CN107083344A - A kind of pseudomonad strain for preventing and treating Prospect on Kiwifruit Bacterial Canker and its application - Google Patents
A kind of pseudomonad strain for preventing and treating Prospect on Kiwifruit Bacterial Canker and its application Download PDFInfo
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Abstract
The present invention relates to microbial technology field, and in particular to a kind of pseudomonad strain of preventing and treating Prospect on Kiwifruit Bacterial Canker and its application.Pseudomonad of the present invention（Pseudomonas sp.）Bacterial strain deposit number is CCTCC NO：M2016556.The pseudomonad of the present invention isolates and purifies what is obtained from the Kiwi berry branch of collection, and separating screening method is simple, and the bacterial strain has very strong inhibitory action to kiwi berry bacterial canker pathogen, can substantially suppress the growth of kiwi berry bacterial canker pathogen.The strain isolation is from health plant tissue; it is inoculated with Kiwi berry branch, blade no abnormality seen; the active metabolite of generation does not have obvious toxic action to plant tissue; and property is stable; squirt conveniently; prevent and treat Kiwi berry ulcer effect significantly, provide an environmental protection, simple, effective approach for the preventing and treating of Kiwi berry ulcer, utilize environmental protection.
Description
Technical field
The present invention relates to microbial technology field, and in particular to a kind of pseudomonad strain of preventing and treating Prospect on Kiwifruit Bacterial Canker and
It is applied.
Background technology
Prospect on Kiwifruit Bacterial Canker is a kind of crushing bacteriosis.The trunk of the sick main harm Kiwi berry, branches and tendrils, young sprout and
Blade, easily causes plant dead.The disease is found for 1980 first in Japan, is found successively in U.S. etc. after nineteen eighty-three.1987
Year pathogen is accredited as the pathogenic mutation Pseudomonas syringae of Bacterial leaf spot Kiwi berry
pv.actinidiae.Before 1984, China does not have kiwi berry bacterial canker distribution, is found in Hunan earliest within 1985
Dongshan peak farm, and in the 10 short years, rapid diffusion spreads to the most Kiwifruit Culture regions of China.The sick oncoming force
Ferociousness, usually breaks out on a large scale, causes large area to ruin garden, causes serious economic loss, directly threatens the hair of Kiwi berry industry
Exhibition.Kiwi berry bacterial canker bacterium in 1996 is put into China's Forest Plant Quarantine Pest list.It is listed within 2013 the " whole nation
Forestry danger harmful organism list " (No. 4 bulletin of the State Administration of Forestry 2013 year).It there is no having for this disease of radical cure both at home and abroad at present
Effect technology, plant, which once catches an illness just to propagate to stretch rapidly, comes, irresistible.Hypopathia orchard death strain rate general 5~10%, weight
The dead strain rate in sick garden is up to 20~60%.Orchard worker is felt simply helpless, or allows its natural death, or excavated by manual work to remove, and garden is abandoned mostly and is replanted
The fruit tree of other kinds.
The preventing and treating for kiwi berry bacterial canker mainly uses the medicament based on streptomysin and copper agent at present
Preventing and treating, prevention effect is not satisfactory.But largely use copper agent and caused the resistance problems of ulcer bacteria, simultaneously
A large amount of field control effects for using brought food-safety problem to drastically influence canker of antibiotic.Thus seek
Change the biological pesticide for sending out new and effective the stability and development to Kiwi berry industry brings glad tidings.
The content of the invention
The present invention provides a kind of new pseudomonad strain for poisoning low preventing and treating Prospect on Kiwifruit Bacterial Canker excellent effect, it is intended to
Biological pesticide new sources are found, a kind of novel biological bactericide that can be used for Prospect on Kiwifruit Bacterial Canker preventing and treating is developed.
The pseudomonad strain of present invention preventing and treating Prospect on Kiwifruit Bacterial Canker, pseudomonad (Pseudomonas sp.) name
Referred to as pseudomonad SPANRS-62, bacterial strain is preserved in China typical culture collection center (abbreviation CCTCC), and deposit number is
CCTCC NO：M2016556 (referred to as pseudomonad CCTCC M2016556), the address of depositary institution is big positioned at Wuhan, China
Learn, preservation date is on October 11st, 2016.
