans strain X M2 treatment fluids are taken 30 μ l respectively and are inoculated in melonidum wound (3 mm × 3 mm), Comparison, for adding equivalent sterilized water, after drying, takes 1 × 10 respectively⁵Individual spore/mL Pear black spot bacterium spore suspension 15 μ l inoculation In melonidum wound, dry, bagging moisturizing, 25 DEG C of constant temperature culture 7 d, 10 melonidums of each process, be repeated 3 times, statistics morbidity feelings Condition.

Pear fruit occurring degree grade scale:

0 grade anosis;

1 grade of scab accounts for less than the 1% of fruit surface area；

3 grades of scabs account for the 2% ~ 3% of fruit surface area；

5 grades of scabs account for the 4% ~ 6% of fruit surface area；

7 grades of scabs account for the 7% ~ 10% of fruit surface area；

9 grades of scabs account for more than the 11% of fruit surface area；

(2) test results test result shows, the pantoea agglomerans XM2 of the different disposal liquid being inoculated in wound is black to pears Pinta bacterium the most protected effect (see figure 2), protection effect by by force to weak successively: bacteria suspension, fermentation liquid, filtrate and heat-killed Liquid.Bacteria suspension preventive effect is up to 97.73%, and the preventive effect of heat-killed liquid is only 56.82%, and pantoea agglomerans XM2 bacterial strain bacterium is described Somatic diseases prevention is best, and metabolite takes second place, and refers to table 2.

The impact on adopting rear Pear black spot of the table 2 pantoea agglomerans XM2 different disposal liquid

Treatment fluid	Disease index	Prevention effect/%
			Bacteria suspension	2.22 d	97.73 a
Fermentation liquid	12.22 c	87.50 b
			Filtrate	17.78 c	81.82 b
Heat-killed liquid	42.22 b	56.82 c

Note: different lower cases represent significant difference between process (P < 0.05), lower same.

2. the pantoea agglomerans strain X M2 bacteria suspension different vaccination concentration controlling experiment to Pear black spot

(1) pantoea agglomerans strain X M2 bacteria suspension concentration is formulated as 1 × 10 by test method respectively⁸cfu/mL、1×10⁶ cfu/mL、1×10⁴Cfu/mL, Pear black spot bacterium spore concentration is formulated as 1 × 10 respectively⁶Individual spore/mL, 1 × 10⁵Individual spore/ mL、1×10⁴Individual spore/mL.The bacteria suspension of variable concentrations respectively taking 30 μ l and adds melonidum wound, after drying, inoculation is different dense Degree Pear black spot bacterium spore suspension 15 μ l, dries, bagging moisturizing, 25 DEG C of constant temperature culture 7 d, and (method is same for statistics incidence Before).10 melonidums of each process, are repeated 3 times.

(2) test results test result shows, the melonidum wound of inoculation pantoea agglomerans XM2 bacteria suspension, its disease index is low In each comparison；XM2 bacteria suspension concentration on wound is the biggest, and pathogen inoculum density is the lowest, and its preventive effect is the highest.Identical During XM2 bacteria suspension concentration, its preventive effect reduces with the increase of pathogen concentration；Identical Pear black spot bacterium spore suspension concentration Time, preventive effect increases (see Fig. 3, Fig. 4, Fig. 5) with the increase of XM2 bacteria suspension concentration.Pear black spot bacterium spore suspension low concentration (1 × 10⁴Cfu/mL), time, XM2 bacteria suspension can effectively suppress disease to occur, the preventive effect of three concentration all more than 95%, and In 0.05 level, difference is the most notable；Pear black spot bacterium spore suspension high concentration (1 × 10⁶Cfu/mL) time, XM2 bacteria suspension Can only reduce the occurrence degree of disease, the preventive effect of three concentration is all below 40%, and difference is not notable, in detail in 0.05 level It is shown in Table 3.

