CN105331561B - One plant of radiation rhizobium for preventing plant root cancer and its application - Google Patents
One plant of radiation rhizobium for preventing plant root cancer and its application Download PDFInfo
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Abstract
The radiation rhizobium for preventing plant root cancer the invention discloses one plant and its application.Radiation rhizobium provided by the present invention are specially radiation rhizobium (Rhizobium radiobacter) PAR, it is CGMCC No.11639 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.The bacterial strain is similar to the nutritional need of Agrobacterium tumefaciens, and grow it is rapid, can plant rhizosphere can rapid, high volume colonize, there is ecological niche advantage；And bacterial strain PAR does not generate antibiotic, can avoid Agrobacterium tumefaciens (A.tumefaciens) and develops immunity to drugs, than the advantage that the radiation rhizobium of production antibiotic have more ecological niche；In addition, the bacterial strain can strong inhibition Agrobacterium tumefaciens (A.tumefaciens) infect susceptible plants sunflower, tomato and peach and form crown gall tumor, have a good application prospect.
Description
Technical field
The invention belongs to agriculture, woods and garden crop protection technique fields, are related to the radiation of one plant of prevention plant root cancer
Rhizobium and its application.
Background technology
Root knot is a kind of common bacterial caused by Agrobacterium tumefaciens (Agrobacterium tumefaciens)
Disease.The host range of Agrobacterium tumefaciens is very wide, can infect 93 sections, 331 643 kinds of the plants belonged to.In Europe, North America, south
Some countries such as non-and Asia generally occur.Have a different degrees of generation in China peach cultivation area, incidence 20%~
90% (horse Deqie, the quick fruit root knots diseases of Wang Hui and its biological control;J]Fruit tree, 02 phase of nineteen ninety-five).More serious
Root knot generation area is the provinces and regions such as Hebei, Shandong, Liaoning, Henan, Hubei, Jiangsu.Early ageing after peach seedling is aggrieved, plant are short
Change, or even dead；At age tree infection this after being ill can undergrowth, tree vigo(u)r dies down, fruit is small, low output, the age of tree shorten, cause
Great economic loss.The generation of China's fruit root knot disease is fairly common, but the apparent harm of this disease is happened at root, therefore very
More orchard workers to the disease lack recognize, to prevent in time and nursery stock external harmonics introduce when quarantine measures do not give attention, cause germ
Propagation and the state of an illness aggravate year by year, preventing and controlling arduous task.It is at present crop rotation and life to the most effective control measure of root knot
Object is prevented.But many orchards or nursery can not carry out long-term crop rotation.Using the method for biological control, it is using bacteria agent in advance
Reduce the effective measures that pathogen enters in plant and interfere it to be metabolized.This technology input is low, efficient, is that a peasant recognizes
Can, operable strong agricultural product production technology.
About the biological control of root knot, although once reporting mycorrhizal fungi, actinomyces, agrobacterium, false list both at home and abroad
Spore bacterium, Bacillus alcaligenes, bacillus extract have crown gall germ certain antagonism, but remove K84 and its genetically engineered bacteria
Strain K1026 (trade name Nogall) outside, is not commercialized.
For the universal root knot caused by A.tumefaciens occurs on fruit tree and other plant, China at present
Have no the Biological agents of registration.Obtain be suitble to China's soil types and with China's independent intellectual property right biocontrol microorganisms and
Its cell-free preparation has important practical significance to the prevention and control of root knot.
Invention content
The radiation rhizobium for preventing plant root cancer the object of the present invention is to provide one plant and its application.
Radiation rhizobium provided by the present invention are specially radiation rhizobium (Rhizobium radiobacter)
PAR, the bacterial strain are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 9th, 2015
(abbreviation CGMCC, address are：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is CGMCC No.11639.
The bacterium that the present invention also protection activity ingredient is the radiation rhizobium (Rhizobium radiobacter) PAR
Agent.
In the microbial inoculum, the viable count of radiation rhizobium (Rhizobium radiobacter) PAR can be 5
Microbial inoculum described in~200 hundred million/g.
It may also include absorption carrier in the microbial inoculum；The absorption carrier can be diatomite, turf, vermiculite, perlite and
At least one of calcium carbonate.
More specifically, in one embodiment of the invention, the absorption carrier is specifically by turf and vermiculite according to body
Product ratio 1:1 proportioning mixes.
Further, the microbial inoculum by the radiation rhizobium (Rhizobium radiobacter) PAR zymotic fluid and
The absorption carrier is according to 1L:2Kg to 1L:4Kg (such as 1L：Proportioning 2Kg) mixes.
In one embodiment of the invention, the radiation rhizobium (Rhizobium radiobacter) PAR is prepared
Zymotic fluid when use nutrient broth, in the zymotic fluid of radiation rhizobium (Rhizobium radiobacter) PAR,
The viable count of the radiation rhizobium (Rhizobium radiobacter) is 50~8,000,000,000/ml (such as 5,000,000,000/ml) described hair
Zymotic fluid.Correspondingly, in the preparation-obtained microbial inoculum, the radiation rhizobium (Rhizobium radiobacter)
The viable count of PAR is 5~1,500,000,000/g (such as 1,000,000,000/g) described microbial inoculum；The water content of the microbial inoculum is within 3%.
