CN107034167A - A kind of lysine bacillus for preventing and treating Prospect on Kiwifruit Bacterial Canker and its application - Google Patents
A kind of lysine bacillus 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 lysine bacillus of preventing and treating Prospect on Kiwifruit Bacterial Canker and its application.Lysine bacillus deposit number of the present invention is CCTCC NO：M2016557.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.The strain isolations of SPANRS 213 of the present invention are from health plant tissue; the active metabolite of generation does not have obvious toxic action to plant tissue; and property is stable; prepare simple; 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 lysine bacillus of preventing and treating Prospect on Kiwifruit Bacterial Canker
And its application.
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
The biological pesticide for sending out new and effective is changed, is allowed to as agricultural production service, by with important theory significance and application value.
The content of the invention
The present invention provides a kind of new lysine bacillus for poisoning low preventing and treating Prospect on Kiwifruit Bacterial Canker excellent effect.
The lysine bacillus of present invention preventing and treating Prospect on Kiwifruit Bacterial Canker, the entitled lysine of lysine bacillus
Bacillus SPANRS-213 (Lysinibacillus sp.SPANRS-213), the bacterial strain is preserved in Chinese Typical Representative culture guarantor
Tibetan center (abbreviation CCTCC), deposit number is CCTCC NO：M2016557 (referred to as lysine bacillus CCTCC
M2016557), the address of depositary institution is located at Wuhan, China university, and preservation date is on October 11st, 2016.
Lysine bacillus CCTCC M2016557 bacterial strains of the present invention are creamy white, colloidal substance, the smooth moistening in surface, in
Cardiac prominence rises, and neat in edge, viscosity is normal.
The lysine bacillus CCTCC M2016557 of the present invention are in Sichuan Province Shifang City Hong Bai towns (north latitude
31.36 °, 104.03 ° of west longitude, 861 meters of height above sea level) kiwi fruit plant is isolated, in May, 2015 in sampling time, its bolter
Choosing 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, until obtaining single bacterium colony, is then transferred into cryopreservation tube, with sweet repeatedly on the KB culture medium flat plates newly prepared
Oil sealing is deposited, and 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 the microbial bacterial agent includes the bad ammonia of above-mentioned preventing and treating Prospect on Kiwifruit Bacterial Canker
Sour bacillus, 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 lysine bacillus in preventing and treating Prospect on Kiwifruit Bacterial Canker.
Further, application of the above-mentioned lysine bacillus in preventing and treating Prospect on Kiwifruit Bacterial Canker, wherein the Kiwi berry
The pathogenic bacteria of canker are kiwi berry bacterial canker pathogen (Pseudomonas syringae pv.Actinidiae).
The present invention also provides a kind of culture of lysine bacillus, fermentation culture, the filtered fluid of fermentation culture.
The present invention also provides a kind of culture of lysine bacillus, fermentation culture, the filtered fluid of fermentation culture
Application in preventing and treating Prospect on Kiwifruit Bacterial Canker.
Further, the filtered fluid of the culture of above-mentioned lysine bacillus, fermentation culture, fermentation culture is anti-
The application in Prospect on Kiwifruit Bacterial Canker is controlled, it includes being cultivated above-mentioned lysine bacillus using KB solid mediums, obtains
To the filtered fluid of culture, fermentation culture and fermentation culture；With the culture of resulting lysine bacillus, hair
Ferment nutrient solution, the filtered fluid of fermentation culture suppress the growth of the pathogenic bacteria of Prospect on Kiwifruit Bacterial Canker.
