CN109234193A - Rahnella aquatilis ZF7 and its application in plant growth-promoting diseases prevention - Google Patents
Rahnella aquatilis ZF7 and its application in plant growth-promoting diseases prevention Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses rahnella aquatilis ZF7 and its applications in plant growth-promoting diseases prevention.The present invention provides rahnella aquatilis (Rahnella aquatilis) ZF7, deposit number is CGMCC NO.16014.The application of above-mentioned rahnella aquatilis (Rahnella aquatilis) ZF7 or its culture solution or its bacteria suspension or its tunning in antibacterial is also the scope of protection of the invention.Prevention and treatment of this test result for rahnella aquatilis for root knot disease provides theoretical foundation, the the isolating and purifying of the bacterial strain antibacterial material, antibacterial Mechanism of disease control and field efficacy will be studied, further from now on to which the bacterial strain is preferably applied for biological control.Bacterial strain ZF7 has preferable environmental stability, strong in the colonization ability of sunflower and Chinese cabbage rhizosphere soil, shows that bacterial strain ZF7 has preferable biological and ecological methods to prevent plant disease, pests, and erosion application prospect.
Description
Technical field
The invention belongs to field of biotechnology more particularly to a kind of rahnella aquatilis ZF7 and its in plant growth-promoting diseases prevention
In application.
Background technique
In plant root, when plant growth-promoting rhizobacteria (Plant-Growth-Promoting Rhizobacteria,
When PGPR) with host's interaction, PGPR is colonized with highly dense group and is promoted plant growth.As surviving in around crop root
Spontaneous type plant beneficial bacteria, PGPR can not only promote plant growth, and have the function of controlling disease, and can be can
Sustainable agriculture development provides new approach.PGPR promotes plant growth to be typically divided into two kinds of differences of direct growth-promoting and indirect growth-promoting
Mode.Direct Promoting bacteria can provide the short nutriment of plant directly to promote the growth and metabolism of plant, such as generate plant
Object hormone (auxin, gibberellin, the basic element of cell division and ethylene etc.), fixed nitrogen, Soluble phosphorus, vitamin and siderophore generation etc..Indirectly
Promoting bacteria then passes through the harmful plant pathogenic microorganisms of prevention and infects, and induces the defense response of plant, enhances the health of plant, from
And promote plant growth indirectly.
Rahnella aquatilis has played important function in terms of plant growth-promoting diseases prevention as one of plant growth-promoting bacteria.Li Gui
Pretty young woman etc. separate from the rhizosphere soil of masson pine obtains the aquatic drawing engler Rahnella aquatilis of 1 plant of rhizosphere growth-promoting endophytic bacteria
JZ-GX1, and find that the bacterium shows significant growth-promoting functions to pine tree and poplar.Research also found bacterial strain JZ-GX1 and its lure
Becoming bacterial strain JZ-GX1-2 and JZ-GX1-10 can effectively degrade phytate, have growth-promoting functions to masson pine seedling.The hair such as Song Fangxu
Existing water draws engler bacterium JZ-GX1 thallus shell capsule spore Cytospora chrysosperma golden yellow to dothiorella gregaria bacterium and quasi- stem point
Mould Phomopsis sp. has apparent fungistatic effect.The discovery such as Hoda H.El-Hendawy is processed through rahnella aquatilis
Tomato seeds the sensibility of xanthomonas campestris is reduced, while the effect of growth-promoting can be played.The discovery such as Jiao Ziwei is aquatic
Draw engler bacterium HX2 that can synthesize pyrroloquinoline quinone (PQQ) and heteroauxin (IAA), these substances can promote the increasing of Corn Biomass
Add, improves the intake of corn nourishment.Liang Zhihong etc. has found that agrobacterium E26 has good preventive effect, while table to Isolated From Grapevine
The generation of detail rhzomorph is a more stable character.Rahnella aquatilis HX 2 is to Agrobacterium tumefaciens Agrobacterium
Tumefaciens, rhizobiaceae A.rhizogenes, grape agrobacterium A.vitis and rice leaf spot bacteria
Xanthomonas oryzae pv.oryzae, cotton bacterial angular leaf spot bacterium Xanthomonas campestris
21 kinds of pathogens such as pv.malvacearum have wide spectrum inhibitory effect, especially have good preventive effect to root knot.Bacterial strain
HX2 thallus and supernatant have different degrees of inhibiting effect to the formation of crown gall nodule, but the preventive effect of fermented liquid supernatant liquid is better than
The preventive effect of thallus.
Summary of the invention
It is an object of the present invention to provide rahnella aquatilis (Rahnella aquatilis) ZF7.
Rahnella aquatilis (Rahnella aquatilis) ZF7, deposit number CGMCC provided by the invention
NO.16014。
Above-mentioned rahnella aquatilis (Rahnella aquatilis) ZF7 or its culture solution or its bacteria suspension or its fermentation
Application of the product in antibacterial is also the scope of protection of the invention.
Above-mentioned rahnella aquatilis (Rahnella aquatilis) ZF7 or its culture solution or its bacteria suspension or its fermentation
Application of the product in preparation plant biological prevention and control agent is also the scope of protection of the invention.
Above-mentioned rahnella aquatilis (Rahnella aquatilis) ZF7 or its culture solution or its bacteria suspension or its fermentation
Application of the product in controlling plant diseases is also the scope of protection of the invention.
Above-mentioned rahnella aquatilis (Rahnella aquatilis) ZF7 or its culture solution or its bacteria suspension or its fermentation
Application of the product in prevention and treatment plant root cancer is also the scope of protection of the invention.