In another embodiment of the present invention, the radiation rhizobium (Rhizobium radiobacter) are prepared
Bulk fermentation culture solution is used when the zymotic fluid of PAR, radiation rhizobium (Rhizobium radiobacter) PAR's
In zymotic fluid, the viable count of the radiation rhizobium (Rhizobium radiobacter) is 500~80,000,000,000/ml (such as 500
Hundred million/ml) zymotic fluid.Wherein, the solvent of the bulk fermentation culture solution is water, and solute and concentration are as follows：Corn flour 0.8~
1.5%, peptone 0.2~0.5%, sucrose 0.8~1.2%, yeast powder 0.2~0.6%, NH4Cl 0.3~0.8%,
MgSO4·7H2O 0.03~0.08%；Wherein, % indicates mass percentage.Correspondingly, in the preparation-obtained microbial inoculum
In, the viable count of radiation rhizobium (Rhizobium radiobacter) PAR be 100~20,000,000,000/g (such as 12,000,000,000/
G) microbial inoculum；The water content of the microbial inoculum is within 3%.
The preparation method of the microbial inoculum also belongs to protection scope of the present invention.
The preparation method of the microbial inoculum, specifically may include following steps：
(1) radiation rhizobium (Rhizobium radiobacter) PAR is cultivated, the radiation root nodule is obtained
The zymotic fluid of bacterium (Rhizobium radiobacter) PAR；In the zymotic fluid, the radiation rhizobium
The viable count of (Rhizobium radiobacter) is 500~80,000,000,000/ml (such as 50,000,000,000/ml)；
(2) by zymotic fluid and the absorption carrier obtained by step (1) according to 1L:2Kg to 1L:4Kg (such as 1L：Matching 2Kg)
Than mixing, the microbial inoculum is obtained.
In step (1), the culture medium for cultivating the radiation rhizobium (Rhizobium radiobacter) PAR is
Bulk fermentation culture solution；The solvent of the bulk fermentation culture solution is water, and solute and concentration are as follows：Corn flour 0.8~1.5%,
Peptone 0.2~0.5%, sucrose 0.8~1.2%, yeast powder 0.2~0.6%, NH4Cl 0.3~0.8%, MgSO4·7H2O
0.03~0.08%；Wherein, % indicates mass percentage.
More specifically, the solvent of the culture medium is water, and solute and concentration are as follows：Corn flour 1.0%, peptone
0.3%, sucrose 1.2%, yeast powder 0.4%, NH4Cl 0.4%, MgSO4·7H2O 0.06%；Wherein, % indicates quality percentage
Content.
In step (1), the condition of culture of the radiation rhizobium (Rhizobium radiobacter) PAR is cultivated
For：The seed liquor of the radiation rhizobium (Rhizobium radiobacter) PAR is inoculated into the bulk fermentation culture
In liquid, inoculum concentration is 0.5~5% (volume ratio), 28~30 DEG C of cultivation temperature, rotating speed 150rpm, initial pH 6.0~7.2, training
Support the time 18~for 24 hours.
The zymotic fluid of radiation rhizobium (Rhizobium radiobacter) PAR also belongs to the protection of the present invention
Range.
The present invention also provides a kind of products for preventing and/or treating plant root cancer.
Product provided by the present invention for preventing and/or treating plant root cancer includes the bacterium of independent packaging
Agent and fungicide and/or disinfectant.
The fungicide and/or disinfectant can be it is following in it is any：Lime sulfur, hydrogen peroxide, Peracetic acid, agricultural chain
Mycin, ferrous sulfate, copper sulphate, potassium permanganate etc..
In one embodiment of the invention, the fungicide and/or disinfectant are specially lime sulfur or hydrogen peroxide.Its
In, the lime sulfur can be 45% (mass percentage) crystal lime polysulfies.In one embodiment of the invention, described
Lime sulfur is specially Hebei Shuangji chemical limited company's product, and agriculture chemical registration card number is PD90105-2, product standard card number
For Q/SJHG04-2005, production licence number HNP13089-D0385.Concretely 1% (quality percentage contains the hydrogen peroxide
Amount) hydrogen peroxide (i.e. hydrogen peroxide：Water (v:V)=1:99).
Radiation rhizobium (Rhizobium radiobacter) the PAR or described microbial inoculums or the zymotic fluid are such as
It is lower 1) or 2) in application also belong to protection scope of the present invention：
1) prevent and/or treat plant root cancer；
2) product for preventing and/or treating plant root cancer is prepared；
The method of the prevention and/or treatment plant root cancer includes and is not limited to：It directly applies such as seed dressing, root dipping, pour
Fill, smear etc., it is used with the mixing of chemical fertilizer, organic fertilizer and organic-inorganic compound fertilizer, with antibiotic, chemical sterilization
What agent, inorganic salts or copper ion preparation and fumigant used is used cooperatively.
