CN103952338A - Pantoea agglomeran new bacterial strain XM2, preparation method for suspension of same, and method for controlling pear black spot - Google Patents
Pantoea agglomeran new bacterial strain XM2, preparation method for suspension of same, and method for controlling pear black spot Download PDFInfo
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Abstract
The invention discloses a new bacterial strain XM2 of pantoea agglomeran, and the new bacterial strain is preserved in China General Microbiology Center of Committee for Culture Collection of Microorganisms at 17th February, 2014, and the preservation number is CGMCC NO. 8822. The invention detailedly describes the morphological characteristics, the physiological and biochemical indexes, 16SrDNA sequencing results and the like of the new bacterial strain XM2 of pantoea agglomeran. A preparation method of a suspension of the new bacterial strain XM2 comprises seed liquid preparation, fermentation broth preparation and bacterial suspension preparation. A method of using the new bacterial XM2 suspension to control pear black spot comprises controlling before pear harvesting, instantly controlling after pear harvesting and controlling after pear pre-cooling, and the controlling effects can respectively reach 90% or more, 82% or more and 70% or more. The controlling opportunities are more and the biocontrol effect is good. The controlling effect is relatively good when controlling operations in different stages are combined.
Description
Technical field
The present invention relates to microorganism field, specifically relate to a kind of pantoea agglomerans (
pantoea agglomeran) new strain X M2 and the method for preventing and treating to Pear black spot thereof.
Background technology
Pear black spot is a kind of serious worldwide gas borne fungus diseases that endangers, by chain lattice spore (
alternaria alternata) cause all there is generation before the operatic circle is adopted He after adopting.China is pears plantations and big export country, adopts infecting of rear pathogenic micro-organism and causes that to adopt rear rotten loss serious, directly affects the operatic circle storing and produces, and effective control of Pear black spot becomes problem anxious to be resolved in pears industry.The post-harvest diseases that uses chemical bactericide to control fruit is considered to the most effective control method always.Yet, along with pathogenic bacteria is drug-fast, increase gradually, and people's raising that fruit quality is required and the reinforcement of environmental consciousness, postharvest fruit and vegetable biological control method as a kind of new way of controlling post-harvest diseases, is approved by people gradually, and is become study hotspot.In recent years have much and report about the research of Pear black spot biological control.Qi Dongmei etc. screened a bacillus subtilis (
bacillus subtilis) H110, the control rate that its fermented liquid is adopted rear black spot to pears reaches 86.6%.Xie Li from soil, be separated to streptomyces aureus (
streptomyces aureus) B-105, the inhibiting rate 67.4% of its fermented liquid to Pear black spot.Benbow etc. have studied Cryptococcus
cryptococcus informominiatuswith Rhodotorula
rhodotorula glutinisfungistatic effect to pears disease,
cryptococcus informominiatuswith
rhodotorula glutinisbefore gathering, 1 d processes pears, can effectively control the rear black spot of adopting of pears.Song Cong etc. be separated to a bacillus subtilis (
bacillus subtilis) J18, the operatic circle is soaked fruit 30 s in bacteria suspension, sprays inoculation alternaric bacteria after drying, and the sickness rate of Pear black spot is only 16.57%, and bacterial strain can surely be grown well on the operatic circle.
Summary of the invention
The object of this invention is to provide a stain of Pantoea agglomerans (
pantoea agglomeran) new strain X M2 and the method for preventing and treating to Pear black spot thereof.
The object of the invention is to be achieved through the following technical solutions.
One stain of Pantoea agglomerans (
pantoea agglomeran) new strain X M2, on February 17th, 2014, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.8822, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Described pantoea agglomerans (
pantoea agglomeran) new strain X M2 is separated from Jin Tan village, Shanxi province Taiyuan city prune fruit surface and purifying obtains.At bacteriolyze meat soup (LB) culture medium culturing 48 h ~ 72 h.Pantoea agglomerans (
pantoea agglomeran) new strain X M2 is Gram-negative bacteria, rod-short, size is (0.5 ~ 0.9) μ m * (1.0 ~ 2.5) μ m, amphitrichous, has mobility (see figure 1).On LB culture medium flat plate after aerobic cultivation 24 h ~ 48 h, produce smooth surface, projection, translucent, neat in edge, diameter is the yellow bacterium colony of 2.0 mm ~ 3.0 mm.Strain X M2 is facultative anaerobe, and growth pH scope is 5.7 ~ 7.5, and growth temperature range is 4 ℃ ~ 40 ℃, NaCl concentration range 0% ~ 7%.Other physiological and biochemical index is as shown in table 1:
Table 1 strain X M2 physiological and biochemical property
Through 16S rDNA sequencing: pcr amplification obtains the 16S rDNA fragment of strain X M2, order-checking length is 1386 bp, and the sequence accession number in GenBank is KF150143.Sequence in 16S rDNA sequence and GenBank database is carried out to BLAST and compare, result shows, strain X M2 and general Pseudomonas
pantoeain pantoea agglomerans (
p. agglomerans) homology reach 99.8%, combining form feature and physiological and biochemical index, determine strain X M2 be pantoea agglomerans (
p. agglomerans).