Above-mentioned rahnella aquatilis (Rahnella aquatilis) ZF7 or its culture solution or its bacteria suspension or its fermentation
Product is also the scope of protection of the invention promoting the application in plant growth.
In above-mentioned application, the disease is the disease that pathogen causes；
The pathogen or the antibacterial bacterium are fungi or bacterium；
And/or the fungi is early blight of tomato Alternaria solani, Tomato gray leaf spot bacterium Stemphylium
Solani Weber, P. capsici Phytophthora capsici, cucumber stick spore leaf spot fungi Corynespora
At least one of cassiicola and Rhizoctonia solani Kuhn Rhizoctonia solani；
And/or the bacterium is bacterial canker of tomato Clavibacter michiganensis
Subsp.michiganensis, Tomato mottle germ Pseudomonas syringae pv.tomato, cauliflower black rot
Xanthomonas campestris pv.campestris, avenae subsp.citrull Pseudomonas syringae
Pv.lachrymans, Cucumis sativus stem Bacteria erwinia Pectobacterium carotovorum subsp.brasiliense, grape
At least one of crown gall germ Agrobacterium vitis.
Above-mentioned rahnella aquatilis (Rahnella aquatilis) ZF7 or its culture solution or its bacteria suspension or its fermentation
Application of the product in degradation phosphorus (Phos) is also the scope of protection of the invention；
Or, above-mentioned rahnella aquatilis (Rahnella aquatilis) ZF7 or its culture solution or its bacteria suspension or its
Tunning is also the scope of protection of the invention preparing the application in IAA.
Another object of the present invention is to provide a kind of method for preventing and treating plant root cancer.
Method provided by the invention, for by above-mentioned rahnella aquatilis (Rahnella aquatilis) ZF7 or its training
Nutrient solution or its bacteria suspension or its tunning act on plant, realize prevention and treatment plant root cancer.
3rd purpose of the invention is to provide a kind of method for promoting plant growth.
Method provided by the invention, for by above-mentioned rahnella aquatilis (Rahnella aquatilis) ZF7 or its training
Nutrient solution or its bacteria suspension or its tunning act on plant, realize and promote plant growth.
The experiment proves that rahnella aquatilis ZF7 is from isolated one plant of oriental cherry rhizosphere soil to crown gall
Disease has good antagonistic effect, while having the biocontrol bacterial strain of growth-promoting functions to plant.The bacterial strain can Soluble phosphorus, produce hormone, from
And show the growth-promoting effect to Chinese cabbage.There is good preventive effect to Crown gall simultaneously.
Plant growth-promoting rhizobacteria plays growth-promoting effect by approach such as minerals dissolution, auximone release, nitrogen fixation
Fruit.Some researches show that the main mechanism of phosphorus-solubilizing bacteria dissolution mineral phosphorus is that direct oxidation reaction is occurred outside cytoplasma membrane to generate
Organic acid, and with the decline of pH value, lead to the dissolution of phosphorus.It is good molten that this research is found by experiment that bacterial strain ZF7 has
Phosphorus effect, Soluble phosphorus diameter is up to 28.21.The pH value variation for having detected bacterial strain ZF7 in 40h simultaneously, as the time increases, bacterial strain ZF7
PH value gradually decrease, in 16h, reach minimum: 3.59.IAA stimulates plant cell hyperplasia and enlarged-area, secrets out of rush
Into the nutriment of bacterial growth.The study find that bacterial strain reaches the length of seedling overground part and the growth-promoting rate of root long respectively
56.47%, 54.74%, have good to the growth-promoting rate of the weight of Chinese cabbage overground part and root weight up to 233.84% and 164.71%
Good growth-promoting effect, this is almost the same with the growth-promoting result of study of reported rahnella aquatilis.This research is it has also been found that bacterial strain
ZF7 has good preventive effect to Crown gall, and the control efficiency to sunflower root knot is 94.52%, does not have with bacterial strain HX2
There is significant difference.Meanwhile bacterial strain ZF7 shows very strong inhibition to 10 kinds of facilities vegetable diseases such as cucumber bacterial angular leaf spot
Effect, certain efficient antibacterial substances can be generated by showing bacterial strain ZF7 also.But it need to also be into one about antibacterial substance and type
Step analysis.
Prevention and treatment of this test result for rahnella aquatilis for root knot disease provides theoretical foundation, from now on will be into one
Step studies the isolating and purifying of the bacterial strain antibacterial material, antibacterial Mechanism of disease control and field efficacy, to by the bacterial strain preferably
Applied to biological control.Bacterial strain ZF7 has preferable environmental stability, colonizes energy in sunflower and Chinese cabbage rhizosphere soil
Power is strong, shows that bacterial strain ZF7 has preferable biological and ecological methods to prevent plant disease, pests, and erosion application prospect.
Preservation explanation
Strain name: rahnella aquatilis
Latin name: Rahnella aquatilis
Strain number: ZF7
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism abbreviation: CGMCC
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date: on June 28th, 2018
Collection is registered on the books number: CGMCC No.16014
Detailed description of the invention
Fig. 1 is systematic evolution tree of the bacterial strain ZF7 based on 16s rDNA sequence.
The growth rate (A) and pH that Fig. 2 is bacterial strain ZF7 change (B).
Fig. 3 is bacterial strain ZF7 to various pathogenic bacteria antibacterial situation.
Fig. 4 is the Soluble phosphorus circle of bacterial strain ZF7 (A), HX2 (B) on NBRIP culture medium.