The root knot is specially the crown gall caused by Agrobacterium tumefaciens (Agrobacterium tumefaciens)
Disease.
The plant is concretely because A.tumefaciens infection causes fruit tree, forest, garden crop or the work of root knot
Object, such as：Peach, tomato, sunflower, cherry, willow, tree peony, rose etc..
The present invention also provides a kind of prevention and/or the methods for the treatment of plant root knot.
The method provided by the present invention for preventing plant root knot, this method can be seed dressing, root dipping, pouring, smearing, microbial inoculum
Using being either made, bio-feritlizer is directly applied or cooperation soil fumigant is applied.
In an embodiment of the present invention, the method for preventing plant root knot is specially following (A) or (B)：
(A) include the following steps：Vegetable seeds and the microbial inoculum are mixed, sowed after seed dressing；
(B) include the following steps：The root system of the plant is placed in the dilution of the microbial inoculum and is impregnated, is planted after immersion
Kind.
In (A), the work of radiation rhizobium described in the microbial inoculum (Rhizobium radiobacter) PAR
Bacterium number is 1,000,000,000/g；The vegetable seeds and the microbial inoculum (the radiation rhizobium (Rhizobium radiobacter)
The viable count of PAR is 1,000,000,000/g) mixing proportioning concretely 1:1 (mass ratio).Further, described dress seed is specially：By institute
Microbial inoculum (viable count of radiation rhizobium (Rhizobium radiobacter) PAR is 1,000,000,000/g) is stated with water according to 1g：
The proportioning of 1ml is blended into paste, then is added is stirred with the vegetable seeds of the quality such as the microbial inoculum thereto.
In (B), radiation rhizobium described in the dilution of the microbial inoculum (Rhizobium radiobacter)
The viable count of PAR is 100,000,000/ml；The time of the immersion is 15min.
The method for the treatment of plant root cancer provided by the present invention, includes the following steps：Cut off root knot morbidity plant
Then carcinoma smears fungicide and/or disinfectant in incision, after drying, the microbial inoculum is smeared, then again with the microbial inoculum
Dilution carries out root irrigation；The fungicide and/or disinfectant be it is following in it is any：Lime sulfur, hydrogen peroxide, peroxide second
Acid, agricultural streptomycin, ferrous sulfate, copper sulphate, potassium permanganate；
More specifically, the method for the treatment plant root cancer can be following (C) or (D)：
(C) include the following steps：The carcinoma of root knot morbidity plant is cut off, then smears 1% (quality percentage in incision
Content) hydrogen peroxide, after drying, the microbial inoculum is smeared, then carries out root irrigation with the dilution of the microbial inoculum again；
Wherein, the viable count of radiation rhizobium described in the microbial inoculum (Rhizobium radiobacter) PAR is 10
Hundred million/g；The viable count of radiation rhizobium described in the dilution of the microbial inoculum (Rhizobium radiobacter) PAR is
0.2 hundred million/g.
(D) include the following steps：The carcinoma of root knot morbidity plant is cut off, then smears the dilute of lime sulfur in incision
Liquid is released, after drying, the microbial inoculum is smeared, then carries out root irrigation with the dilution of the microbial inoculum again.
Wherein, the viable count of radiation rhizobium described in the microbial inoculum (Rhizobium radiobacter) PAR is 10
Hundred million/g；The viable count of radiation rhizobium described in the dilution of the microbial inoculum (Rhizobium radiobacter) PAR is
0.2 hundred million/g.The effective content of lime sulfur described in the dilution of the lime sulfur is 0.15% (mass percentage.
In one embodiment of the invention, the lime sulfur can be that 45% (mass percentage) crystal stone sulphur closes
Agent.In one embodiment of the invention, the lime sulfur is specially Hebei Shuangji chemical limited company's product, and pesticide is stepped on
Note card number is PD90105-2, and product standard card number is Q/SJHG04-2005, production licence number HNP13089-D0385.Phase
It answers, the dilution of the lime sulfur is 300 times of liquid of the lime sulfur.
In (C) and (D), the liniment of daubing medicament (dilution of the microbial inoculum or the lime sulfur)
Amount is 50ml.Further include the steps that covering l Water Paper (can pesticide control evaporation failure) after having smeared medicament.The tool of the pouring root
Body processing method is as follows：A circular furrow are done around morbidity plant rootstock, prevention liquid sprawling, every plant fills around rootstock
Medicine 2L.
The plant is concretely because A.tumefaciens infection causes fruit tree, forest, garden crop or the work of root knot
Object, such as：Peach, tomato, sunflower, cherry, willow, tree peony, rose etc..
Radiation rhizobium (Rhizobium radiobacter) PAR CGMCC No.11639 tools provided by the present invention
There is following advantage：
1, it is located away from the selective medium tablet D grown suitable for Agrobacterium tumefaciens1M culture medium (bibliography：
Schaad,N.W.2001.Laboratory Guide for the Identification of Plant Pathogenic
Bacteria,3rd Edition.The American Phytopathological Society,St.Paul,MN.(pp 6,
9)), similar to the nutritional need of Agrobacterium tumefaciens, and grow it is rapid, can plant rhizosphere can rapid, high volume colonize, have
There is ecological niche advantage.