Described bacteriolyze meat soup (LB) substratum is comprised of the raw material of following mass/volume part: yeast powder 5, and peptone 10, NaCl 5, agar powder 18, distilled water 1000; PH 7.4-7.6.The preparation method of the new strain X M2 bacteria suspension of pantoea agglomerans, comprises the steps:
(1) seed liquor preparation, from test tube slant with the mono-bacterium colony of the inoculating needle picking one fresh pantoea agglomerans XM2 of ring, is inoculated in LB nutrient solution, and 28 ℃, 160 rpm/min shake constant temperature culture 24 h, make the seed liquor of pantoea agglomerans XM2;
(2) fermented liquid preparation is by the seed liquor of pantoea agglomerans XM2 according in 1% ratio access LB nutrient solution, and 28 ℃ of 160 rpm/min shakes constant temperature culture 72 h, makes the fermented liquid of pantoea agglomerans XM2;
(3) collecting cells, by the fermented liquid of pantoea agglomerans XM2 through centrifugal 10 min of 10000 rpm/min, is abandoned supernatant liquor; In precipitation, add aqua sterilisa, again, through centrifugal 10 min of 10000 rpm/min, abandon supernatant liquor; Again in precipitation, add sterilized water, mix, obtain the bacteria suspension of pantoea agglomerans XM2.
the control test of the new strain X M2 of pantoea agglomerans to Pear black spot
1. the control test of the different treatment bacterium liquid of the new strain X M2 of pantoea agglomerans to Pear black spot
(1) test method is mixed with following 4 kinds of bacterium liquid by the new strain X M2 of pantoea agglomerans:
A: being mixed with concentration is 1 * 10
6the fermenation raw liquid of cfu/mL;
B: the filtered liquid of fermenation raw liquid after 0.22 μ m membrane filtration;
C: after fermenation raw liquid is centrifugal, abandon supernatant liquor, precipitation is cleaned 2 times through aqua sterilisa, and being prepared into concentration is 1 * 10
6the bacteria suspension of cfu/mL;
D: fermenation raw liquid is at 121 ℃ of 100KPa autoclaving 20 min, and being prepared into concentration is 1 * 10
6the dead liquid of heat kill of cfu/mL;
The preparation of Pear black spot bacterium spore suspension:
In fresh Pear black spot bacterium flat board, add 10 mL sterilized waters, with sterilizing spreader, scrape gently and wash bacterium cake surface, wash the conidium of lower fresh pathogenic bacteria, collect washing lotion.By four layers of filtered through gauze of sterilizing, make spore suspension.Opticmicroscope microscopy suspension miospore situation, is configured to desired concn.
The new strain X M2 treatment solution of above-mentioned 4 kinds of pantoea agglomerans is got respectively to 30 μ l and be inoculated in the operatic circle wound (3 mm * 3 mm), contrast as adding equivalent sterilized water, after drying, get respectively 1 * 10
5individual spore/mL Pear black spot bacterium spore suspension 15 μ l are inoculated in the operatic circle wound, dry, and bagging moisturizing, 25 ℃ of constant temperature culture 7 d, each processes 10 the operatic circles, repeats statistics incidence 3 times.
Pear fruit occurring degree grade scale:
0 grade---anosis;
1 grade---scab accounts for below 1% of surface-area of fruit;
3 grades---scab accounts for 2% ~ 3% of fruit surface-area;
5 grades---scab accounts for 4% ~ 6% of fruit surface-area;
7 grades---scab accounts for 7% ~ 10% of fruit surface-area;
9 grades---scab accounts for fruit the more than 11% of surface-area;
(2) test results test result shows, the pantoea agglomerans XM2 that is inoculated in the different treatment liquid of wound all has provide protection (see figure 2) to Pear black spot bacterium, and protection effect is by by force to successively weak: the dead liquid of bacteria suspension, fermented liquid, filtered liquid and heat kill.It is 97.73% that bacteria suspension preventive effect is up to, and the preventive effect of the dead liquid of heat kill is only 56.82%, illustrates that the diseases prevention of pantoea agglomerans XM2 bacterial strain somatic cells is best, and meta-bolites takes second place, and refers to table 2.
Table 2 pantoea agglomerans XM2 different treatment liquid is on adopting the impact of rear Pear black spot
| Treatment solution | Disease index | Prevention effect/% |
| Bacteria suspension | 2.22? d | 97.73? a |
| Fermented liquid | 12.22? c | 87.50? b |
| Filtered liquid | 17.78? c | 81.82? b |
| The dead liquid of heat kill | 42.22? b | 56.82 ?c |
Note: significant difference between different lowercases represent to process (
p<0.05), lower same.