Fig. 5 be bacterial strain ZF7, HX2 in DF+, DF culture medium with solution S olution I, II reacting phenomenon of Solution.
Fig. 6 is bacterial strain ZF7, HX2 and IAA not growth-promoting effect of the same amount to Chinese cabbage.
Fig. 7 is the growth-promoting effect of bacterial strain ZF7, HX2 and IAA to Chinese cabbage.
Fig. 8 is that the greenhouse bacterial strain ZF, HX2 prevents and treats Crown gall effect detection.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Rahnella aquatilis HX 2 is provided by College of Resources and Environmental Sciences, China Agricultural University associate professor Guo Yanbin.
In document, " Jiao Ziwei, position be gentle, Li Lei, Zhang Xiang for rahnella aquatilis (Rahnella aquatilis) HX2 bacterial strain
The refined .PQQ and IAA growth-promoting factors of cutting edge of a knife or a sword, Guo Yan are to Rahnella aquatilis HX2 bacterial strain growth-promoting mechanism Xinjiang Agricultural Sciences
2017,54 (5): disclosing in 907-917. ", and the public can obtain from College of Resources and Environmental Sciences, China Agricultural University；It can also be from application
People obtains.
Sunflower (kind Ba Kui 118) purchased from Gansu Xian Nong International Agriculture Development Co., Ltd, Chinese cabbage (new No. 3 of Beijing) by
Vegetable & Flower Inst., Chinese Academy of Agriculture Science provides.
It is as follows for examination culture medium in following embodiments:
Tryptone fluid nutrient medium (LB): yeast powder 5g, tryptone 10g, NaCl 10g, distilled water 1000mL.Pancreas
Peptone solid medium (LB): yeast powder 5g, tryptone 10g, NaCl 10g, agar 15g, distilled water 1000mL.Ma Ling
Potato dextrose culture-medium (PDA): potato 200g, glucose 20g, agar 15g, distilled water 1000mL.Water agar (WA):
Agar 5g, distilled water 1000mL.Dispense 5mL test tube.Peptone powdered beef fluid nutrient medium (NB): peptone 10g, powdered beef
3g, NaCl 5g, distilled water 1000mL, pH7.0.Peptone powdered beef culture medium (NA): peptone 10g, powdered beef 3g, NaCl
5g, agar 15g, distilled water 1000mL, pH7.0.Agrobacterium selective medium (MW): mannitol 10g, biotin 100ug,
0.1% crystal violet 2mL, 0.1%Fe-EDTA 2mL, sodium nitrate (NaNO3) 5g, dipotassium hydrogen phosphate (K2HPO4) 0.3g, NaCl
0.2g, magnesium sulfate (MgSO4·7H2O) 0.1g, agar 12g, distilled water 1000mL, pH7.0-7.2.DF(Dworkin and
Foster) minimal medium (1L): peptone 5.0g, yeast extract 1.5g, beef extract 1.5g, NaCl 5.0g, pH 7.0.
NBRIP solid medium: glucose 10g, tricalcium phosphate (Ca3(PO4)2) 5g, magnesium chloride (MgCl2·7H2O) 5g, magnesium sulfate
(MgSO4·7H2O) 0.25g, potassium chloride (KCl) 0.2g, ammonium sulfate ((NH4)2SO4) 0.1g, agar 15g, distilled water 1000mL,
pH 7.0。
Embodiment 1, bacterial strain ZF7 separation, purifying and identification
1, bacterial strain ZF7 is separated
The strain isolation is from oriental cherry rhizosphere soil, and after acquiring soil sample, using plate dilution method, 99mL is added in 1g soil,
In distilled water, soil suspension is prepared, is coated on MW culture medium, 28 DEG C of cultures are for 24 hours.It is pure that picking single bacterium falls within MW plate streaking
Change, 28 DEG C of culture 48h, 4 DEG C of refrigerators save backup.
2, the identification of bacterial strain ZF7
The bacterial strain ZF7 of above-mentioned screening is identified as follows:
1) bacterial strain ZF7 morphologic observation and physiological property measurement
By isolated strains streak inoculation in LB culture medium, 28 DEG C of culture 48h observe the colonial morphology of the bacterial strain.According to " primary
Outstanding Bacteria Identification handbook " the 8th edition and " common bacteria system identification handbook ", after 28 DEG C of 24~48h of culture, to Physiology and biochemistry spy
Sign is by being identified.
2) Biolog microorganism automatic identifying system is identified
It is utilized using BIOLOG GENIII kit (being operated according to kit specification) measurement sole carbon source.All instrument
Device consumptive material is all Biolog Products.
Bacterial strain ZF7 is creamy white on LB culture medium, is translucent, and periphery of bacterial colonies is wet, in older bacterium colony (after 10d)
The heart is in light red.Thallus takes on a red color after Gram's staining, is negative bacterium, and elongated rod shape, major diameter is about 4 μm, and minor axis is about 1 μm, and two
It holds rounded.The biochemical reactions (table 1) of bacterial strain ZF7 are detected according to " common bacteria system identification handbook ".Bacterial strain ZF7 is leather
Lan Shi negative bacillus, glucose fermentation produce acid and produce gas, and oxidase negative contacts enzyme positive, are typical Enterobacteriaceae strains.Bacterium
Strain ZF7 bacterial strain can be using 45 kinds of carbon sources such as N- acetyl-D-glucose amine, Beta-methyl-D-Glucose glycosides, D- cellobioses, no
44 kinds of carbon sources such as N- acetyl group-D galactosamine, erythritol, xylitol can be utilized.