The impact on adopting rear Pear black spot of the table 3 XM2 variable concentrations bacteria suspension

3. the pantoea agglomerans strain X M2 bacteria suspension different vaccination time controlling experiment to Pear black spot

(1) test method is 1 × 10 in inoculation 15 μ l concentration⁵Before the Pear black spot bacterium spore suspension of individual spore/mL 24 h, front 12 h, front 4 h, 0 h, rear 4 h, rear 12 h, the different time of rear 24 h are distinguished at melonidum wound (3 mm × 3 mm) Inoculating 30 μ l concentration is 1 × 10⁸Cfu/mL pantoea agglomerans XM2 bacteria suspension, compares as first meeting 30 μ l sterilized water, 3 h at wound Rear access Pear black spot bacterium suspension, dries, bagging moisturizing, 25 DEG C of constant temperature culture 7 d, statistics incidence (method is the same). 10 melonidums of each process, are repeated 3 times.

(2) result of the test pantoea agglomerans strain X M2 bacteria suspension is inoculated in melonidum wound (3 in difference relative to inoculation time Mm × 3 mm), the relative inoculation time impact all having prevention effect, pantoea agglomerans and Pear black spot bacterium spore to Pear black spot is anti- Controlling effect, XM2 bacterial suspension inoculation time process preventive effect the most early is significantly better than the process in relative time evening, and pantoea agglomerans is described Strain X M2 bacteria suspension preventive effect is better than therapeutic effect.Along with XM2 bacteria suspension is relative to the postponement of inoculation time, preventive effect is decline Trend (see figure 6).The process preventive effect of 24 h inoculation XM2 bacteria suspensions is the highest in advance, reaches 94.94%；Inoculation XM2 bacteria suspension simultaneously 70.85% is had dropped down to the process preventive effect of Pear black spot bacterium spore suspension；XM2 bacteria suspension is relative to Pear black spot bacterium spore suspension The process of liquid inoculation in evening, preventive effect is all about 50%, and in 0.05 level, difference is not notable, refers to table 4.

Table 4 XM2 and the impact on Pear black spot of the Spores inoculation time

Inoculation time	Disease index	Prevention effect %
			B 24h	5 d	94.94 a
B 12h	12.22 c	87.64 b
			B 4h	17.56 c	82.24 b
0	28.89 c	70.85 b
			A 4h	46.33 b	53.15 c
A 12h	46.78 b	52.69 c
			A 24h	48.89 b	50.56 c
CK	98.89 a	-

4. pantoea agglomerans strain X M2 Growth trends on melonidum wound

(1) test method takes 30 μ l1 × 10⁸The XM2 fermentation liquid of cfu/mL is inoculated in melonidum wound (3 mm × 3 mm) In, the XM2 bacterial population measured after l h is initial value (0 h).The melonidum moisturizing respectively of inoculation XM2 is stored in 28 DEG C, 20 DEG C, 4 DEG C With at 0 DEG C.Often processing 10 fruits, test is repeated 3 times.Melonidum at 28 DEG C measures once every day, every 3 days of the melonidum at 20 DEG C Measuring once, the melonidum at 4 DEG C and 0 DEG C measures once for every 6 days.Assay method is from melonidum wound cut-off with aseptic card punch Footpath and the degree of depth are the pulp organization of 10mm, are put in oneself and add the aseptic of the phosphate buffer of the 0.05 mol/L pH7.0 of 1.0 mL In mortar, count with dilution-plate method after grinding in aseptic operating platform.The cfu of gained Antagonistic Fungi is changed into log₁₀ ^cfu。

(2) result of the test is at 28 DEG C, 20 DEG C, 4 DEG C and 0 DEG C, pantoea agglomerans strain X M2 at melonidum wound in first increasing Breed trend the most smoothly, all the most surely can grow at wound.Pantoea agglomerans XM2 is 2.7 × 10 at the initial number of melonidum wound⁷ Cfu/mL, when inoculating 1 d at 28 DEG C, melonidum bacterial wound number is initial 3 × 10⁸Times ~ 2.7 × 10⁹Times, when inoculating 4 d, pears Really bacterial wound number reaches maximum, is initial 3 × 10¹⁶Times ~ 1.27 × 10¹⁸Times, when inoculating 12 d, melonidum bacterial wound Number is still the 1.3 × 10 of starter bacteria number¹⁰Times ~ 4 × 10¹¹Again (Fig. 7)；When inoculating 3 d at 20 DEG C, melonidum bacterial wound number reaches Maximum, is the 3 × 10 of starting bacteria number¹⁴Times ~ 1.25 × 10¹⁵Times, held stationary after 12 d, when inoculating 36 d, melonidum wound Bacterial population is the 6 × 10 of starting bacteria number⁹Times ~ 1 × 10¹¹Again (Fig. 8)；When inoculating 6 d at 4 DEG C, melonidum bacterial wound number reaches Maximum, maintains level the most substantially, and when inoculating 66 d, melonidum bacterial wound number is the 3.2 × 10 of starting bacteria number¹²Times ~ 5 × 10¹⁰Times；At 0 DEG C, melonidum bacterial wound number increasing degree is little, also inhibits agglomerating while low temperature suppression Pear black spot growth The growth of general bacterium XM2, but during to 66 d, melonidum bacterial wound number remains to keep 1.08 × 10⁸cfu/mL~5.4×10⁸ Cfu/mL, is 4 times ~ 20 times (Fig. 9) of starting bacteria number.