2. the control test of the new strain X M2 bacteria suspension of pantoea agglomerans different vaccination concentration to Pear black spot
(1) test method is formulated as respectively 1 * 10 by the new strain X M2 bacteria suspension of pantoea agglomerans concentration
8cfu/mL, 1 * 10
6cfu/mL, 1 * 10
4cfu/mL, Pear black spot bacterium spore concentration is formulated as respectively 1 * 10
6individual spore/mL, 1 * 10
5individual spore/mL, 1 * 10
4individual spore/mL.The bacteria suspension of different concns is respectively got to 30 μ l and add the operatic circle wound, after drying, inoculation different concns Pear black spot bacterium spore suspension 15 μ l, dry bagging moisturizing, 25 ℃ of constant temperature culture 7 d, statistics incidence (method is the same).Each processes 10 the operatic circles, repeats 3 times.
(2) test results test result shows, the operatic circle wound of inoculation pantoea agglomerans XM2 bacteria suspension, and its disease index is lower than contrast separately; XM2 bacteria suspension working concentration on wound is larger, and pathogenic bacteria inoculum density is lower, and its preventive effect is higher.During identical XM2 bacteria suspension concentration, its preventive effect reduces with the increase of pathogenic bacteria concentration; During identical Pear black spot bacterium spore suspension concentration, preventive effect increases (seeing Fig. 3, Fig. 4, Fig. 5) with the increase of XM2 bacteria suspension concentration.Pear black spot bacterium spore suspension lower concentration (1 * 10
4cfu/mL) time, XM2 bacteria suspension can effectively suppress disease and occur, and the preventive effect of three concentration is all more than 95%, and difference is not remarkable in 0.05 level; Pear black spot bacterium spore suspension high density (1 * 10
6cfu/mL) time, XM2 bacteria suspension can only reduce the occurrence degree of disease, and the preventive effect of three concentration is all below 40%, and in 0.05 level, difference is not remarkable, refers to table 3.
Table 3 XM2 different concns bacteria suspension is on adopting the impact of rear Pear black spot
3. the control test of new strain X M2 bacteria suspension different vaccination time of pantoea agglomerans to Pear black spot
(1) test method is 1 * 10 in inoculation 15 μ l concentration
5it is 1 * 10 that the different time of front 24 h of Pear black spot bacterium spore suspension of individual spore/mL, front 12 h, front 4 h, 0 h, rear 4 h, rear 12 h, rear 24 h is inoculated respectively 30 μ l concentration at the operatic circle wound (3 mm * 3 mm)
8cfu/mL pantoea agglomerans XM2 bacteria suspension, contrasts as first connecing 30 μ l sterilized waters at wound, accesses Pear black spot bacterium suspension after 3 h, dries bagging moisturizing, 25 ℃ of constant temperature culture 7 d, statistics incidence (method is the same).Each processes 10 the operatic circles, repeats 3 times.
(2) the new strain X M2 bacteria suspension of test-results pantoea agglomerans is inoculated in the operatic circle wound (3 mm * 3 mm) at the relative inoculation time of difference, Pear black spot is all had to prevention effect, the relative inoculation time effects prevention effect of pantoea agglomerans and Pear black spot bacterium spore, XM2 bacterial suspension inoculation time processing preventive effect relatively early is significantly better than the relative time processing in evening, illustrates that the new strain X M2 bacteria suspension of pantoea agglomerans preventive effect is better than result for the treatment of.Along with the postponement of the relative inoculation time of XM2 bacteria suspension, preventive effect (see figure 6) on a declining curve.The processing preventive effect of 24 h inoculation XM2 bacteria suspensions is the highest in advance, reaches 94.94%; The processing preventive effect of simultaneously inoculating XM2 bacteria suspension and Pear black spot bacterium spore suspension has dropped to 70.85%; The processing of the relative Pear black spot bacterium of XM2 bacteria suspension spore suspension inoculation in evening, preventive effect is all in 50% left and right, and in 0.05 level, difference is not remarkable, refers to table 4.
Table 4 XM2 and the impact of Spores inoculation time on Pear black spot
| 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. the growth of the new strain X M2 of pantoea agglomerans on the operatic circle wound is dynamic
(1) test method is got 30 μ l1 * 10
8the XM2 fermented liquid of cfu/mL is inoculated in the operatic circle wound (3 mm * 3 mm), and the XM2 bacterial count of measuring after l h is initial value (0 h).By inoculation XM2 the operatic circle respectively moisturizing store at 28 ℃, 20 ℃, 4 ℃ and 0 ℃.10 fruits of every processing, test repeats 3 times.Measure once the operatic circle every day at 28 ℃, and the operatic circle at 20 ℃ is measured once for every 3 days, and the every 6 days mensuration of the operatic circle at 4 ℃ and 0 ℃ once.Measuring method is with aseptic punch tool, from cut-off footpath, the operatic circle wound and the degree of depth, to be the pulp organization of 10mm, be put in oneself and add in the aseptic mortar of phosphoric acid buffer of 0.05 mol/L pH7.0 of 1.0 mL, after grinding in aseptic operating platform, with dilution-plate method, count.The cfu of gained Antagonistic Fungi is changed into log
10 cfu.