The physiological and biochemical property of 1 bacterial strain ZF7 of table
3, Molecular Identification
The DNA that bacterial strain ZF7 is extracted using DNA of bacteria kit, as the template DNA of PCR reaction, primer is using thin
Bacterium 16S rDNA universal primer: upstream primer PrimerA:5'-AGAGTTTGATCCTGGCTCAG-3'；Downstream primer Primer
B:5'-AAGGAGGTGATCCAGCCGCA-3'.PCR reaction condition: 95 DEG C of initial denaturation 10min；95 DEG C of denaturation 30s, 55 DEG C of primers
Renaturation 30s, 72 DEG C of extension 45s, 34 circulations；Last 72 DEG C of extensions 8min.PCR product is detected with 1% agarose gel electrophoresis,
Using DNA Marker BM5000 as control test molecular weight of product.
The 16S rDNA purpose band of 1 1376bp is obtained, by sequencing, the nucleotides sequence of 16S rDNA is classified as sequence
1。
Chadogram between bacterial strain ZF7 and other close bacterial strains is constructed using the maximum likelihood method in 6.0 software of MEGA,
16S rDNA identification mark and phylogenetic tree (Fig. 1), discovery bacterial strain ZF7 and rahnella aquatilis Rahnella aquatilis
Affinity reaches 100%.Combining form observation of characteristics and eco-physiological characteristics analysis, it is basic to determine that bacterial strain ZF7 is enterobacteria
Engler Pseudomonas rahnella aquatilis R.aquatilis draws in section.
Strain ZF7 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms on June 28th, 2018
Center, deposit number are CGMCC NO.16014, and classification naming is rahnella aquatilis.
4, bacterial strain ZF7 growth rate and pH value measurement
Bacterial strain ZF7 is seeded in liquid PD culture medium, shaken cultivation 16h at 28 DEG C.The bacteria suspension for cultivating 16h is added
In PD culture medium, unified initial OD values are 0.8 (OD600), 3 repetitions are set altogether, place 28 DEG C of shaking table cultures.It was surveyed every 4 hours
1 time, 11 points are surveyed altogether, and record measures OD value and pH value every time.
Bacterial strain ZF7 growth curve and pH change curve are as shown in Figure 2.Bacterial strain ZF7 is in Exponential growth stage, 12h before 12h
When absorbance reach 0.64, for 24 hours afterwards enter the steady growth phase.PH value is gradually decreased as the time increases, and in 16h, pH value reaches
3.58, and keep stable.
5, bacterial strain Z7 antibiotic measures
1) different antibiotic resistance measurements
By ampicillin (Ampicillin), carbenicillin (Carbenicillin), vancomycin
(Vancomycin), chloramphenicol (Chloramphenicol), gentamicin (Gentamicin), tetracycline
(Tetracycline), spectinomycin (Spectinomycin), kanamycins (Kanamycin), streptomysin
(Streptomycin) 9 kinds of antibiotic are in the LB liquid medium of 0.1% ratio access 4mL.Picking bacterial strain ZF7 single colonie,
It is inoculated into LB liquid medium of the 4mL added with different antibiotic, 28 DEG C are swayed culture for 24 hours.
2) various concentration ampicillin resistant measures
It is 50,100,150,200,250,300,350,400 μ gml by concentration-1Ampicillin with 0.1% ratio
It accesses in the LB liquid medium of 4mL.Picking bacterial strain ZF7 single colonie is inoculated into 4mL added with the ampicillin of various concentration
In LB liquid medium, 28 DEG C are swayed culture for 24 hours.
Bacterial strain ZF7 can be resistant to ampicillin, carbenicillin and vancomycin；Highest can be resistant to 250 μ g
ml-1Ampicillin.Measurement result is fitted curve, and lethal final concentration (EC is calculated50) it is 238.58 μ g
ml-1。
The functional study of embodiment 2, bacterial strain ZF7
One, application of the bacterial strain ZF7 in antibacterial
1, the measurement of bacterial strain ZF7 fungi antimicrobial spectrum
Using tablet face-off method, fungal target bacterium piece selected by 5mm is inoculated with respectively at 90mm PDA plate center, 25 DEG C
Lower culture 2d.Bacterial strain ZF7 is seeded in LB liquid medium, shaken cultivation 16h at 28 DEG C.Apart from culture dish edge 10mm
Locate opposite 4 points to be punched with 10 μ L, 5 μ LZF7 bacteria suspensions are added wherein, to be inoculated with LB liquid medium as blank control, 25
5d is cultivated at DEG C.When blank control will cover with entire culture dish, the control increment (colony radius) of measurement target drone bacterium and
It handles increment (the growth radius after inoculated bacteria), is indicated with bacteriostasis rate.
Bacteriostasis rate (%)=(control increment-processing increment)/control increment × 100%
Disease fungus source is as follows:
Phytophthora capsici (Phytophthora capsici) document " Jiang Houchun, Li Baoju, Shi Yanxia, Xie Xuewen,
The Primary Study North China Agricultural Journal 2011,26 (supplementary issue) that China of Lu Kingdom capsicum epidemic disease soil and seeds are carried disease germs: public in 233-237. "
It opens, the public can obtain from Vegetable & Flower Inst., Chinese Academy of Agriculture Science.
Cucumber stick spore leaf spot fungi (Corynespora cassiicola) document " Gao Wei, Li Baoju, Shi Yanxia,
Thank to the differentiation gardening journal 2011,38 (3) for learning the how main stick spore bacterium of text pathogenicity on cucumber, tomato and eggplant host: 465-
It is disclosed in 470. ", the public can obtain from Vegetable & Flower Inst., Chinese Academy of Agriculture Science.