Further, the filtered fluid of the culture of above-mentioned lysine bacillus, fermentation culture, fermentation culture is anti-
The application in Prospect on Kiwifruit Bacterial Canker is controlled, 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 lysine bacillus are preventing and treating Kiwi berry
Application in canker, the KB solid mediums constituent is：Counted on the basis of 1L KB nutrient solutions, contain new peptone
10.0g, glycerine 7.5g, MgSO4·7H2O 1.5g, K2HPO41.0g, agar 16.0g, H2O 1.0L；
The LB fluid nutrient mediums constituent is：Counted on the basis of 1L LB nutrient solutions, contain new peptone 10.0g, third
Triol 7.5g, MgSO4·7H2O 1.5g, K2HPO41.0g, H2O 1.0L。
Lysine bacillus CCTCC M2016557 of the present invention isolate and purify what is obtained from the Kiwi berry branch of collection, point
Simple from screening technique, 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 mycelia.The strain isolation is from health plant tissue, thus the activity produced
Metabolite does not have obvious toxic action to plant tissue, and property is stable, squirts conveniently, and preventing and treating Kiwi berry ulcer effect shows
Write, provide an environmental protection, simple, effective approach for the preventing and treating of Kiwi berry ulcer, utilize environmental protection.
Brief description of the drawings
Fig. 1 is lysine bacillus CCTCC M2016557 strain morphology figures of the present invention；
Fig. 2 is lysine bacillus CCTCC M2016557 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 lysine bacillus CCTCC M2016557 of embodiment 1 separation and screening
Lysine bacillus CCTCC M2016557 bacterium sources of the present invention come from Sichuan Province Shifang City Hong Bai towns (north latitude
31.36 °, 104.03 ° of west longitude, 861 meters of height above sea level), in May, 2015 in sampling time.
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 nutrient solution flat boards newly prepared, the single bacterium colony until obtaining purifying will be pure
The bacterial strain of change 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 preserves bacterium
Kind.
The lysine bacillus CCTCC M2016557 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：Lysine bacillus CCTCC M2016557 bacterial strains of the present invention are creamy white, colloidal substance, surface light
Sliding moistening, central protuberance, neat in edge, viscosity is normal；Its strain form is as shown in Figure 1；Its scanning electron microscope (SEM) photograph such as sequence table institute
Show.
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 lysine bacillus.
The screening of the Prospect on Kiwifruit Bacterial Canker Antagonistic Fungi of embodiment 3
3.1.1 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 |
| 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.Comprise the following steps that：
1. by Antagonistic Fungi seed liquor in 10% ratio (V:V LB nutrient solutions) are mixed, 28 DEG C, 150r/min is put into and is placed in perseverance
Warm shaking table, shakes 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；
5. whether there is inhibition zone in observation flat board after culture 2d at room temperature, and inhibition zone diameter；
6th, 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 nutrient solutions, contain new 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 1LLB nutrient solutions, contain new peptone 10.0g, glycerine
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 nutrient solutions equipped with triangular flask, is put into and is placed in constant-temperature table, is shaken three days；Experimental group and control group are set,
Wherein experimental group sterile saline dilution is made containing 107Cfu/ml suspension, with atomizers spray kiwi fruit plant leaf
Piece and limb；Control group sprays kiwi fruit plant blade and limb with commercialization agricultural chemicals " agricultural streptomycin " as medicament.Every
Surrounding is sprayed once, and 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 lysine bacillus CCTCC M2016557 incidences of disease are below agricultural chain with disease index in table
Mycin, shows that the Antagonistic Fungi field efficacy effect is better than agricultural streptomycin, there is certain preventive effect effect to Prospect on Kiwifruit Bacterial Canker.
Described above is only the preferred embodiment of the present invention, it should be understood that the present invention is not limited to described herein
Form, is not to be taken as the exclusion to other embodiment, and available for various other combinations, modification and environment, and can be at this
In the text contemplated scope, it is modified by the technology or knowledge of above-mentioned teaching or association area.And those skilled in the art are entered
Capable change and change does not depart from the spirit and scope of the present invention, then all should appended claims of the present invention protection domain
It is interior.