(2) test-results is at 28 ℃, 20 ℃, 4 ℃ and 0 ℃, and the new strain X M2 of pantoea agglomerans is first and breeds stably trend after growth at the operatic circle wound, all can better surely grow at wound.Pantoea agglomerans XM2 is 2.7 * 10 at the initial number of the operatic circle wound
7cfu/mL, while inoculating 1 d at 28 ℃, the operatic circle wound bacterial count is initial 3 * 10
8doubly ~ 2.7 * 10
9doubly, during inoculation 4 d, the operatic circle wound bacterial count reaches maximum value, is initial 3 * 10
16doubly ~ 1.27 * 10
18doubly, during inoculation 12 d, the operatic circle wound bacterial count is still 1.3 * 10 of starter bacteria number
10doubly ~ 4 * 10
11doubly (Fig. 7); While inoculating 3 d at 20 ℃, it is maximum that the operatic circle wound bacterial count reaches, and is 3 * 10 of initial bacterial count
14doubly ~ 1.25 * 10
15doubly, held stationary after 12 d, during inoculation 36 d, the operatic circle wound bacterial count is 6 * 10 of initial bacterial count
9doubly ~ 1 * 10
11doubly (Fig. 8); While inoculating 6 d at 4 ℃, it is maximum that the operatic circle wound bacterial count reaches, and substantially maintains afterwards level, and during inoculation 66 d, the operatic circle wound bacterial count is 3.2 * 10 of initial bacterial count
12doubly ~ 5 * 10
10doubly; At 0 ℃, the operatic circle wound bacterial count increasing degree is little, and low temperature has also suppressed the growth of pantoea agglomerans XM2 when suppressing Pear black spot growth, but during to the 66th d, the operatic circle wound bacterial count still can keep 1.08 * 10
8cfu/mL ~ 5.4 * 10
8cfu/mL is initial bacterial count 4 times ~ 20 times (Fig. 9).
5. the impact of the new strain X M2 fermented liquid of pantoea agglomerans on Pear black spot bacterium mycelia form
(1) test method is by 10
6individual/mL pears alternaric bacteria spore suspension 1 mL is inoculated in 100 mL potato glucose (PDB) substratum, 25 ℃ of standing constant temperature culture.After pears alternaric bacteria spore grows uniform mycelia suspension, add 1 * 10
8cfu/mL pantoea agglomerans XM2 bacteria suspension 1mL; Contrast, for to add 1mL sterilized water in pears alternaric bacteria silk suspension, is observed and is processed and the mycelia metamorphosis contrasting respectively after 72 h under opticmicroscope.
(2) test-results visual inspection is found, alternaric bacteria mycelia is evenly distributed in control group, the vigorous (see figure 10) of growing; Alternaric bacteria mycelia skewness in treatment group, shrinkage is agglomerating, grow sparse (seeing Figure 11).By observation by light microscope, find, alternaric bacteria mycelia mycelia in control group is sturdy, continuous, smooth surface (seeing Figure 12); Alternaric bacteria mycelia is part fracture in treatment group, and cell content is excessive, and torsional deformation is assembled agglomerating (seeing Figure 13, Figure 14).
In a word, pantoea agglomerans XM2 adopts rear black spot biological control for pears new bacterial strain is provided, by test, reach a conclusion: the antifungal mechanism of pantoea agglomerans XM2 is the common effect of thalline and meta-bolites, has both had the competition in nutrition and space, have again pair destruction of pathogenic bacteria mycelia; The protection effect of pantoea agglomerans XM2 is relevant with concentration, and it is lower that pantoea agglomerans XM2 concentration is larger, pears are adopted rear blackspot pathogenic bacteria inoculum density, and its preventive effect is better; The inoculation time that pantoea agglomerans XM2 and pears are adopted rear black spot protospore affects biological control effect, the fungistatic effect of first inoculating pantoea agglomerans XM2 be better than significantly simultaneously or after in the effect of germ inoculation.
With the bacteria suspension of the new strain X M2 of pantoea agglomerans, to the method for preventing and treating of Pear black spot, be:
(1) the operatic circle before gathering, prevent and treat method the operatic circle and gather before 3 d ~ 5 d, with atomizer, at fruit surface spraying concentration, be 1 * 10
6cfu/mL ~ 1 * 10
8the pantoea agglomerans XM2 bacteria suspension of cfu/mL, the preventive effect of pears being adopted to rear black spot reaches more than 97%;
(2) the operatic circle is 1 * 10 in concentration immediately after preventing and treating immediately method the operatic circle after gathering and gathering
6cfu/mL ~ 1 * 10
8in the pantoea agglomerans XM2 bacteria suspension of cfu/mL, soak 2 min ~ 3 min, enter fresh-keeping warehouse storage after fruit dries naturally, the preventive effect of pears being adopted to rear black spot reaches more than 82%;
(3) after the operatic circle precooling, preventing and treating after the precooling of method the operatic circle, is 1 * 10 in concentration
6cfu/mL ~ 1 * 10
8in the pantoea agglomerans XM2 bacteria suspension of cfu/mL, soak 2 min ~ 3 min, after naturally drying, carry out the operatic circle vanning work, the preventive effect of pears being adopted to rear black spot reaches more than 70%.