2, antibiotic is not generated, Agrobacterium tumefaciens (A.tumefaciens) is can avoid and develops immunity to drugs, than producing antibiotic
Radiation rhizobium have more ecological niche advantage, which is one plant of potential Antagonistic Fungi.
3, can strong inhibition Agrobacterium tumefaciens (A.tumefaciens) infect susceptible plants sunflower, tomato and peach
And crown gall tumor is formed, preventive effect respectively reaches 89.1%, 90.0% and 56.1%.
Radiation rhizobium (Rhizobium radiobacter) PAR CGMCC No.11639 provided by the present invention are
The characteristics of peach rhizosphere soil is separated to one plant of bacterium from Shandong, the bacterial strain be：When being cultivated on nutrient medium tablet, bacterium colony
Protrusion is sticky, and quickly, this is one of the precondition that biocontrol microorganisms can be mass-produced and apply to bacterium colony expansion rate.It is outstanding with its bacterium
Liquid mixes inoculation sunflower with Agrobacterium tumefaciens bacteria suspension, can obviously control the formation of carcinoma, be identified as radiation root nodule
Bacterium (Rhizobium radiobacter), names as PAR.Multiple field diseases prevention experiments have shown that, prepared with the bacterial strain fermentation liquor
Microbial inoculum in field to the protection effect of peach root knot up to 43.7%~56.1%, the diseases preventions of the K84 preparations used with peasant is imitated
Fruit is suitable.Since the bacterial strain does not generate inhibition zone in bacteriostatic test plate, which can not generate antibiotic, can
Caused protection effect may be caused to decline even forfeiture because Agrobacterium tumefaciens develop immunity to drugs after avoiding prolonged application
Problem.When PAR strain fermentation cultures, the speed of growth can be completed to ferment soon, within 20~24, and viable count can reach after fermentation
500~80,000,000,000/ml, it is at low cost at producing.Therefore PAR is one plant of very potential fruit tree or other plant root knot biocontrol microorganisms,
To propose the present invention.
Preservation explanation
Strain name：Radiation rhizobium
Latin name：(Rhizobium radiobacter)
Strain number：PAR
Preservation mechanism：China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is referred to as：CGMCC
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date：On November 9th, 2015
Collection is registered on the books number：CGMCC No.11639
Specific implementation mode
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.
The separation and identification of embodiment 1, radiation rhizobium (Rhizobium radiobacter) PAR
One, the screening of radiation rhizobium (R.radiobacter) bacterial strain PAR
Radiation rhizobium (R.radiobacter) PAR of the present invention is that dilution plate is used from peach rhizosphere soil
What method and plate streak separation obtained, separation method is：
(1) separation of radiation rhizobium：The acquisition of soil sample acquires peach respectively in Shandong Province Tai'an Xiyuan garden spot
The fibrous root of different cultivars health peach and the Rhizosphere Soil of adhesion in garden indicate collecting location, time and acquisition people.Weigh 1g peach
Fibrous root is put into 99ml sterile waters, and oscillation shakes up 30min.Then 1ml is drawn from triangular flask with liquid-transfering gun, another Sheng is added
In the test tube for having 9ml sterile waters, be mixed evenly, and so on be respectively prepared 10-3、10-4、10-5The soil of different dilutions is molten
Liquid.100 μ L Soil Slurries are taken to be coated on Selective agar medium D1On M tablets, three culture dishes of each gradient coating, 27-30 DEG C
Culture 2-4 days.The bacterium of picking different shape is transferred on nutrient agar (i.e. NA solid mediums) purifying of crossing.
Wherein, the Selective agar medium D1M components and proportioning are：Cellobiose 5.0g, NH4Cl 1.0g, MgSO4·7H2O
0.3g, K2HPO43H2O 3.0g, NaH2PO4·H2O 1.0g, peacock green 0.01g, agar 18g, distilled water 1000mL；pH
7.0~7.2.
The NA solid mediums component and proportioning are：Glucose 10g, peptone 10g, beef extract 3g, yeast extract 1g, fine jade
Fat 18g, distilled water 1000ml, pH are adjusted to 7.0,121 DEG C of high pressure sterilizations 30 minutes.
(2) screening of the efficient antagonism rhizobium of Peach crown gall
1. pathogenic identification：There are 3 regions related with tumorigenesis in crown gall germ：The areas Vir participate in certain morning of tumor inducing
Phase process.The areas T-DNA, the generation of the growth and crown gall alkali (opine) of the gene control tumour of carrying, is most important DNA pieces
Section.3rd area then controls bacterium and absorbs and utilize the crown gall alkali generated in tumor tissues.Using virD2A/virD2C primers (ginseng
Examine document：Haas J H,Moore L W,Ream W,et al.Universal PCR primers for the
detection of phytopathogenic Agrobacterium strains.Applied Environment
Microbiology,1995,61(8):2879~2884) and Tip6F/Tip 6R primer (bibliography：Yakabe L E,
Maccree M M,Sudarshana P,et al.Novel PCR primers for detection of genetically
diverse virulent Agrobacterium tumefaciens biovar 1strains.Journal of General
Plant Pathology,2012,78:121~126), the areas vir of separated bacterium and the areas T-DNA are measured respectively, selected
It selects and does not expand the bacterial strain to 224bp and 243bp target fragments as non-pathogenic strain to be tested.
virD2A：5′-ATGCCCGATCGAGCTCAAGT-3′；
virD2C：5′-TCGTCTGGCTGACTTTCGTCATAA-3′.