In document, " Chai Ali, Shi Yanxia, Xie Xuewen, iwan are ancient for tomato early blight bacterium (Alternaria solani)
Beautiful, the precious poly- processing tomato early blight pathogen identification North China Agricultural Journal 2015,30 (supplementary issue) of Lee: disclosing in 316-320. ", public
Crowd can obtain from Vegetable & Flower Inst., Chinese Academy of Agriculture Science.
Tomato gray leaf spot bacterium (Stemphylium solani Weber) is in document " Li Baoju, Zhou Yanfang, Li Jin
Duckweed thanks the diagnosis and treatment China's Vegetable 2010 for learning text S. lycopersici leaf spot (gray leaf spot), (23): in 24-26. "
Open, the public can obtain from Vegetable & Flower Inst., Chinese Academy of Agriculture Science.
Rhizoctonia solani Kuhn (Rhizoctonia solani) is in document " Gao Wei, Li Baoju, Sun Junde, Shi Yanxia, gold
Antagonism China's Vegetable 2008 of the red Trichoderma viride to cucumber Rhizoctonia solani Kuhn and Fusarium oxysporum, (6): public in 9-12. "
It opens, the public can obtain from Vegetable & Flower Inst., Chinese Academy of Agriculture Science.
Fusarinm solani (Fusarium solani) is in document " Ben Haiyan, Shi Yanxia, Xie Xuewen, Chai Ali, Li Bao
Control effect gardening journal 2016,43 (11) of the poly- nitrolim soil disinfection to cucumber root rot and soil surface characters:
It is disclosed in 2173-2181. ", the public can obtain from Vegetable & Flower Inst., Chinese Academy of Agriculture Science.
2, bacterium antimicrobial spectrum measures
Using Stonie Double layer culture method, bacterial strain ZF7 is seeded in LB liquid medium, shaken cultivation 16h at 25 DEG C.
At 90mm PDA plate center, is punched with 10 μ L pipette tips, 5 μ L ZF7 bacteria suspensions are added, wherein to be inoculated with LB liquid medium
For blank control, cultivated for 24 hours at 25 DEG C.Culture dish is inverted, 3mL chloroform will be added in each culture dish in draught cupboard, it is quiet
12h is set to be evaporated chloroform and inactivate ZF7 bacterial strain.By the pathogenetic bacteria of selection at 25 DEG C shaken cultivation 36h in NB culture medium,
100 μ L bacteria suspensions are added in 4mL 5% (m/v) WA culture medium, PDA plate are poured into after mixing, as upper layer.It is cultivated in incubator
48h is observed and is measured inhibition zone size.
Pathogenetic bacteria source is as follows:
The bacillary stem Bacteria erwinia of cucumber (Pectobacterium carotovorum subsp.brasiliense) is
In document " Feng Zhi qin carrot soft rot Pectinatus Brazil subspecies (Pectobacterium carotovorum
In China China plant protection guide 2017,37 (7) occur for cucumber bacterial soft rot caused by subsp.brasiliense): 58-
It is disclosed in 62. ", the public can obtain from Vegetable & Flower Inst., Chinese Academy of Agriculture Science.
In document, " stone has prolonged cucumber bacterial angular leaf spot bacterium (Pseudomonas syringae pv.lachrymans)
Rosy clouds, Zhang Nan, the precious poly- Mechanism of Systemic Resistance in Cucmber Induced by Pseudomonas syringae pv. lachrymans of Lee research gardening journal 2008,
35 (2): disclosing in 221-226. ", and the public can obtain from Vegetable & Flower Inst., Chinese Academy of Agriculture Science.
Pseudomonas syringae pv.tomato (Pseudomonas syringae pv.tomato) is in document " Li Baoju, Zhu
Brightness, stone prolong identification and the prevention and treatment the Changjiang river the vegetables 2008 of rosy clouds bacterial spot of tomato, disclose in 9:23-24. ", and the public can be from
Vegetable & Flower Inst., Chinese Academy of Agriculture Science obtains.
Isolated From Grapevine germ (Agrobacterium vitis) is in document " Li Jinyun, Wang Huimin, Wang Jianhui root knot
The Primary Study Scientia Agricultura Sinica 2004,37 (12) of biocontrol microorganisms E26 bacterial strain generation bacteriocin: disclosing in 1860-1865. ",
The public can obtain from College of Resources and Environmental Sciences, China Agricultural University.
Cauliflower black rot (Xanthomonas campestris pv.campestris) " has been made widely known, Lee in document
The pests occurrence rule and prevention and treatment China's Vegetable 2011,17 of Jin Ping, Zhou Huimin, the precious poly- bacillary black rot of brassicaceous vegetable of Lee:
It is disclosed in 23-25. ", the public can obtain from Vegetable & Flower Inst., Chinese Academy of Agriculture Science.
Bacterial canker of tomato (Clavibacter michiganensis subsp.michiganensis) is in document
" Li Huanling, Shi Yanxia, Xie Xuewen, the precious poly- canker of tomato of Lee pests occurrence rule and Prevention Technique China's Vegetable 2011,23:
It is disclosed in 24-27. ", the public can obtain from Vegetable & Flower Inst., Chinese Academy of Agriculture Science.
Above-mentioned bacterial strains can also be obtained from applicant.