Above three kinds of methods cut both ways: the method for the operatic circle preharvest spraying pantoea agglomerans XM2 bacteria suspension is simple, reduce and adopt rear workload, prevention effect is best, but the bacteria suspension of fruit face may skewness in spraying process, to pathogen infection, bring chance, also have the possibility that infects other diseases in field simultaneously; The method bacteria suspension that the operatic circle is soaked with the new strain X M2 bacteria suspension of pantoea agglomerans after adopting is immediately evenly distributed fruit face, and the preventive effect of pears being adopted to rear black spot is higher, but more concentrated because of the operatic circle collecting time in actual production, may increase labour intensity every day; The method of soaking with the new strain X M2 bacteria suspension of pantoea agglomerans after the operatic circle precooling is upper abundant many compared with the first two method time, but the colonization ability of pantoea agglomerans XM2 is not so good as at normal temperatures at low temperatures, therefore it is low compared with first two method pears to be adopted to the preventive effect of rear black spot.When black spot is carried out biological control after production application pantoea agglomerans XM2 bacteria suspension is adopted pears, should suit measures to local conditions, select the applicable method of preventing and treating or three kinds of ways that method combines, make pantoea agglomerans XM2 bring into play maximum Biocontrol Effect.
Beneficial effect of the present invention
(1) the various processing of pantoea agglomerans XM2 fermented liquid of the present invention all can suppress Pear black spot, and the processing of wherein inoculating bacteria suspension presses down disease effect and is significantly better than other processing, and pantoea agglomerans XM2 is the high efficient strain that pears are adopted rear black spot biological control;
(2) pantoea agglomerans XM2 of the present invention is that Multiple components and factor play a role to the control of Pear black spot, disease is difficult to develop immunity to drugs: the antifungal mechanism of pantoea agglomerans XM2 is that the effect that control pears are adopted rear black spot is played in viable bacteria body and meta-bolites acting in conjunction, in addition, according to bacteriogenic Substance (as bacteriocin), can affect the multiple pathways metabolism of sensitive organism simultaneously, infer because of pathogenic bacteria and undergo mutation the possibility of bacteriocin generation resistance is not easy to occur;
(3) the collecting cells technique of pantoea agglomerans XM2 of the present invention is simple, and the fermentation condition of pantoea agglomerans XM2 is not harsh, can realize suitability for industrialized production, can be made into dry bacteria and is applied in the middle of suitability for industrialized production, and commercial value is large;
(4) with the microbial inoculum of pantoea agglomerans XM2 of the present invention to the prevention effect of Pear black spot more lastingly because pantoea agglomerans XM2 separates from prune fruit surface, have with pears and adopt ecological suitability and the affinity to host plant that rear black spot is identical or close, field planting is easy, add bacterial reproduction rapid, the preventive effect that makes pantoea agglomerans XM2 adopt rear black spot to pears is more lasting;
(5) the high pantoea agglomerans XM2 of Biocontrol Effect efficiency belongs to biocontrol bacteria, and reproduction speed is fast, and under optimum conditions, dozens of minutes fissiparity successively, has very high biological and ecological methods to prevent plant disease, pests, and erosion efficiency;
(6) being conducive to maintain the antibacterial substance that beneficial microorganism eubiosis pantoea agglomerans XM2 produces directly works for Pear black spot, high specificity, can not have a negative impact to other beneficial microorganism in agroecosystem successively, contribute to maintain the beneficial microorganism eubiosis.
(7) with the microbial inoculum control Pear black spot of pantoea agglomerans XM2 of the present invention can before the operatic circle is gathered, prevent and treat, the operatic circle prevents and treats after gathering immediately and the operatic circle precooling after prevent and treat three kinds of methods, prevention effect reaches respectively more than 90%, more than 82% and more than 70%.Prevent and treat chance many, biocontrol effect is good.If different times is better in conjunction with prevention effect.
Accompanying drawing explanation
Fig. 1 is the morphological specificity of pantoea agglomerans XM2 of the present invention under transmission electron microscope.
Fig. 2 is the different treatment bacterium liquid of the new strain X M2 of the pantoea agglomerans 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 * 10
4cfu/mL) prevention effect.
Fig. 4 is that pantoea agglomerans XM2 bacteria suspension different vaccination concentration of the present invention is to Pear black spot (1 * 10
5cfu/mL) prevention effect
Fig. 5 is that pantoea agglomerans XM2 bacteria suspension different vaccination concentration of the present invention is to Pear black spot (1 * 10
6cfu/mL) prevention effect
Fig. 6 is the prevention effect of pantoea agglomerans XM2 bacteria suspension different vaccination time of the present invention to Pear black spot.
Fig. 7 is the growth of pantoea agglomerans XM2 of the present invention on the operatic circle wound dynamically (28 ℃).
Fig. 8 is the growth of pantoea agglomerans XM2 of the present invention on the operatic circle wound dynamically (20 ℃).
Fig. 9 is the growth of pantoea agglomerans XM2 of the present invention on the operatic circle wound dynamically (4 ℃ and 0 ℃).
Figure 10 is Pear black spot bacterium mycelium culture form.