5. the pantoea agglomerans strain X M2 fermentation liquid impact on Pear black spot bacterium hypha form

(1) test method is by 10⁶Individual/mL pears alternaric bacteria spore suspension 1 mL is inoculated in 100 mL Rhizoma Solani tuber osi Fructus Vitis viniferaes In sugar (PDB) culture medium, 25 DEG C stand constant temperature culture.After pears alternaric bacteria spore grows uniform hyphal suspension, add 1×10⁸Cfu/mL pantoea agglomerans XM2 bacteria suspension 1mL；Comparison is addition 1mL sterilized water in pears rod method hyphal suspension, After 72 h, observation processes and the hypha form change compareed the most respectively.

(2) result of the test perusal find, alternaric bacteria mycelia is evenly distributed in matched group, grow vigorous (see Figure 10)；Alternaric bacteria mycelia is skewness in process group, and shrinkage is agglomerating, grows sparse (see Figure 11).Pass through optical microscope It has been observed that alternaric bacteria mycelia mycelia in matched group is sturdy, continuous, smooth surface (see Figure 12)；Alternaric bacteria mycelia exists Portion fractures in process group, cell inclusion is excessive, torsional deformation, assembles agglomerating (see Figure 13, Figure 14).

In a word, pantoea agglomerans XM2 is that pears are adopted rear melasma Biological control and provided new bacterial strain, draws knot by test Opinion: the antifungal mechanism of pantoea agglomerans XM2 is thalline and the common effect of metabolite, both there is the competition of nutrition and space, again There is the destruction to pathogen mycelia；The protection effect of pantoea agglomerans XM2 is relevant with concentration, pantoea agglomerans XM2 concentration is the biggest, It is the lowest that rear black speck pathogen inoculum density adopted by pears, and its preventive effect is the best；The inoculation of rear melasma archesporium adopted by pantoea agglomerans XM2 and pears Time effects Biological control effect, first the fungistatic effect of inoculation pantoea agglomerans XM2 be significantly better than simultaneously or after inoculate in pathogenic bacteria Effect.

With the bacteria suspension of pantoea agglomerans strain X M2 to the prevention and controls of Pear black spot it is:

(1) 3 d ~ 5 d before prevention and controls melonidum is gathered before melonidum is gathered, with aerosol apparatus at fruit surface spraying concentration be 1×10⁶cfu/mL~1×10⁸The pantoea agglomerans XM2 bacteria suspension of cfu/mL, pears are adopted the preventive effect of rear melasma reach 97% with On；

(2) it is 1 × 10 in concentration immediately after after melonidum is gathered, prevention and controls melonidum is gathered immediately⁶cfu/mL~1×10⁸ The pantoea agglomerans XM2 bacteria suspension of cfu/mL soaks 2 min ~ 3 min, after fruit dries naturally, enters fresh-keeping warehouse storage, to pears The preventive effect adopting rear melasma reaches more than 82%；

(3), after melonidum pre-cooling after prevention and controls melonidum pre-cooling, it is 1 × 10 in concentration⁶cfu/mL~1×10⁸cfu/mL Pantoea agglomerans XM2 bacteria suspension in soak 2 min ~ 3 min, after naturally drying, carry out melonidum vanning work, after pears are adopted The preventive effect of melasma reaches more than 70%.