Pseudomonad CCTCC M2016556 bacterial strains of the present invention are creamy white, colloidal substance, the smooth moistening in surface, middle cardiac prominence
Rise, neat in edge, viscosity is normal.
The pseudomonad CCTCC M2016556 of the present invention are in the wild Fructus actinidiae chinensis that Jiangxi Province Jinggang Mountain height above sea level is 1414m
Plant is isolated, and its separating screening method is：
1st, the Kiwi berry branch of collection is rinsed well with clear water, 75% alcohol surface sterilization, room temperature is air-dried, with sterilization
Blade afterwards is cut for sample sheet in right amount, is put into grind bag, adds appropriate physiological saline, grinding, immersion 3min take its plant
The μ L of supernatant 100 of lapping liquid are added in the KB culture mediums newly prepared, and are coated with oese, line is handled.
2nd, sample culture dish will be connect to cultivate after certain time at room temperature, and will treat to grow bacterium colony in ware, the sharp-edged bacterium of picking
Fall, in line purifying repeatedly on the KB culture medium flat plates newly prepared, can obtain SPANRS-62 single bacterium colonies, be then transferred into and freeze
Guan Zhong, is sealed up for safekeeping with glycerine, is positioned in -80 DEG C of ultra low temperature freezers and is preserved strain, the antagonistic experiment for carrying out canker.
The present invention also provides a kind of microbial bacterial agent, and vacation of the microbial bacterial agent comprising above-mentioned preventing and treating Prospect on Kiwifruit Bacterial Canker is single
Born of the same parents' bacteria strain, if necessary, the carrier and auxiliary material commonly used in also being prepared comprising microbial inoculum.
The present invention also provides application of the above-mentioned pseudomonad strain in preventing and treating Prospect on Kiwifruit Bacterial Canker.
Further, application of the above-mentioned pseudomonad strain in preventing and treating Prospect on Kiwifruit Bacterial Canker, wherein the Kiwi berry is burst
The pathogenic bacteria of ulcer disease are kiwi berry bacterial canker pathogen (Pseudomonas syringae pv.Actinidiae).
The present invention also provides a kind of culture of pseudomonad strain, fermentation culture, the filtered fluid of fermentation culture.
The present invention also provides a kind of culture of pseudomonad strain, fermentation culture, the filtered fluid of fermentation culture and existed
Prevent and treat the application in Prospect on Kiwifruit Bacterial Canker.
Further, the filtered fluid of the culture of above-mentioned pseudomonad strain, fermentation culture, fermentation culture is in preventing and treating
Application in Prospect on Kiwifruit Bacterial Canker, it is included being cultivated above-mentioned pseudomonad strain using KB solid mediums, trained
Support thing, fermentation culture, the filtered fluid of fermentation culture；With the culture of resulting pseudomonad strain, fermentation culture,
The filtered fluid of fermentation culture suppresses the growth of the pathogenic bacteria of Prospect on Kiwifruit Bacterial Canker.
Further, the filtered fluid of the culture of above-mentioned pseudomonad strain, fermentation culture, fermentation culture is in preventing and treating
Application in Prospect on Kiwifruit Bacterial Canker, the pathogenic bacteria of the Prospect on Kiwifruit Bacterial Canker are kiwi berry bacterial canker pathogen
(Pseudomonas syringae pv.Actinidiae)。
Culture, fermentation culture, the filtered fluid of fermentation culture of above-mentioned pseudomonad strain are burst in preventing and treating Kiwi berry
Application in ulcer disease, the KB solid mediums constituent is：Counted on the basis of 1L KB solid mediums, contain new albumen
Peptone 10.0g, glycerine 7.5g, MgSO4·7H2O 1.5g, K2HPO41.0g, agar 16.0g, H2O 1.0L。
Pseudomonad CCTCC M2016556 of the present invention isolate and purify what is obtained from the Kiwi berry branch of collection, separation screening
Method is simple, and the bacterial strain has very strong inhibitory action to kiwi berry bacterial canker pathogen, can substantially suppress Kiwi berry
The growth of bacterial canker pathogen mycelia.The strain isolation is from health plant tissue, thus the active metabolism production produced
Thing does not have obvious toxic action to plant tissue, and property is stable, squirts conveniently, and preventing and treating Kiwi berry ulcer effect is Mi significantly
The preventing and treating of monkey peach ulcer provides environmental protection, a simple, effective approach, utilizes environmental protection.