Figure 11 is the impact of pantoea agglomerans XM2 bacteria suspension of the present invention on Pear black spot bacterium mycelia form.
Figure 12 is Pear black spot bacterium mycelia form under microscope.
Figure 13 is the affect I of pantoea agglomerans XM2 bacteria suspension of the present invention on Pear black spot bacterium mycelia form under microscope.
Figure 14 is the affect II of pantoea agglomerans XM2 bacteria suspension of the present invention on Pear black spot bacterium mycelia form under microscope.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
embodiment 1
One stain of Pantoea agglomerans (
pantoea agglomeran) new strain X M2, on February 17th, 2014, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), Classification And Nomenclature be pantoea agglomerans (
pantoea agglomeran), preserving number is CGMCC No.8822.
Pantoea agglomerans of the present invention (
pantoea agglomeran) new strain X M2 is separated from Jin Tan village, Shanxi province Taiyuan city prune fruit surface and purifying obtains.At bacteriolyze meat soup (LB) substratum (yeast powder 5 g, peptone 10 g, NaCl 5 g, agar powder 18 g, distilled water 1000mL, pH 7.4-7.6), cultivate 48 h ~ 72 h.Pantoea agglomerans (
pantoea agglomeran) new strain X M2 is Gram-negative bacteria, rod-short, size is (0.5 ~ 0.9) μ m * (1.0 ~ 2.5) μ m, amphitrichous, has mobility (see figure 1).On bacteriolyze broth culture flat board after aerobic cultivation 24 h ~ 48 h, produce smooth surface, projection, translucent, neat in edge, diameter is the yellow bacterium colony of 2.0 mm ~ 3.0 mm.Strain X M2 is facultative anaerobe, and growth pH scope is 5.7 ~ 7.5, and growth temperature range is 4 ℃ ~ 40 ℃, NaCl concentration range 0% ~ 7%; Other physiological and biochemical indexs are 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 | - |
| Rhamnosyl Rhamnose | + |
| Wood sugar Xylose | + |
| Starch Starch | - |
| Seminose Mannitose | + |
| Inositol Inositol | - |
| N.F,USP MANNITOL mannitol | + |
| Gelatine liquefication Gelatin liquification | - |
| Indole test Indole production | - |
| H 2S tests H 2S production | - |
| Citrate trianion Utilization of citrate | + |
| Phenylalanine deaminase Phenylalanine deaminase | - |
| Methionin deshydroxy enzyme Lysine deaminase | - |
| Catalase Catalase test | + |
| Oxydase Oxidase test | - |
| Arginine dihydrolase Arginine dihndrolase | - |
| Nitrate reduction Nitrate reduction | + |
| KOH reaction KOH test | - |
| Litmus milk Litmus milk | Peptonize, produce acid |
| Methyl red test Methyl red test | - |
Through 16S rDNA sequencing: pcr amplification obtains the 16S rDNA fragment of strain X M2, order-checking length is 1386 bp, and the sequence accession number in GenBank is KF150143.Sequence in 16S rDNA sequence and GenBank database is carried out to BLAST and compare, result shows, strain X M2 and general Pseudomonas
pantoeain pantoea agglomerans (
p. agglomerans) homology reach 99.8%, combining form feature and physiological and biochemical index, determine strain X M2 be pantoea agglomerans (
p. agglomerans).
embodiment 2
Adopt pantoea agglomerans of the present invention (
pantoea agglomeran) the bacteria suspension control Pear black spot of new strain X M2.
Pantoea agglomerans (
pantoea agglomeran) newly the preparation method of strain X M2 bacteria suspension is as follows:
Under aseptic condition, from test tube slant, with the mono-bacterium colony of the inoculating needle picking one fresh pantoea agglomerans XM2 of ring, be inoculated in the triangular flask that 20mL LB nutrient solution is housed, after 28 ℃ of 160rpm/min concussion cultivation 24h, make the seed liquor of pantoea agglomerans XM2; Seed liquor is equipped with in the fermenting container of LB nutrient solution according to the access of 1% ratio, and 28 ℃, 160rpm/min concussion are cultivated 72h, make the fermented liquid of pantoea agglomerans XM2; Fermented liquid, through centrifugal 10 min of 10000 rpm/min, is abandoned to supernatant liquor, then added aqua sterilisa, again, through centrifugal 10 min of 10000 rpm/min, abandon supernatant liquor, in precipitation, add sterilized water, mix, obtain the bacteria suspension of pantoea agglomerans XM2; It is 1 * 10 that this bacteria suspension is configured to concentration
6cfu/mL ~ 1 * 10
8the bacteria suspension of the pantoea agglomerans XM2 of cfu/mL, adopts rear black spot for preventing and treating pears.
Before the operatic circle is gathered, 3 ~ 5 d are 1 * 10 with atomizer at fruit surface spraying concentration
6cfu/mL ~ 1 * 10
8the bacteria suspension of the pantoea agglomerans XM2 of cfu/mL, select the time period fine, sunshine is sufficient to implement control.This method can effectively prevent the operatic circle field pathogenic bacteria to the infecting of postharvest storage phase, and reduces storage period pears and adopts the generation of rear black spot.Through sample survey, the prevention effect of pears being adopted to rear black spot reaches more than 97%.
embodiment 3
Adopt pantoea agglomerans of the present invention (
pantoea agglomeran) the bacteria suspension control Pear black spot of new strain X M2.