Three of the above method cuts both ways: the method for melonidum preharvest spraying pantoea agglomerans XM2 bacteria suspension is simple, reduces Adopting rear workload, prevention effect is best, but during spraying the bacteria suspension in fruit face may skewness, to pathogen infection Bring chance, there is also the probability infecting other diseases in field simultaneously；Melonidum uses pantoea agglomerans strain X M2 bacterium after adopting immediately Method bacteria suspension being evenly distributed in fruit face that suspension soaks, the preventive effect that pears are adopted rear melasma is higher, but because of in actual production Melonidum collecting time is relatively concentrated, and may increase labor intensity every day；Soak with pantoea agglomerans strain X M2 bacteria suspension after melonidum pre-cooling The method of bubble is compared with first two method time upper abundant many, but the colonization ability of pantoea agglomerans XM2 is not as at room temperature at low temperatures Under, therefore the preventive effect that pears are adopted rear melasma is low compared with first two method.At production application pantoea agglomerans XM2 bacteria suspension, pears are adopted When rear melasma carries out Biological control, answer treatment in accordance with local conditions, select the way that the prevention and controls being suitable for or three kinds of methods combine, Pantoea agglomerans XM2 is made to play maximum Biocontrol Effect.

Beneficial effects of the present invention

(1) the various process of the pantoea agglomerans XM2 fermentation liquid of the present invention all can suppress Pear black spot, and wherein inoculation bacterium is hanged The process of liquid presses down disease effect and is significantly better than other process, and pantoea agglomerans XM2 is the high efficient strain that rear melasma Biological control adopted by pears；

(2) the pantoea agglomerans XM2 of the present invention to the preventing and treating of Pear black spot is Multiple components and factor plays a role, disease It is difficult to develop immunity to drugs: the antifungal mechanism of pantoea agglomerans XM2 is viable bacteria body and metabolite jointly acts on and plays after preventing and treating pears adopt The effect of melasma, additionally, can affect the multiple of sensitive organism according to bacteriogenic Substance (such as bacteriocin) simultaneously Metabolic pathway, infers that the probability that bacteriocin produces resistance because pathogen is undergone mutation is not easy appearance；

(3) the bacteria suspension preparation technology of the pantoea agglomerans XM2 of the present invention is simple, and the fermentation condition of pantoea agglomerans XM2 is the most severe Carving, can realize industrialized production, can be made into dry bacteria and be applied in the middle of industrialized production, commercial value is big；

(4) more lasting due to agglomerating general to the prevention effect of Pear black spot with the microbial inoculum of the pantoea agglomerans XM2 of the present invention Bacterium XM2 is to separate from Prunus mume Sieb. Et Zucc. fruit surface, has and adopts the same or like annidation of rear melasma and to posting with pears The affinity of main plant, field planting is easy, adds bacterial reproduction rapid so that pears are adopted rear melasma and prevented by pantoea agglomerans XM2 It is more lasting to imitate；

(5) Biocontrol Effect efficiency height pantoea agglomerans XM2 belongs to biocontrol bacteria, and reproduction speed is fast, under optimum conditions, several Schizogamy in ten minutes successively, has the highest biological and ecological methods to prevent plant disease, pests, and erosion efficiency；

(6) be conducive to maintaining the direct pin of antibacterial substance produced by beneficial microorganism ecological balance pantoea agglomerans XM2 Pear black spot is worked, high specificity, other beneficial microbe in Agro-ecological System will not be had a negative impact successively, Contribute to maintaining beneficial microorganism ecological balance.

(7) with the microbial inoculum preventing and treating of the pantoea agglomerans XM2 of the present invention, Pear black spot can be prevented and treated before melonidum is gathered, melonidum is gathered After immediately preventing and treating and melonidum pre-cooling after prevent and treat three kinds of methods, prevention effect respectively reaches more than 90%, more than 82% and more than 70%. Preventing and treating chance many, biocontrol effect is good.If it is more preferable that different times is combined prevention effect.

Accompanying drawing explanation

Fig. 1 is the pantoea agglomerans XM2 of the present invention morphological characteristic under transmission electron microscope.

Fig. 2 is the different disposal bacterium solution of the pantoea agglomerans strain X M2 of the present invention prevention effect to Pear black spot.

Fig. 3 is that pantoea agglomerans XM2 bacteria suspension different vaccination concentration of the present invention is to Pear black spot (1 × 10⁴Cfu/mL) anti- Control effect.