Brief description of the drawings
Fig. 1 is pseudomonad CCTCC M2016556 strain morphology figures of the present invention；
Fig. 2 is pseudomonad CCTCC M2016556 of the present invention bacterial strain scanning electron microscope (SEM) photograph；
Fig. 3 is negative control group result of the test in the control efficacy experiment of Antagonistic Fungi of the present invention；
Fig. 4 tests positives control group result of the test for the control efficacy of Antagonistic Fungi of the present invention；
Fig. 5 is experimental group result of the test in the control efficacy experiment of Antagonistic Fungi of the present invention.
Embodiment
Technical scheme is described in further detail with reference to specific embodiment, but protection scope of the present invention is not
It is confined to as described below.
The pseudomonad CCTCC M2016556 of embodiment 1 separation and screening
Wild Fructus actinidiae chinensis branch of the pseudomonad CCTCC M2016556 bacterium sources of the present invention from Jinggangshan County, Jiangxi Province, sampled point
Height above sea level be 1414m, longitude and latitude be N26 ° 36 ' 30, E114 ° 07 ' 33；
Separation side is：1st, the Kiwi berry branch of collection is rinsed well with clear water, 75% alcohol surface sterilization, room temperature wind
It is dry, cut for sample sheet in right amount, put into grind bag with the blade after sterilization, add appropriate physiological saline, grinding, immersion
3min, takes the μ L of supernatant 100 of its plant mill liquid to add in the KB culture mediums newly prepared, is coated, rule with oese
Processing.
The method of purifying is：Sample culture dish will be connect to cultivate after certain time at room temperature, treat to grow bacterium colony in ware, picking side
Edge clearly bacterium colony, in purifying for 3 generations repeatedly on the KB solid medium flat boards newly prepared, the single bacterium colony until obtaining purifying will
Purified bacterial strain is numbered, and is then transferred into cryopreservation tube, is sealed up for safekeeping with glycerine, is positioned in -80 DEG C of ultra low temperature freezers and protects
Deposit strain.
The pseudomonad CCTCC M2016556 of embodiment 2 identification
1. cultivation conditions are observed
Strain will be preserved to activate on KB solid mediums, cultivated at room temperature, colony growth situation is observed, record bacterium colony
Flora feature, including color, the speed of growth, shape, quality etc..
It finally found that：Pseudomonad CCTCC M2016556 bacterial strains of the present invention are creamy white, and colloidal substance, surface is smooth wet
Profit, central protuberance, neat in edge, viscosity is normal；Its strain form is as shown in Figure 1；Its scanning electron microscope (SEM) photograph is as shown in Figure 2.
2. molecular biology identification
DNA is extracted：With bacteria PCR universal primer 27F, (primer sequence is：
Eubac27F [106] 5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (primer sequence is：
Eubac1492R [106] 5'-TACGGYTACCTTGTTACGACT-3') bacterial strain is subjected to 16SrDNA amplifications, obtain
Expanding fragment length be about 250bp, then carry out 16S r DNA sequencing, Molecular Identification result is as shown in sequence table；Survey
The sequence obtained carries out homology search in GenBank, and similarity analysis is carried out with sequence in database, finds 16S r DNA's
Sequencing results are just pseudomonad.
The screening of the Prospect on Kiwifruit Bacterial Canker Antagonistic Fungi of embodiment 3
3.1 test material
Hunan Province Dong Shanfeng, Jiangxi Province Jinggang Mountain and Sichuan Province's collection Kiwi berry sample it is separated go out Kiwi berry in it is raw
Bacterium.