Tip6F：5′-GGTCTAATGCGCAGAGGTGT-3′；
Tip 6R：5′-CGGCTCAAGGATTAGACAGG-3′.
Wherein, the amplification system (total volume in the areas bacterium vir：25.0 μ L) be：0.6 μ L (s < of DNA profiling;1 μ g), upstream and downstream draws
Each 1.0 μ L of object (5pmol/ μ L), 10 × buffer solution, 2.5 μ L, dNTP Mixture (2.5mM each) 1.5 μ L, Taq polymerase
(2.5U/ μ L) 0.5 μ L, 18.0 μ L of ultra-pure water.
PCR amplification condition：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 30s；56 DEG C of annealing 30s；72 DEG C of extension 40s；35 follow
Ring；72 DEG C of supplements extend 10min.PCR product is detected with 1.2% agarose electrophoresis.
The amplification system in the areas bacterium T-DNA is identical as the areas vir amplification system, and PCR amplification condition is：95 DEG C of pre-degenerations
5min；95 DEG C of denaturation 30s；56 DEG C of annealing 30s；72 DEG C of extension 30s；35 cycles；72 DEG C of supplements extend 10min.PCR product is used
1.2% agarose electrophoresis detection.
2. bioassay：Potting diseases prevention experiment is carried out with easy diseased plant sunflower and tomato, and is with peach-pit and peach seedling
It tries material and carries out field diseases prevention experiment.Specific method is with reference to embodiment 3-5.
By screening above, one plant of radiation rhizobium (R.radiobacter) is finally obtained, which is denoted as PAR.
Two, bacterial strain is identified
1, Microbiological Characteristics are identified
The colonial morphology formed on nutrient agar NA culture mediums is round convexity, and transparent slightly faint yellow, surface is smooth
It is sticky, there are mucus, neat in edge, positive and negative solid colour.It is cultivated two days later at 28 DEG C on NA culture mediums, somatic cells are in bar
Shape can move.The single presence of cell exists in pairs, and size is about 1.5-3.0 μm of 0.6-1.0 μ ms.Gram-negative,
Without gemma.PAR bacterial strains are grown in pH 7.0-7.4,26-29 DEG C of well-grown of temperature using several kinds of carbon source.
2, molecular biological characteristic
The genomic DNA for extracting bacterial strain PAR expands 16S rRNA sequences, by the sequence with universal primer 27f and 1492r
Sequencing.
27f：5'-AGA GTT TGA TCC TGG CTC AG-3'；
1492r：5'-TAC GGC TAC CTT GTT ACG ACT T-3'.
The sequence of the 16S rRNA of bacterial strain PAR refers to sequence 1 in sequence table.
In view of mentioned microorganism CHARACTERISTICS IDENTIFICATION and molecular biological characteristic qualification result, step 1 is detached and purified
To bacterial strain PAR be accredited as radiation rhizobium (Rhizobium radiobacter).The radiation rhizobium
(R.radiobacter) PAR is preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life on November 9th, 2015
(abbreviation CGMCC, address are at object center：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number CGMCC
No.11639。
Embodiment 2, the Antibacterial Activity of radiation rhizobium (Rhizobium radiobacter) PAR and microbial inoculum
It prepares
One, the Antibacterial Activity of radiation rhizobium (Rhizobium radiobacter) PAR
Agrobacterium tumefaciens (A.tumefaciens) ATCC 23308 is inoculated in NA (formula sees above) inclined-plane, 28 DEG C
Cultivate 48h.Then appropriate amounts of sterilized water is added in inclined-plane, prepares pathogen bacteria suspension.Take 1ml bacteria suspensions (108cfu/ml)
It is placed in sterilized culture dish, pours into the YEB culture mediums of 40-50 DEG C of thawing, and shake up, it is flat that the antagonism measurement carried disease germs is made
Plate.Radiation rhizobium (Rhizobium radiobacter) PAR CGMCC that the embodiment 1 of picking culture for 24 hours obtains
No.11639, point are connected on antagonism assay plate.After 28 DEG C of culture 1-2d, the formation feelings of colonial morphology and inhibition zone are observed
Condition, vernier caliper measurement antibacterial circle diameter (mm).
The YEB nutrient media components and proportioning are：Peptone 5g, yeast extract 1g, beef extract 5g, MgSO40.4936g, sugarcane
Sugared 5g, agar 15g, water 1000ml.Adjust pH to 7.0,121 DEG C of high pressure sterilizations 30 minutes.