Pantoea agglomerans (
pantoea agglomeran) preparation method of new strain X M2 bacteria suspension is with embodiment 2.
By pantoea agglomerans (
pantoea agglomeran) to be configured to concentration be 1 * 10 to new strain X M2 bacteria suspension
6cfu/mL ~ 1 * 10
8the bacteria suspension of the pantoea agglomerans XM2 of cfu/mL, the operatic circle is soaked 2 min ~ 3 min with this bacteria suspension after plucking immediately, is placed in plastic box, reenters fresh-keeping warehouse precooling, storage after fruit dries naturally.Attention, in natural dry process, is avoided the operatic circle to contact with other materials that may carry pathogenic bacteria such as soil.This method also can effectively prevent the operatic circle field pathogenic bacteria to the infecting of postharvest storage phase, and reduces storage period pears and adopts the generation of rear black spot.Through sample survey, the prevention effect of pears being adopted to rear black spot reaches more than 82%.
embodiment 4
Adopt pantoea agglomerans of the present invention (
pantoea agglomeran) the bacteria suspension control Pear black spot of new strain X M2.
Pantoea agglomerans (
pantoea agglomeran) preparation method of new strain X M2 bacteria suspension is with embodiment 2.
First by pantoea agglomerans (
pantoea agglomeran) to be configured to concentration be 1 * 10 to new strain X M2 bacteria suspension
6cfu/mL ~ 1 * 10
8the bacteria suspension of the pantoea agglomerans XM2 of cfu/mL, when the operatic circle is after fresh-keeping warehouse precooling 24 h, by not having the operatic circle of mechanical injury to soak 2 min ~ 3 min with this bacteria suspension, is placed in plastic box, carries out the vanning work of the operatic circle after fruit dries naturally again.Notice that whole immersion and dry process all need to carry out in fresh-keeping warehouse, control the operatic circle temperature raises, and occurs water of condensation.This method can effectively prevent the operatic circle field pathogenic bacteria to the infecting of postharvest storage phase, and reduces storage period pears and adopts the generation of rear black spot.Sample survey result is that the prevention effect that pears are adopted to rear black spot reaches more than 70%.
Claims (5)
- One stain of Pantoea agglomerans ( pantoea agglomeran) new strain X M2, it is characterized in that on February 17th, 2014, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.8822.
- Pantoea agglomerans according to claim 1 ( pantoea agglomeran) new strain X M2, it is characterized in that: the new strain X M2 of described pantoea agglomerans is separated from Jin Tan village, Shanxi province Taiyuan city prune fruit surface and purifying obtains, on bacteriolyze broth culture, cultivate 48 h ~ 72 h, the new strain X M2 of pantoea agglomerans is Gram-negative bacteria, rod-short, size is 0.5 μ m * 1.0, μ m ~ 0.9 μ m ~ 2.5 μ m, amphitrichous; On bacteriolyze broth culture flat board after aerobic cultivation 24 h ~ 48 h, produce smooth surface, projection, translucent, neat in edge, diameter is the yellow bacterium colony of 2.0 mm ~ 3.0 mm; The new strain X M2 of pantoea agglomerans is facultative anaerobe, and growth pH scope is 5.7 ~ 7.5, and growth temperature range is 4 ℃ ~ 40 ℃, NaCl concentration range 0% ~ 7%; Other physiological and biochemical indexs are as shown in the table:Strain X M2 physiological and biochemical property
Feature Result Glucose Glucose + L-arabinose L-arabinose + Sucrose Sucrose + Trehalose Trehalose - Rhamnosyl Rhamnose + Wood sugar Xylose + Starch Starch - Seminose Mannitose + Inositol Inositol - N.F,USP MANNITOL mannitol + Gelatine liquefication Gelatin liquification - Indole test Indole production - H 2S tests H 2S production - Citrate trianion Utilization of citrate + Phenylalanine deaminase Phenylalanine deaminase - Methionin deshydroxy enzyme Lysine deaminase - Catalase Catalase test + Oxydase Oxidase test - Arginine dihydrolase Arginine dihndrolase - Nitrate reduction Nitrate reduction + KOH reaction KOH test - Litmus milk Litmus milk Peptonize, produce acid Methyl red test Methyl red test - Through 16S rDNA sequencing, pcr amplification obtains the 16S rDNA fragment of strain X M2, and order-checking length is 1386 bp, and the sequence accession number in GenBank is KF150143; Sequence in 16S rDNA sequence and GenBank database is carried out to BLAST and compare, result shows, strain X M2 and general Pseudomonas pantoeain pantoea agglomerans ( p. agglomerans) homology reach 99.8%, combining form feature and physiological and biochemical index, determine strain X M2 be pantoea agglomerans ( p. agglomerans). - Pantoea agglomerans according to claim 2 ( pantoea agglomeran) new strain X M2, it is characterized in that: described bacteriolyze broth culture is that the one-tenth by following mass/volume part is grouped into: 5 parts of yeast powders, 10 parts of peptones, 5 parts of NaCl, 18 parts of agar powders, 1000 parts of distilled water; PH 7.4 ~ 7.6.