Fig. 4 is that pantoea agglomerans XM2 bacteria suspension different vaccination concentration of the present invention is to Pear black spot (1 × 10⁵Cfu/mL) anti- Control effect

Fig. 5 is that pantoea agglomerans XM2 bacteria suspension different vaccination concentration of the present invention is to Pear black spot (1 × 10⁶Cfu/mL) anti- Control effect

Fig. 6 is the pantoea agglomerans XM2 bacteria suspension different vaccination time of the present invention prevention effect to Pear black spot.

Fig. 7 is the pantoea agglomerans XM2 of the present invention Growth trends (28 DEG C) on melonidum wound.

Fig. 8 is the pantoea agglomerans XM2 of the present invention Growth trends (20 DEG C) on melonidum wound.

Fig. 9 is the pantoea agglomerans XM2 of the present invention Growth trends (4 DEG C and 0 DEG C) on melonidum wound.

Figure 10 is Pear black spot bacterium mycelia culture form.

Figure 11 is the pantoea agglomerans XM2 bacteria suspension of the present invention impact on Pear black spot bacterium hypha form.

Figure 12 is Pear black spot bacterium hypha form under microscope.

Figure 13 is that under microscope, pantoea agglomerans XM2 bacteria suspension of the present invention affects I to Pear black spot bacterium hypha form.

Figure 14 is that under microscope, pantoea agglomerans XM2 bacteria suspension of the present invention affects II to Pear black spot bacterium hypha form.

Detailed description of the invention

Below in conjunction with embodiment, the present invention is further illustrated.

Embodiment 1

One stain of Pantoea agglomerans (Pantoea agglomeran) strain X M2, is preserved in Chinese micro-life on February 17th, 2014 CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 (are called for short in thing culture presevation administration committee's common micro-organisms center Number, Institute of Microorganism, Academia Sinica, postcode 100101), Classification And Nomenclature is pantoea agglomerans (Pantoea agglomeran), Preserving number is CGMCC No.8822.

Pantoea agglomerans (Pantoea agglomeran) strain X M2 of the present invention is from Jin Tan village, Shanxi province Taiyuan city Prunus mume Sieb. Et Zucc. Fruit surface separates and purification obtains.At bacteriolyze meat soup (LB) culture medium (yeast powder 5 g, peptone 10 g, NaCl 5 g, fine jade Cosmetics 18 g, distilled water 1000mL, pH 7.4-7.6) cultivate 48 h ~ 72 h.Pantoea agglomerans (Pantoea agglomeran) bacterium Strain XM2 is gram negative bacteria, rod-short, and size is (0.5 ~ 0.9) μ m (1.0 ~ 2.5) μm, and amphitrichous has Mobility's (see figure 1).On bacteriolyze broth bouillon flat board after aerobic cultivation 24 h ~ 48 h, produce smooth surface, projection, Translucent, neat in edge, the yellow color colonies of a diameter of 2.0 mm ~ 3.0 mm.Strain X M2 is facultative anaerobe, grows pH model Enclosing is 5.7 ~ 7.5, and growth temperature range is 4 DEG C ~ 40 DEG C, NaCl concentration scope 0% ~ 7%；Other physiological and biochemical indexs As shown in table 1:

Table 1 strain X M2 physiological and biochemical property

Feature	Result
		Glucose Glucose	+
L-arabinose L-arabinose	+
		Sucrose Sucrose	+
Trehalose Trehalose	-
		Rhamnose Rhamnose	+
Xylose Xylose	+
		Starch Starch	-
Mannose Mannitose	+
		Inositol Inositol	-
Mannitol mannitol	+
		Gelatin liquefaction Gelatin liquification	-
Indole test Indole production	-
		H₂S tests H₂S production	-
Citrate Utilization of citrate	+
		Phenylalanine deaminase Phenylalanine deaminase	-
Lysine deshydroxy enzyme Lysine deaminase	-
		Catalase Catalase test	+
Oxidase Oxidase test	-
		Arginine dihydrolase Arginine dihndrolase	-
Nitrate reduction Nitrate reduction	+
		KOH reacts KOH test	-
Litmus milk Litmus milk	Peptonize, produce acid
		Methyl red test Methyl red test	-

Embodiment 2

Use the bacteria suspension preventing and treating Pear black spot of pantoea agglomerans (Pantoea agglomeran) strain X M2 of the present invention.