As a result as shown in Table 2 and Fig. 3, A: bacterial canker of tomato B: bacterial spot of tomato bacterium C: cauliflower black rot
D: cucumber bacterial angular leaf spot bacterium E: cucumber bacterial soft rot bacterium F: Isolated From Grapevine germ G: tomato early blight bacterium H: grey tikka
Germ I: P. capsici J: cucumber stick spore leaf spot fungi K: Rhizoctonia solani Kuhn L: Fusarinm solani；Show that bacterial strain ZF7 can
Significantly inhibit bacterial canker of tomato, bacterial spot of tomato bacterium, cauliflower black rot, cucumber bacterial angular leaf spot bacterium, Huang
Melon stem Bacteria erwinia and Isolated From Grapevine germ, meanwhile, also it is able to suppress tomato early blight bacterium, gray leaf spot bacterium, Phytophthora capsici
Germ, how main stick spore germ, but Rhizoctonia solani and Fusarinm solani inhibitory effect are poor, and it is wider to illustrate that ZF7 has
Antimicrobial spectrum.
Fungistatic effect of the 2 bacterial strain ZF7 of table to 12 kinds of phytopathogens
Note: +++ indicate that bacterium bacteriostatic diameter is more than or equal to 5.0cm or fungi bacteriostasis rate is more than or equal to 60%；++ indicate thin
Bacterium bacteriostatic diameter is more than or equal to 4.0cm and is less than 5.0cm or fungi bacteriostasis rate more than or equal to 40% less than 60%；+ indicate bacterium suppression
Bacterium diameter is less than 4.0cm or fungi bacteriostasis rate less than 40%；Indicate unrestraint effect.
Two, bacterial strain ZF7 growth-promoting effect detection
1, Soluble phosphorus plate qualitative detection
By rahnella aquatilis ZF7 and Rahnella aquatilis HX 2 respectively in PDA culture medium, carried out after 28 DEG C of culture 48h
Activation, is then inoculated in the test tube equipped with 5mL LB liquid medium, 28 DEG C, after 170r/min shakes training for 24 hours, is prepared into
108CFU ml-1Bacteria suspension；The qualitative filter paper piece (diameter D=0.5cm) of sterilizing is put in NBRIP solid medium tablets
Centre is connected on filter paper with the bacteria suspension point that liquid-transfering gun draws each bacterial strain processing of 10 μ L, is dried up under aseptic condition.7d is cultivated, so
The Soluble phosphorus circle of each processing is observed afterwards, and measures its diameter.Each processing is repeated 3 times.
As a result as shown in table 3 and fig. 4, it can be seen that rahnella aquatilis ZF7, HX2 in NBRIP culture medium culture 7d,
Soluble phosphorus loop diameter is respectively 28.2mm, 27.83；Show that rahnella aquatilis ZF7 has dissolving P capacity, with rahnella aquatilis
HX2 is without significant difference.
3 bacterial strain ZF7, HX2 Soluble phosphorus loop diameter of table and IAA yield
Note: being the IAA yield of bacterial strain in DF+ culture medium outside one column bracket of IAA yield, is bacterium in DF culture medium in bracket
The IAA yield of strain.
2, IAA (heteroauxin) is measured
Bacterial strain ZF7 is activated through PDA solid medium, LB culture medium shake training 1-2d, and with 0.1% inoculum concentration (bacterium solution amount/
Cultivate base unit weight, v/v) it is inoculated in DF, DF+ culture medium respectively, in 28 DEG C of culture 7d, 12000r/min centrifugation 5min, take on 1mL
Clear liquid is drawn 1mL titer from prepared IAA aqueous solution 5,10,15,20,25 and 30 μ g/mL respectively and is set in test tube
In test tube, it is separately added into 50 μ L Solution I (10mmol/L phosphoric acid) and (the 1mL 0.5mol/L of 2ml Solution II
FeCl3It is dissolved in 50mL 35%HClO4) reaction solution, 25min is reacted after mixing in room temperature, absorbance at 530nm is detected, to go
Ionized water does blank control, draws IAA content standard curve and calculates its amount (table 3) for producing IAA.3 weights are arranged in each processing
It is multiple.Bacterial strain ZF7 and HX2 strain inoculated have detected two in 7d in the DF culture medium without or with 500 μ g/mL tryptophans
The IAA yield of strain bacterium.
As a result as shown in figure 5, A: bacterial strain ZF7 is in DF+ culture medium；B: bacterial strain HX2 is in DF+ culture medium；C:DF+ culture
Base；D: bacterial strain ZF7 is in DF culture medium；E: bacterial strain HX2 is in DF culture medium；F:DF culture medium；Bacterial strain ZF7, HX2 are in DF+, DF
In culture medium with solution S olution I, II reacting phenomenon of Solution；Show bacterial strain ZF7 containing 500 μ g/mL tryptophans
Training 7d is shaken in DF culture medium can largely generate IAA, and concentration reaches 31.032 μ g/mL.And in the DF culture medium for not containing tryptophan
In, when 7d, the IAA concentration that bacterial strain ZF7 is generated is equal less than 4 μ g/mL.As a result illustrate that tryptophan is conducive to bacterial strain ZF7 generation
IAA.Find that bacterial strain ZF7 produces IAA ability and is better than bacterial strain HX2 simultaneously.