- 4. the preparation method of the new strain X M2 bacteria suspension of pantoea agglomerans, comprises the steps:(1) seed liquor preparation, from test tube slant with the mono-bacterium colony of the inoculating needle picking one fresh pantoea agglomerans XM2 of ring, is inoculated in bacteriolyze cultured solution of broth, and 28 ℃, 160 rpm/min shake constant temperature culture 24 h, make the seed liquor of pantoea agglomerans XM2;(2) fermented liquid preparation is by the seed liquor of pantoea agglomerans XM2 according in 1% ratio access bacteriolyze cultured solution of broth, and 28 ℃ of 160 rpm/min shakes constant temperature culture 72 h, makes the fermented liquid of pantoea agglomerans XM2;(3) collecting cells, by the fermented liquid of pantoea agglomerans XM2 through centrifugal 10 min of 10000 rpm/min, is abandoned supernatant liquor; In precipitation, add aqua sterilisa, again, through centrifugal 10 min of 10000 rpm/min, abandon supernatant liquor; Again in precipitation, add sterilized water, mix, obtain the bacteria suspension of pantoea agglomerans XM2.
- 5. with the bacteria suspension of the new strain X M2 of pantoea agglomerans, prevent and treat the method for Pear black spot, it is characterized in that:Before the operatic circle is gathered, 3 ~ 5 d are 1 * 10 with atomizer at fruit surface spraying concentration 6cfu/mL ~ 1 * 10 8the bacteria suspension of the pantoea agglomerans XM2 of cfu/mL, select the time period fine, sunshine is sufficient to implement control;OrIt is 1 * 10 that the new strain X M2 bacteria suspension of pantoea agglomerans is configured to concentration 6cfu/mL ~ 1 * 10 8the bacteria suspension of the pantoea agglomerans XM2 of cfu/mL, the operatic circle is soaked 2 ~ 3 min with this bacteria suspension after plucking immediately, is placed in plastic box, reenters fresh-keeping warehouse precooling, storage after fruit dries naturally; Attention, in natural dry process, is avoided the operatic circle to contact with other materials that may carry pathogenic bacteria such as soil;OrIt is 1 * 10 that the new strain X M2 bacteria suspension of pantoea agglomerans is configured to concentration 6cfu/mL ~ 1 * 10 8the bacteria suspension of the pantoea agglomerans XM2 of cfu/mL, when the operatic circle is after fresh-keeping warehouse precooling 24 h, by not having the operatic circle of mechanical injury to soak 2 ~ 3 min with this bacteria suspension, is placed in plastic box, carries out the vanning work of the operatic circle after fruit dries naturally again; Notice that whole immersion and dry process all need to carry out in fresh-keeping warehouse, control the operatic circle temperature raises, and occurs water of condensation.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695353A (en) * | 2016-02-01 | 2016-06-22 | 山西省农业科学院农产品贮藏保鲜研究所 | Bacillus new strain, preparation method of fermentation product of Bacilus amyloliquefaciens new strain, and application of fermentation product in controlling pear black spot |
| CN114790431A (en) * | 2022-05-06 | 2022-07-26 | 山东省林业科学研究院 | Pantoea agglomerans and application thereof in prevention and treatment of pepper diseases |
| CN116064303A (en) * | 2022-09-26 | 2023-05-05 | 黑龙江省农业科学院经济作物研究所 | Pantoea agglomerans for producing trans-cinnamic acid and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695353A (en) * | 2016-02-01 | 2016-06-22 | 山西省农业科学院农产品贮藏保鲜研究所 | Bacillus new strain, preparation method of fermentation product of Bacilus amyloliquefaciens new strain, and application of fermentation product in controlling pear black spot |
| CN105695353B (en) * | 2016-02-01 | 2020-08-25 | 山西省农业科学院农产品贮藏保鲜研究所 | Novel bacillus strain, preparation method of fermentation product of novel bacillus strain and application of fermentation product in preventing and treating pear black spot |
| CN114790431A (en) * | 2022-05-06 | 2022-07-26 | 山东省林业科学研究院 | Pantoea agglomerans and application thereof in prevention and treatment of pepper diseases |
| CN114790431B (en) * | 2022-05-06 | 2023-06-20 | 山东省林业科学研究院 | Pantoea agglomerans and application thereof in aspect of controlling pepper diseases |
| CN116064303A (en) * | 2022-09-26 | 2023-05-05 | 黑龙江省农业科学院经济作物研究所 | Pantoea agglomerans for producing trans-cinnamic acid and application thereof |
| CN118165900A (en) * | 2024-05-16 | 2024-06-11 | 东北林业大学 | Pantoea agglomerans SM-1-GJ and application thereof |
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| CN103952338B (en) | 2016-08-17 |
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