For examination Prospect on Kiwifruit Bacterial Canker pathogen：The kiwi berry bacterial of separation identification is studied through Sichuan Province's science of natural resources
Pathogenic Fungus of Canker Pseudomonas syringae pv.Actinidiae, it is specific as shown in table 1：
Table 1 is for examination pathogen
| Numbering | For examination canker (Pseudomonas syringae pv.actinidiae.Psa) bacterial strain |
| Pc | psa2012DJY |
| P1 | psa2013DJY |
| P6 | psa2013PJ |
| 155 | psa2014EB |
| 185 | psa2014YJ |
| 206 | psa2014QL |
Raw Antagonistic Fungi screening in Prospect on Kiwifruit Bacterial Canker：
This experiment carries out the screening of raw Antagonistic Fungi in Prospect on Kiwifruit Bacterial Canker with Odontothrips loti, comprises the following steps that：
1. by Kiwi berry Antagonistic Fungi seed liquor in 10% ratio (V:V LB nutrient solutions) are mixed, 28 DEG C, 150r/min is put into
Constant-temperature table is placed in, is shaken three days；
2. drawing 100 microlitres of Pathogenic Fungus of Canker bacterium solutions with liquid-transfering gun, it is applied to spreading rod is flat on KB culture dishes, carries out mark
Note；
3. again on the culture medium of coating cause of disease, place Oxford cup；Each culture dish places three Oxford cups, and does three groups
Repeat；
4. drawing 200 microlitres of endophyte liquid with liquid-transfering gun again, add in Oxford cup；At room temperature after culture 5.2d, observation
Whether there is inhibition zone in flat board, and inhibition zone diameter；
5th, by Antagonistic Fungi to being divided into relatively strong (antibacterial circle diameter ＞ 20mm) for trying the inhibitory activity of canker bacterial strain, it is medium
(10.1~20mm) and weaker (0.1~10mm) 3 grades, are expressed as relatively strong (+++), medium (++), weaker (+).
KB solid medium constituents of the invention used are：Counted on the basis of 1L KB solid mediums, contain new albumen
Peptone 10.0g, glycerine 7.5g, MgSO4·7H2O 1.5g, K2HPO41.0g, agar 16.0g, H2O 1.0L。
LB fluid nutrient medium constituents are：Counted on the basis of 1L LB fluid nutrient mediums, contain new peptone 10.0g, third
Triol 7.5g, MgSO4·7H2O 1.5g, K2HPO41.0g, H2O 1.0L。
Antibacterial result is as shown in table 2 below：
The Antagonistic Fungi of table 2 is to the different inhibitions for trying canker bacterial strain
The interior preventive effect experiment of 3.2 Antagonistic Fungis and safety experiment
3.2.1 the fresh braches of red kiwi fruit, are chosen as vegetable material progress control efficacy experiment
The Antagonistic Fungi that pathogen and preliminary screening go out is connected to fresh KB solid medium flat boards, 24 are cultivated at room temperature small
When.The suspension containing 107cfu/ml is made with sterile saline dilution.The annual red kiwi fruit branch of clip, every is cut
Into 10cm trifles, 6 trifles (wherein 1 trifle connects physiological saline as control) are cut per branch, are put into after sodium hypochlorite surface sterilization
It is covered with moisturizing in the food box of filter paper standby；2 minor wounds are made at each trifle two ends with sterile file, wax liquor closes two sections.
Same branch, canker bacterial strain 155 (psa2014EB) is connected in the wound of 6 trifles, and remaining 1 trifle connects physiological saline work
For control；5 trifle wounds are randomly selected after 1 day and meet Antagonistic Fungi pseudomonad SPANRS-62.6 groups are set to repeat experiment, in 20
Placed at DEG C after 3 weeks, the epidermis of wound is gently scratched with sterilizing blade, observation ulcer scab and bacterial ooze overflow situation, with
Evaluate the biocontrol effect of Antagonistic Fungi.