The preparation method of pantoea agglomerans (Pantoea agglomeran) strain X M2 bacteria suspension is as follows:

Aseptically, with the Inoculating needle picking one ring fresh pantoea agglomerans mono-bacterium colony of XM2 from test tube slant, it is inoculated in Equipped with the triangular flask of 20mL LB culture fluid, 28 DEG C of 160rpm/min concussions prepare the seed of pantoea agglomerans XM2 after cultivating 24h Liquid；Seed liquor is accessed in round equipped with LB culture fluid according to the ratio of 1%, 28 DEG C, 160rpm/min shake cultivation 72h, prepares the fermentation liquid of pantoea agglomerans XM2；Fermentation liquor 10000 rpm/min is centrifuged 10 min, abandons supernatant, add Aquesterilisa, is again centrifuged 10 min through 10000 rpm/min, abandons supernatant, adds sterilized water, mixing, it is thus achieved that become in precipitation The bacteria suspension of the general bacterium XM2 of group；It is 1 × 10 that this bacteria suspension is configured to concentration⁶cfu/mL~1×10⁸The pantoea agglomerans of cfu/mL The bacteria suspension of XM2, is used for preventing and treating pears and adopts rear melasma.

Before melonidum is gathered, 3 ~ 5 d aerosol apparatus are 1 × 10 at fruit surface spraying concentration⁶cfu/mL~1×10⁸ The bacteria suspension of the pantoea agglomerans XM2 of cfu/mL, will select the time period fine, that sunshine is sufficient to implement preventing and treating.This method can have The postharvest storage phase is infected by pathogen in effect prevention melonidum field, reduces storage period pears and adopts the generation of rear melasma.Sampled investigation, The prevention effect that pears are adopted rear melasma reaches more than 97%.

Embodiment 3

The preparation method of pantoea agglomerans (Pantoea agglomeran) strain X M2 bacteria suspension is with embodiment 2.

Pantoea agglomerans (Pantoea agglomeran) strain X M2 bacteria suspension is configured to concentration is 1 × 10⁶cfu/mL~1 ×10⁸The bacteria suspension of the pantoea agglomerans XM2 of cfu/mL, melonidum is soaked 2 min ~ 3 min with this bacteria suspension immediately after plucking, puts In plastic box, after fruit dries naturally, reenter fresh-keeping warehouse pre-cooling, storage.Note in natural dry process, it is to avoid will Melonidum contacts with other materials that may carry pathogen such as soil.After this method also can effectively prevent melonidum field pathogen to adopting Infecting of storage period, reduces storage period pears and adopts the generation of rear melasma.Sampled investigation, the prevention effect that pears are adopted rear melasma reaches To more than 82%.

Embodiment 4

First pantoea agglomerans (Pantoea agglomeran) strain X M2 bacteria suspension being configured to concentration is 1 × 10⁶cfu/ mL~1×10⁸The bacteria suspension of the pantoea agglomerans XM2 of cfu/mL, when melonidum is after fresh-keeping warehouse pre-cooling 24 h, will not have mechanical injury Melonidum soaks 2 min ~ 3 min with this bacteria suspension, is placed in plastic box, carries out the dress of melonidum after fruit dries naturally again Case works.Noticing that whole immersion and dry process are required in fresh-keeping warehouse carry out, preventing and treating melonidum temperature raises, and condensation occurs Water.This method can effectively prevent melonidum field pathogen to infect the postharvest storage phase, reduces storage period pears and adopts the generation of rear melasma. Sample survey results is that the prevention effect that pears are adopted rear melasma reaches more than 70%.