The results show that radiation rhizobium (Rhizobium radiobacter) PAR CGMCC that embodiment 1 obtains
No.11639 does not generate inhibition zone to Agrobacterium tumefaciens (A.tumefaciens) ATCC 23308.Result explanation, radiation
Rhizobium (Rhizobium radiobacter) PAR CGMCC No.11639 do not generate antibiotic.
Two, the preparation of radiation rhizobium (Rhizobium radiobacter) PAR microbial inoculums
1, radiation rhizobium (Rhizobium radiobacter) the PAR CGMCC obtained embodiment 1
The lawn of No.11639 is transplanted in nutrient broth, and 28~30 DEG C of shaking table shaken cultivation 22h obtain seed liquor.
Wherein, the formula of nutrient broth is：Glucose 10g, peptone 10g, beef extract 3g, yeast extract 1g, distilled water
1000ml, pH are adjusted to 7.0~7.2,121 DEG C of high pressure sterilizations 30 minutes.
2, the seed liquor that step 1 obtains is inoculated into nutrient broth in 0.8% ratio (volume ratio), cultivation temperature 28~
30 DEG C, rotating speed 180rpm, initial pH 6.0~7.0, incubation time is for 24 hours.Zymotic fluid is obtained after fermentation, is radiated in zymotic fluid
The viable count of shape rhizobium (Rhizobium radiobacter) PAR CGMCC No.11639 is up to 5,000,000,000/mL.
3, the radiation root nodule fermented liquid and absorption carrier obtained step 2 --- turf and vermiculite (volume ratio 1:1)
It is 1 according to volume mass ratio:2 (i.e. 1L：Ratio 2Kg) is mixed evenly to get microbial inoculum.Radiation rhizobium in the microbial inoculum
The viable count of (Rhizobium radiobacter) PAR CGMCC No.11639 is up to 1,000,000,000/g, and water content is within 3%.
Embodiment 3, radiation rhizobium PAR bacterial strains infect sunflower shape to Agrobacterium tumefaciens (A.tumefaciens)
At the inhibiting effect of crown gall tumor
It will be cultivated in nutrient broth (formula sees above) to the radiation rhizobium (Rhizobium of logarithmic phase
Radiobacter) PAR CGMCC No.11639 and Agrobacterium tumefaciens (A.tumefaciens) ATCC 23308 are with volume ratio
10:1 (V/V) is mixed, in mixed liquor, radiation rhizobium (Rhizobium radiobacter) PAR CGMCC No.11639
Viable count be 106The viable count of a/ml, Agrobacterium tumefaciens (A.tumefaciens) ATCC 23308 are 105A/ml.It will
Mixed liquor is seeded at the sunflower cotyledon of emergence 10d or so, and inoculum concentration is 20 μ L.Specific method is with reference to Wang Hongyan (with reference to text
It offers：Wang Hongyan cherries and oriental cherry root knot cause of disease and biological control research:[Shuo Shilunwen ]China Agricultural University, 1998, p7.)
Method.
Radiation rhizobium (Rhizobium radiobacter) PAR CGMCC No.11639 are replaced with equivalent sterile water
Bacteria suspension, with Agrobacterium tumefaciens (A.tumefaciens) ATCC 23308 mix as a contrast (CK).
10 plants of sunflower seedlings, totally 3 repetitions are often handled, quantitative result is averaged ± standard deviation.It is positioned over 28 DEG C of training
Interior is supported, sees whether knob occur after a week, vernier caliper measurement knob diameter and sunflower stem thickness.Count incidence
And disease index, calculate the protection effect of Antagonistic Fungi.State of an illness partition of the level (table 1) refers to Tao Tie men and the bright (bibliography that rises：
Tao Tienan, the bright staple crops Disaster Assessments that rise;M]Beijing:Scientia Agricultura Sinica technology publishing house, 2010) method.Disease
Feelings index=Σ (sick grade strain number × represents numerical value) × 100/ investigation total strain number × superlative degree typical value.
The root knot state of an illness criteria for classifying on 1 sunflower stem of table
The result shows that after inoculation sunflower 7d, control group (CK groups) morbidity is serious, and root nodule is significantly greater than radiation rhizobium
PAR processing group (PAR groups) root nodule, disease index are 83.3 (tables 2).The disease index of PAR bacterial strains processing is relatively low, and preventive effect reaches
89.1%.
2 radiation rhizobium PAR bacterial strains of table form A.tumefaciens the inhibiting effect of crown gall tumor on sunflower
Preventive effect (%)=(CK group disease indexs-PAR organizes disease index)/CK group disease index × 100%.
Embodiment 4, radiation rhizobium PAR bacterial strains infect tomato to Agrobacterium tumefaciens (A.tumefaciens) and are formed
The inhibiting effect of crown gall tumor
After emergence 30d tomato seedling rhizome base portions are scratched and hinder root 10, with radiation rhizobium (Rhizobium
Radiobacter) the bacteria suspension (10 of PAR CGMCC No.116396Cfu/ml then) root dipping is transplanted to a diameter of 12cm's
Pouring root after in flowerpot, every plant of pouring 100ml bacteria suspension, the clear water of control (CK) equivalent are handled.Use Agrobacterium tumefaciens within 2nd day
(A.tumefaciens) 23308 bacteria suspensions (10 of ATCC6Cfu/ml) pouring root, every plant of pouring 100ml.Often handle 15 plants of tomatoes
Seedling, 3 repetitions, quantitative result are averaged ± standard deviation.