Result of the test is as follows：Negative control group (experimental result is as shown in Figure 3), cut wound mouthful the inside is dried, no ulcer bacteria
Pyorrhea goes out (in figure at A)；Cut wound mouthful outside does not change colour, sterile pyorrhea goes out (in figure at B) without canker scab；
Positive controls (experimental result is as shown in Figure 4), cut wound mouthful the inside has bacterial ooze to overflow (in figure at A)；Cut wound mouthful is outer
There is canker scab in face, discoloration, has bacterial ooze to overflow (in figure at B)；
Experimental group (experimental result is as shown in Figure 5), cut wound mouthful the inside is dried, and sterile pyorrhea goes out (in figure at A)；Cut wound mouthful is outer
Face disease-free spot, does not change colour, and sterile pyorrhea goes out (in figure at B).
3.2.2 red kiwi fruit plant, which is chosen, as vegetable material carries out field efficiency test
Healthy plant is chosen in the orchard fallen ill in Prospect on Kiwifruit Bacterial Canker, and the winter is pruned after branch, the antagonism that preliminary screening is gone out
Bacterium is activated in the LB fluid nutrient mediums equipped with triangular flask, is put into and is placed in constant-temperature table, is shaken three days；Experimental group and control are set
The dilution of group, wherein experimental group sterile saline is made containing 107Cfu/ml suspension, uses atomizers spray kiwi fruit plant
Blade and limb；Control group sprays kiwi fruit plant blade and limb with commercialization agricultural chemicals " agricultural streptomycin " as medicament.Often
Sprayed once every surrounding, investigation statisticses are carried out in Second Year canker in the April high-incidence season.Statistical result is as shown in table 3 below：
The field efficiency test result of table 3
It can be seen that the pseudomonad CCTCC M2016556 incidences of disease are below agricultural streptomycin, table with disease index in table
The bright Antagonistic Fungi field efficacy effect is better than agricultural streptomycin, there is certain preventive effect effect to Prospect on Kiwifruit Bacterial Canker.
<110>Sichuan Natural Resources Research Institute
<120>A kind of pseudomonad strain for preventing and treating Prospect on Kiwifruit Bacterial Canker and its application
<160> 1
<210> 1
<211> 1405
<212> DNA
<213>Pseudomonad（Pseudomonas sp.）
<400> 1
ggtaccgtcc tcccgaaggt tagactagct acttctggtg caacccactc ccatggtgtg 60
acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg acattctgat tcgcgattac 120
tagcgattcc gacttcacgc agtcgagttg cagactgcga tccggactac gatcggtttt 180
atgggattag ctccacctcg cggcttggca accctctgta ccgaccattg tagcacgtgt 240
gtagcccagg ccgtaagggc catgatgact tgacgtcatc cccaccttcc tccggtttgt 300
caccggcagt ctccttagag tgcccaccat tacgtgctgg taactaagga caagggttgc 360
gctcgttacg ggacttaacc caacatctca cgacacgagc tgacgacagc catgcagcac 420
ctgtctcaat gttcccgaag gcaccaatcc atctctggaa agttcattgg atgtcaaggc 480
ctggtaaggt tcttcgcgtt gcttcgaatt aaaccacatg ctccaccgct tgtgcgggcc 540
cccgtcaatt catttgagtt ttaaccttgc ggccgtactc cccaggcggt caacttaatg 600
cgttagctgc gccactaaaa gctcaaggct tccaacggct agttgacatc gtttacggcg 660
tggactacca gggtatctaa tcctgtttgc tccccacgct ttcgcacctc agtgtcagta 720
ttagtccagg tggtcgcctt cgccactggt gttccttcct atatctacgc atttcaccgc 780
tacacaggaa attccaccac cctctaccat actctagtca gtcagttttg aatgcagttc 840
ccaggttgag cccggggatt tcacatccaa cttaacaaac cacctacgcg cgctttacgc 900
ccagtaattc cgattaacgc ttgcaccctc tgtattaccg cggctgctgg cacagagtta 960
gccggtgctt attctgtcgg taacgtcaaa acaatcacgt attaggtaac tgcccttcct 1020
cccaacttaa agtgctttac aatccgaaga ccttcttcac acacgcggca tggctggatc 1080
aggctttcgc ccattgtcca atattcccca ctgctgcctc ccgtaggagt ctggaccgtg 1140
tctcagttcc agtgtgactg atcatcctct cagaccagtt acggatcgtc gccttggtga 1200
gccattacct caccaactag ctaatccgac ctaggctcat ctgatagcgc aaggcccgaa 1260
ggtcccctgc tttctcccgt aggacgtatg cggtattagc gttcgtttcc gaacgttatc 1320
ccccactacc aggcagattc ctaggcatta ctcacccgtc cgccgctctc aagagaagca 1380
agcttctctc taccgctcga ctgca 1405
Claims (8)
1. a kind of pseudomonad strain for preventing and treating Prospect on Kiwifruit Bacterial Canker, it is characterised in that the pseudomonad（Pseudomonas
sp.）Entitled pseudomonad SPANRS-62, bacterial strain is preserved in China typical culture collection center, and deposit number is CCTCC
NO：M2016556.