3, Chinese cabbage growth-promoting is tested
Under greenhouse experiment, consistent, the healthy Chinese cabbage seeds of random selection full grains degree are sowed, to seedling
It grows 2-4 piece true leaf and carries out root irrigation.4 processing of test setting: space management, clear water pouring root, every young plant fill 20mL；Configuration
Concentration is 1 × 10-7The IAA solution of μ g/mL, pouring root amount are respectively 10,20,30mL；The processing of bacterial strain ZF7 bacterium solution: every young plant is with dense
Degree is 108The bacterial strain ZF7 bacteria suspension pouring root of cfu/mL, pouring root bacterium solution amount are respectively 10,20,30mL.10 repetitions of each processing.
The processing of HX2 bacterium solution: method is the same as bacterial strain ZF7 processing.Chinese cabbage plant above ground and underground part length and fresh weight are measured after 7 days, and are counted
Calculate growth-promoting rate.
Growth-promoting rate=(processing strain ground and underground part length and fresh weight-control strain ground and underground part length and fresh weight)/
Compare strain ground and underground part length and fresh weight × 100%
Bacterial strain ZF7, HX2 and IAA not same amount to the growth-promoting effectiveness results of Chinese cabbage as shown in fig. 6, A: bacterial strain ZF7, HX2 and
Growth-promoting effect B of the IAA different amounts to Chinese cabbage overground part length: bacterial strain ZF7, HX2 and IAA different amounts are to Chinese cabbage root long
Growth-promoting effect C: bacterial strain ZF7, HX2 and IAA different amounts to growth-promoting effect D: bacterial strain ZF7, HX2 of Chinese cabbage overground part weight
And IAA different amounts are to the growth-promoting effect of Chinese cabbage root weight；
Bacterial strain ZF7, HX2 and IAA are to the growth-promoting effectiveness results of Chinese cabbage as shown in fig. 7, A: pouring root 10mL；B: pouring root
20mL；C: pouring root 30mL (every figure is from left to right followed successively by clear water control, IAA, HX2, ZF7).
The above results show that bacterial strain ZF7 has good growth-promoting effect to Chinese cabbage, and bacterial strain ZF7 is to Seedling of Chinese Cabbage
The growth-promoting rate of overground part length and root long is respectively up to 56.47%, 54.74%；To the growth-promoting of Chinese cabbage overground part weight and root weight
Rate is respectively up to 233.84% and 164.71%.
Three, bacterial strain ZF7 prevents and treats root knot effect detection
Bacterial strain ZF7 is inoculated in LB liquid medium, and 28 DEG C, 150r/min shake culture 28h, concentration, which is made, is
108Bacteria suspension (the OD of CFU/mL600For 0.6).For the Isolated From Grapevine bacterium K308 (Isolated From Grapevine germ (Agrobacterium of examination
Vitis) in document, " Li Jinyun, Wang Huimin, Wang Jianhui root knot biocontrol microorganisms E26 bacterial strain generate the Primary Study of bacteriocin
Scientia Agricultura Sinica 2004,37 (12): disclosing in 1860-1865. ", and the public can be from College of Resources and Environmental Sciences, China Agricultural University
Obtain) be also configured to concentration be 108Bacteria suspension (the OD of CFU/mL600To be 0.6) spare, with bacterial strain ZF7 thallus suspension liquid 1:1 (body
Product ratio) mixing, while being individually inoculated with bacterial strain ZF7 thallus suspension liquid and being compared with sterile water inoculation.With disinfection inoculation pin to
Needle thorn causes wound in day certain herbaceous plants with big flowers stem section, draws 10 μ L prepare liquids and is inoculated in wound, wraps up wound with sterilized non-fat cotton, for 24 hours after
Absorbent cotton is removed, observe dross situation after 15-20d is cultivated in greenhouse and claims knurl weight.Every kind of processing sets 10 repetitions, and test repeats
Three times.
Preventive effect=(control crown gall tumor fresh weight-processing crown gall tumor fresh weight)/control crown gall tumor fresh weight × 100%
Meanwhile pathogen K308 preventive effect is compared with rahnella aquatilis bacterial strain HX2 has been reported.
As a result such as table 4 and Fig. 8 (A: clear water control；B: individually it is inoculated with K308；C: individually it is inoculated with ZF7；D: bacterial strain HX2 bacterium is outstanding
Liquid and K308 are with 1: 1 ratio combined inoculation；E: bacterial strain ZF7 bacteria suspension and K308 are with 1: 1 ratio combined inoculation) shown in, bacterium
Strain ZF7 reaches 94.52% to the preventive effect of Isolated From Grapevine germ K308, and bacterial strain HX2 reaches the preventive effect of root knot germ K308
94.59%.Bacterial strain ZF7 is not significantly different the preventive effect of Crown gall with bacterial strain HX2.