It uproots after 2 months, checks whether there is crown gall tumor.It investigates dross number on root and measures root nodule size and stem thickness, unite
Incidence and disease index are counted, the protection effect of Antagonistic Fungi is calculated.State of an illness partition of the level (table 3) is with reference to Tao Tie men and bright rises
(bibliography：Tao Tienan, the bright staple crops Disaster Assessments that rise;M]Beijing:Scientia Agricultura Sinica technology publishing house,
2010) method.Disease index=Σ (sick grade strain number × represents numerical value) × 100/ investigation total strain number × superlative degree typical value.
The root knot state of an illness criteria for classifying on 3 tomato seedling of table
The result shows that control group (CK groups) tomato root basal part of stem has a crown gall tumor of apparent enlargement, disease index 88.8, and
Do not occur crown gall tumor with the tomato seedling of PAR bacterial strain processing group (PAR groups) or crown gall tumor is smaller, disease index is substantially reduced, preventive effect
Reach 90.0% (table 4).
4 radiation rhizobium PAR bacterial strains of table form A.tumefaciens the inhibiting effect of crown gall tumor on tomato
Preventive effect (%)=(CK group disease indexs-PAR organizes disease index)/CK group disease index × 100%.
Embodiment 5, radiation rhizobium PAR microbial inoculums are to the inhibiting effect of peach root knot
One, inhibiting effect of the radiation rhizobium PAR microbial inoculums processing peach-pit to peach root knot
PAR microbial inoculums (viable count is 1,000,000,000/g) seed dressing prepared by conventional lamination peach-pit embodiment 2, first by PAR bacterium
Agent is with water according to 1g：It is paste that the proportioning of 1ml, which mixes, then peach-pit is added thereto, is stirred, PAR microbial inoculums and peach-pit
Proportioning is 1:1 (w/w, i.e. mass ratio).Then it is sowed March to the peach continuous cropping nursery lot in the Shandong Province Tai'an towns Cu Lai
In.
Control treatment (CK groups) is done with the peach-pit directly sowing dressed seed without PAR microbial inoculums.
150-200 seeds, 4 repetitions are often handled, quantitative result is averaged ± standard deviation.Making ridge point between each processing
Every ridge side 1-2 rows are gone as protection.Line-spacing 40cm, spacing in the rows 10cm.It uproots in 9-10 months autumn current year, checks whether there is root
Carcinoma.It investigates dross number on root and measures root nodule size and stem thickness, count incidence and disease index, calculate the anti-of PAR microbial inoculums
Sick effect.State of an illness partition of the level (table 5) refers to Tao Tie men and the bright (bibliography that rises：Tao Tienan, bright staple crops of rising
Zai Haipinggu [M]Beijing:Scientia Agricultura Sinica technology publishing house, 2010) method.Disease index=Σ (sick grade strain number × generation
Table numerical value) × 100/ investigation total strain number × superlative degree typical value.
The root knot state of an illness criteria for classifying in 5 peach seedling of table
The results show that can obviously reduce peach to peach-pit Dressing using radiation rhizobium PAR microbial inoculums (PAR groups)
The incidence and disease index of root knot, to the control effect of Peach crown gall up to 56.1% (table 6).
6 radiation rhizobium PAR microbial inoculums of table handle inhibiting effect of the peach-pit to peach root knot
Preventive effect (%)=(CK group disease indexs-PAR organizes disease index)/CK group disease index × 100%.
Two, inhibiting effect of the radiation rhizobium PAR microbial inoculums to peach seedling root knot
PAR microbial inoculums (viable count is 1,000,000,000/g) prepared by embodiment 2 are diluted with 10 times of water, then by healthy peach seedling
Root is immersed in above-mentioned diluted PAR bacterium solutions, is taken out after 15 minutes, is planted March to Shandong Province Tai'an pine nursery
In peach second crop soil.
Control treatment (CK groups) is done with the peach seedling (being impregnated without microbial inoculum) directly planted.
100-150 seedling, 4 repetitions are often handled, quantitative result is averaged ± standard deviation.Making ridge point between each processing
Every ridge side 1-2 rows are gone as protection.Line-spacing 50cm, spacing in the rows 30cm.It uproots in 9-10 months autumn current year, checks whether there is root
Carcinoma.It investigates dross number on root and measures root nodule size and stem thickness, count incidence and disease index, calculate the anti-of PAR microbial inoculums
Sick effect.State of an illness partition of the level (table 5) refers to Tao Tie men and the bright (bibliography that rises：Tao Tienan, bright staple crops of rising
Zai Haipinggu [M]Beijing:Scientia Agricultura Sinica technology publishing house, 2010) method.Disease index=Σ (sick grade strain number × generation
Table numerical value) × 100/ investigation total strain number × superlative degree typical value.