Claims

1. a stain of Pantoea agglomerans (Pantoea agglomeran) strain X M2, is preserved in Chinese micro-life on February 17th, 2014 Thing culture presevation administration committee's common micro-organisms center, preserving number is CGMCC No.8822；Described pantoea agglomerans bacterial strain XM2 is to obtain from the separation of Jin Tan village, Shanxi province Taiyuan city Prunus mume Sieb. Et Zucc. fruit surface purification, cultivates 48 on bacteriolyze broth bouillon H ~ 72 h, pantoea agglomerans strain X M2 is gram negative bacteria, rod-short, and size is 0.5 μm ~ 0.9 μ m 1.0 μm ~ 2.5 μm, amphitrichous；On bacteriolyze broth bouillon flat board after aerobic cultivation 24 h ~ 48 h, produce smooth surface, projection, semi-transparent Bright, neat in edge, the yellow color colonies of a diameter of 2.0 mm ~ 3.0 mm；Pantoea agglomerans strain X M2 is facultative anaerobe, growth PH scope is 5.7 ~ 7.5, and growth temperature range is 4 DEG C ~ 40 DEG C, NaCl concentration scope 0% ~ 7%；Other Physiology and biochemistries Index is as shown in the table:

Strain X M2 physiological and biochemical property

Through 16S rDNA sequencing, PCR amplification obtains the 16S rDNA fragment of strain X M2, and check order a length of 1386 bp, Sequence accession number in GenBank is KF150143；16S rDNA sequence is carried out with the sequence in GenBank data base BLAST comparison, result shows, the homology of the pantoea agglomerans (P. agglomerans) in strain X M2 and general Pseudomonas (Pantoea) Property reaches 99.8%, and combining form feature and physiological and biochemical index determine that strain X M2 is pantoea agglomerans (P. Agglomerans)；Described bacteriolyze broth bouillon is to be grouped into by the one-tenth of following masses/parts by volume: yeast powder 5 parts, egg White peptone 10 parts, NaCl 5 parts, agar powder 18 parts, distilled water 1000 parts；pH 7.4 ~ 7.6.

The preparation method of pantoea agglomerans strain X M2 bacteria suspension the most according to claim 1, comprises the steps:

(1) seed liquor preparation is with the Inoculating needle picking one ring fresh pantoea agglomerans mono-bacterium colony of XM2 from test tube slant, is inoculated in molten In bacterium cultured solution of broth, 28 DEG C, 160 rpm/min concussion constant temperature culture 24 h, prepare the seed liquor of pantoea agglomerans XM2；

(2) seed liquor of pantoea agglomerans XM2 is accessed in bacteriolyze cultured solution of broth according to the ratio of 1% by fermentation liquid preparation, 28 DEG C 160 rpm/min shake constant temperature culture 72 h, prepare the fermentation liquid of pantoea agglomerans XM2；

(3) fermentation liquor 10000 rpm/min of pantoea agglomerans XM2 is centrifuged 10 min by bacteria suspension preparation, abandons supernatant；To Precipitation adds aquesterilisa, is again centrifuged 10 min through 10000 rpm/min, abandons supernatant；Again add aseptic in precipitation Water, mixing, it is thus achieved that the bacteria suspension of pantoea agglomerans XM2.

3., by the method for the bacteria suspension preventing and treating Pear black spot of the pantoea agglomerans strain XM2 described in claim 1, it is characterized in that:

Before melonidum is gathered, 3 ~ 5 d aerosol apparatus are 1 × 10 at fruit surface spraying concentration⁶cfu/mL~1×10⁸Cfu/mL's The bacteria suspension of pantoea agglomerans XM2, will select the time period fine, that sunshine is sufficient to implement preventing and treating；

Or

Pantoea agglomerans strain X M2 bacteria suspension is configured to concentration is 1 × 10⁶cfu/mL~1×10⁸The pantoea agglomerans XM2 of cfu/mL Bacteria suspension, melonidum pluck after immediately with this bacteria suspension immersion 2 ~ 3 min, be placed in plastic box, after fruit dries naturally Reenter fresh-keeping warehouse pre-cooling, storage；In natural dry process, it is to avoid by melonidum and soil etc., other may carry the thing of pathogen Matter contacts；

Or

Pantoea agglomerans strain X M2 bacteria suspension is configured to concentration is 1 × 10⁶cfu/mL~1×10⁸The pantoea agglomerans XM2 of cfu/mL Bacteria suspension, when melonidum is after fresh-keeping warehouse pre-cooling 24 h, will not have mechanical injury this bacteria suspension of melonidum soak 2 ~ 3 min, put In plastic box, after fruit dries naturally, carry out the vanning work of melonidum again；Notice that whole immersion and dry process are both needed to Fresh-keeping warehouse to be carried out, to prevent melonidum temperature from raising, condensed water occurs.