4 bacterial strain ZF7 and HX2 of table is to root knot preventive effect result
Sequence table
<110>Vegetable & Flower Inst., Chinese Academy of Agriculture Science
<120>rahnella aquatilis ZF7 and its application in plant growth-promoting diseases prevention
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1376
<212> DNA
<213> Rahnella aquatilis
<400> 1
gcggaaagta gcttgctact ttgccggcga gcggcggacg ggtgagtaat gtctgggaaa 60
ctgcctgatg gagggggata actactggaa acggtagcta ataccgcatg acctcgaaag 120
agcaaagtgg gggatcttcg gacctcacgc catcggatgt gcccagatgg gattagctag 180
taggtgaggt aatggctcac ctaggcgacg atccctagct ggtctgagag gatgaccagc 240
cacactggaa ctgagacacg gtccagactc ctacgggagg cagcagtggg gaatattgca 300
caatgggcgc aagcctgatg cagccatgcc gcgtgtgtga agaaggcctt agggttgtaa 360
agcactttca gcgaggagga aggcatcaca cttaatacgt gtggtgattg acgttactcg 420
cagaagaagc accggctaac tccgtgccag cagccgcggt aatacggagg gtgcaagcgt 480
taatcggaat tactgggcgt aaagcgcacg caggcggttt gttaagtcag atgtgaaatc 540
cccgcgctta acgtgggaac tgcatttgaa actggcaagc tagagtcttg tagagggggg 600
tagaattcca ggtgtagcgg tgaaatgcgt agagatctgg aggaataccg gtggcgaagg 660
cggccccctg gacaaagact gacgctcagg tgcgaaagcg tggggagcaa acaggattag 720
ataccctggt agtccacgct gtaaacgatg tcgacttgga ggttgtgccc ttgaggcgtg 780
gcttccggag ctaacgcgtt aagtcgaccg cctggggagt acggccgcaa ggttaaaact 840
caaatgaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgatgcaacg 900
cgaagaacct tacctactct tgacatccac ggaattcgcc agagatggct tagtgccttc 960
gggaaccgtg agacaggtgc tgcatggctg tcgtcagctc gtgttgtgaa atgttgggtt 1020
aagtcccgca acgagcgcaa cccttatcct ttgttgccag cacgtaatgg tgggaactca 1080
aaggagactg ccggtgataa accggaggaa ggtggggatg acgtcaagtc atcatggccc 1140
ttacgagtag ggctacacac gtgctacaat ggcatataca aagagaagcg aactcgcgag 1200
agcaagcgga cctcataaag tatgtcgtag tccggattgg agtctgcaac tcgactccat 1260
gaagtcggaa tcgctagtaa tcgtagatca gaatgctacg gtgaatacgt tcccgggcct 1320
tgtacacacc gcccgtcaca ccatgggagt gggttgcaaa agaagtaggt agctta 1376
Claims (10)
- Rahnella aquatilis 1. (Rahnella aquatilis) ZF7, deposit number is CGMCC NO.16014.
- 2. rahnella aquatilis (Rahnella aquatilis) ZF7 described in claim 1 or its culture solution or its bacteria suspension Or application of its tunning in antibacterial.
- 3. rahnella aquatilis (Rahnella aquatilis) ZF7 described in claim 1 or its culture solution or its bacteria suspension Or application of its tunning in preparation plant biological prevention and control agent.
- 4. rahnella aquatilis (Rahnella aquatilis) ZF7 described in claim 1 or its culture solution or its bacteria suspension Or application of its tunning in controlling plant diseases.
- 5. rahnella aquatilis (Rahnella aquatilis) ZF7 described in claim 1 or its culture solution or its bacteria suspension Or application of its tunning in prevention and treatment plant root cancer.
- 6. rahnella aquatilis (Rahnella aquatilis) ZF7 described in claim 1 or its culture solution or its bacteria suspension Or its tunning is promoting the application in plant growth.
- 7. according to the application any in claim 2-6, it is characterised in that:The disease is the disease that pathogen causes；The pathogen or the antibacterial bacterium are fungi or bacterium；And/or the fungi is early blight of tomato Alternaria solani, Tomato gray leaf spot bacterium Stemphylium Solani Weber, P. capsici Phytophthora capsici, cucumber stick spore leaf spot fungi Corynespora At least one of cassiicola and Rhizoctonia solani Kuhn Rhizoctonia solani；And/or the bacterium is bacterial canker of tomato Clavibacter michiganensis Subsp.michiganensis, Tomato mottle germ Pseudomonas syringae pv.tomato, cauliflower black rot Xanthomonas campestris pv.campestris, avenae subsp.citrull Pseudomonas syringae Pv.lachrymans, Cucumis sativus stem Bacteria erwinia Pectobacterium carotovorum subsp.brasiliense, grape At least one of crown gall germ Agrobacterium vitis.
- 8. rahnella aquatilis (Rahnella aquatilis) ZF7 described in claim 1 or its culture solution or its bacteria suspension Or application of its tunning in degradation phosphorus；Or, rahnella aquatilis (Rahnella aquatilis) ZF7 described in claim 1 or its culture solution or its bacteria suspension Or its tunning is preparing the application in IAA.
- 9. a kind of method for preventing and treating plant root cancer, for by rahnella aquatilis (Rahnella described in claim 1 Aquatilis) ZF7 or its culture solution or its bacteria suspension or its tunning act on plant, realize prevention and treatment plant root cancer.
- 10. a kind of method for promoting plant growth, for by rahnella aquatilis (Rahnella described in claim 1 Aquatilis) ZF7 or its culture solution or its bacteria suspension or its tunning act on plant, realize and promote plant growth.
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| CN113293108A (en) * | 2021-05-08 | 2021-08-24 | 四川农业大学 | Preparation method of microbial agent for cleaning orchid pot soil, obtained biological agent and application |
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| CN109576194A (en) * | 2018-09-26 | 2019-04-05 | 湖北大学 | One plant of multifunctional water draws engler bacterium MEM40 and its application |
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| CN112961807A (en) * | 2021-03-30 | 2021-06-15 | 中国科学院成都生物研究所 | Microbial composition and application thereof in promoting germination and growth of highland barley seeds |
| CN112961807B (en) * | 2021-03-30 | 2023-01-20 | 中国科学院成都生物研究所 | Microbial composition and application thereof in promoting germination and growth of highland barley seeds |
| CN113293108A (en) * | 2021-05-08 | 2021-08-24 | 四川农业大学 | Preparation method of microbial agent for cleaning orchid pot soil, obtained biological agent and application |
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