<110>Sichuan Natural Resources Research Institute
<120>A kind of lysine bacillus for preventing and treating Prospect on Kiwifruit Bacterial Canker and its application
<160> 1
<210> 1
<211> 1411
<212> DNA
<213>Lysine bacillus（Lysinibacillus sp.）
<400> 1
ctggctccaa aggttacctc accgacttcg ggtgttaaaa ctctcgtggt gtgacgggcg 60
gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct gatccgcgat tactagcgat 120
tccggcttca tgtaggcgag ttgcagccta caatccgaac tgagaacgac tttatcggat 180
tagctccctc tcgcgagttg gcaaccgttt gtatcgtcca ttgtagcacg tgtgtagccc 240
aggtcataag gggcatgatg atttgacgtc atccccacct tcctccggtt tgtcaccggc 300
agtcacctta gagtgcccaa ctaaatgatg gcaactaaga tcaagggttg cgctcgttgc 360
gggacttaac ccaacatctc acgacacgag ctgacgacaa ccatgcacca cctgtcaccg 420
ttgcccccga aggggaaact atatctctac agtggtcaac gggatgtcaa gacctggtaa 480
ggttcttcgc gttgcttcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca 540
attcctttga gtttcagtct tgcgaccgta ctccccaggc ggagtgctta atgcgttagc 600
tgcagcacta aggggcggaa accccctaac acttagcact catcgtttac ggcgtggact 660
accagggtat ctaatcctgt ttgctcccca cgctttcgcg cctcagtgtc agttacagac 720
cagatagtcg ccttcgccac tggtgttcct ccaaatctct acgcatttca ccgctacact 780
tggaattcca ctatcctctt ctgcactcaa gtctcccagt ttccaatgac cctccacggt 840
tgagccgtgg gctttcacat cagacttaag aaaccacctg cgcgcgcttt acgcccaata 900
attccggaca acgcttgcca cctacgtatt accgcggctg ctggcacgta gttagccgtg 960
gctttctaat aaggtaccgt caaggtacag ccagttacta ctgtacttgt tcttccctta 1020
caacagagtt ttacgaaccg aaatccttct tcactcacgc ggcgttgctc catcaggctt 1080
tcgcccattg tggaagattc cctactgctg cctcccgtag gagtctgggc cgtgtctcag 1140
tcccagtgtg gccgatcacc ctctcaggtc ggctacgcat cgtcgccttg gtgagccgtt 1200
acctcaccaa ctagctaatg cgccgcgggc ccatcctata gcgacagccg aaaccgtctt 1260
tcagtgtttc accatgaggt gaaacagatt attcggtatt agccccggtt tcccggagtt 1320
atcccaaact ataaggtagg ttgcccacgt gttactcacc cgtccgccgc taacgtcgaa 1380
ggagcaagct ccttctctgt tcgctcgact g 1411
Claims (8)
1. a kind of lysine bacillus for preventing and treating Prospect on Kiwifruit Bacterial Canker, it is characterised in that the lysine bacillus title
For lysine bacillus SPANRS-213（Lysinibacillus sp. SPANRS-213）, the bacterial strain is preserved in Chinese allusion quotation
Type culture collection, deposit number is CCTCC NO：M2016557.
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 lysine bacillus of ulcer disease.
3. application of the lysine bacillus 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 culture of the lysine bacillus described in claim 1, fermentation culture, the filtered fluid of 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 lysine bacillus in claim 1 is adopted
Cultivated with KB solid mediums, obtain the filtered fluid of culture, fermentation culture, fermentation culture；Relied with resulting
Culture, fermentation culture, the filtered fluid of fermentation culture of propylhomoserin bacillus suppress the pathogenic bacteria of Prospect on Kiwifruit Bacterial Canker
Growth.
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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| CN108841754A (en) * | 2018-07-09 | 2018-11-20 | 东北农业大学 | The fermentation culture method of biocontrol bacteria bacillus WXCDD105 |
| CN109136132A (en) * | 2018-08-27 | 2019-01-04 | 田璨熙 | One pseudomonas Pseudomonas sp.KXJ-B7 and its cultural method and application |
| CN113925064A (en) * | 2021-11-01 | 2022-01-14 | 海南大学 | Application of B-lysine-resistant bacillus in inhibition of growth of pitaya soft rot germs |
| WO2023005129A1 (en) * | 2021-07-29 | 2023-02-02 | 海南大学 | Fungicide for dragon fruit canker and use thereof |
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| CN113925064A (en) * | 2021-11-01 | 2022-01-14 | 海南大学 | Application of B-lysine-resistant bacillus in inhibition of growth of pitaya soft rot germs |
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