2. a kind of microbial bacterial agent, it is characterised in that the preventing and treating Kiwi berry that the microbial bacterial agent contains described in claim 1 is burst
The pseudomonad strain of ulcer disease.
3. application of the pseudomonad strain in preventing and treating Prospect on Kiwifruit Bacterial Canker described in claim 1.
4. application according to claim 3, it is characterised in that the pathogenic bacteria of the Prospect on Kiwifruit Bacterial Canker are Kiwi berry bacterium
Property Pathogenic Fungus of Canker（Pseudomonas syringae pv. Actinidiae）.
5. the filtered fluid of the culture of the pseudomonad strain described in claim 1, fermentation culture, fermentation culture.
6. the filtered fluid of culture according to claim 5, fermentation culture, fermentation culture is in preventing and treating Kiwi berry ulcer
Application in disease.
7. application according to claim 6, it is characterised in that including the pseudomonad strain in claim 1 is used
KB solid mediums are cultivated, and obtain the filtered fluid of culture, fermentation culture, fermentation culture；With resulting vacation list
Culture, fermentation culture, the filtered fluid of fermentation culture of born of the same parents' bacteria strain suppress the growth of the pathogenic bacteria of Prospect on Kiwifruit Bacterial Canker.
8. application according to claim 7, it is characterised in that the pathogenic bacteria of the Prospect on Kiwifruit Bacterial Canker are Kiwi berry bacterium
Property Pathogenic Fungus of Canker（Pseudomonas syringae pv. Actinidiae）.
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| CN109136132A (en) * | 2018-08-27 | 2019-01-04 | 田璨熙 | One pseudomonas Pseudomonas sp.KXJ-B7 and its cultural method and application |
| CN111197016A (en) * | 2020-01-10 | 2020-05-26 | 渭南职业技术学院 | Screening method of kiwifruit canker antagonistic strain |
| CN111280183A (en) * | 2020-02-18 | 2020-06-16 | 青岛农业大学 | Apple anthracnose leaf blight biocontrol microbial inoculum and preparation method and application thereof |
| CN112322516A (en) * | 2020-05-19 | 2021-02-05 | 陕西省生物农业研究所 | Preparation method and application of novel biocontrol microbial inoculum for kiwi fruit leaf blight |
| WO2022099837A1 (en) * | 2020-11-14 | 2022-05-19 | 菲吉乐科(南京)生物科技有限公司 | Pseudomonas syringae phage and composition thereof, test kit and use thereof |
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| CN111280183A (en) * | 2020-02-18 | 2020-06-16 | 青岛农业大学 | Apple anthracnose leaf blight biocontrol microbial inoculum and preparation method and application thereof |
| CN111280183B (en) * | 2020-02-18 | 2021-08-03 | 青岛农业大学 | Apple anthracnose leaf blight biocontrol microbial inoculum and preparation method and application thereof |
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| CN117946948B (en) * | 2024-03-22 | 2024-06-07 | 四川省自然资源科学研究院(四川省生产力促进中心) | Pseudomonas strain SCMHT-110 and application thereof |
| CN117965405A (en) * | 2024-04-02 | 2024-05-03 | 西北农林科技大学深圳研究院 | Pseudomonas eastern lake and application thereof in plant disease control |
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