The results show that handling peach seedling using radiation rhizobium PAR microbial inoculums (PAR groups), peach root can obviously reduce
The disease index of carninomatosis, to the control effect of Peach crown gall up to 43.7% (table 7).
7 radiation rhizobium PAR microbial inoculums of table infect peach seedling to Agrobacterium tumefaciens A.tumefaciens and form crown gall tumor
Inhibiting effect
Preventive effect (%)=(CK group disease indexs-PAR organizes disease index)/CK group disease index × 100%.
Three, Peach crown gall therapeutic effect comparative test
1, experimental method
Test site：Chinese Academy of Agricultural Sciences Langfang proving ground peach root knot sick nursery.
Test material：2 years raw susceptible 60 plants of wild peach of artificial infection；Diseased plant line-spacing 2m, spacing in the rows 30cm.
Treatment agent：Hydrogen peroxide (H2O2)；Lime sulfur (active ingredient：Lime sulfur 45% crystallizes, manufacturer：Hebei
Shuan Ji Chemical Co., Ltd.s；Agriculture chemical registration is demonstrate,proved：PD90105-2；Product standard is demonstrate,proved：Q/SJHG04-2005；Production licence：
HNP13089-D0385)；PAR microbial inoculums (viable count is 1,000,000,000/g) prepared by embodiment 2, hereinafter referred to as PAR microbial inoculums.
Treatment conditions：In August, 2015, when 25 DEG C -35 DEG C of ground temperature, soil types sandy loam, pH value 7.2.
The diameter for measuring carcinoma at the stem thickness and rootstock of 60 plants of morbidity (root knot) peach, records data.Carcinoma is cut off
Protection processing is carried out to wound using different agents afterwards, processing is divided into 6 groups：
(1) 1% hydrogen peroxide, i.e. hydrogen peroxide are smeared：Water (v:V)=1:99；
(2) 1% hydrogen peroxide is smeared, after drying 1h, smears PAR microbial inoculum stostes, with the 50 times of liquid irrigating root processing of PAR microbial inoculums；
(3) 300 times of liquid of lime sulfur are smeared；
(4) 300 times of liquid of lime sulfur are smeared, after drying 1h, PAR microbial inoculum stostes are smeared, at 50 times of liquid irrigating roots of PAR microbial inoculums
Reason；
(5) clear water is smeared；
(6) carcinoma is not cut off.
Each treatment agent is uniformly applied to wound excision position (pharmaceutical quantities of smearing are 50mL or so) using hairbrush, is applied
Wet newspaper, pesticide control evaporation failure are covered after smearing medicament；The diseased plant of root irrigation is cooked a circular furrow around rootstock, is prevented
Control liquid sprawling, every plant of perfusion 2L around rootstock.
First month is investigated weekly once after processing, observes plant growing way and overground part state, and record peach restores normal
The growth time used.Third month measures plant height, counts the plant death rate, investigates the recurrence of carcinoma at rootstock, if any
Recurrence measures carcinoma diameter, diseased plant stem thickness.
Total tree of tree/processing of recurrence carcinoma after calculating recurrence rate=processing
Carcinoma diameter × 100% before therapeutic effect=(carcinoma diameter after carcinoma diameter-processing before processing)/processing.
Field management is conventionally carried out, but to avoid broad irrigation, is changed to single plant border irrigation, prevention crown gall bacterium is climing
Prolong.Special weather or other situations are met, please records and notifies in time.
2, result and analysis
(1) extent of injury of the different agents to plant
Peach grows normal, nothing after carcinoma is cut off and medicament, biocontrol agent handle ((1)-more than i.e. (5) processing group)
It wilts, yellow leaf phenomenon, no peach is dead, it was demonstrated that the above therapy will not damage peach tree body.
(2) control effect of different agents and processing mode to root knot
Investigation of uprooting is carried out to 3 months experimental field peach of growth, root nodule recurrence and therapeutic effect are shown in Table 8.
The therapeutic effect of 8 different agents of table and processing mode to root knot
Note：" control " in table is processing (5).
The result shows that carcinoma excision is a kind of method for the crown gall tumor that effective and feasible treatment has occurred, carcinoma is relied only on
When excision, although carcinoma recurrence rate is still up to 100%, recurrence knurl footpath is far smaller than the root nodule diameter before cutting off, treatment effect
Fruit is up to 62.99%.It is only handled with hydrogen peroxide oxidation after carcinoma excision, effect unobvious；And being handled with lime sulfur has centainly
Therapeutic effect, therapeutic effect is up to 77.98%.Chemical agent cooperation PAR microbial inoculums treatment can then significantly reduce Peach crown gall
Recurrence.After wherein being disinfected with hydrogen peroxide, reuses PAR microbial inoculums and smear protection and root irrigation, carcinoma recurrence rate is
80%, preventive effect is up to 83.42%；And lime sulfur+PAR processing therapeutic effect is more preferable, carcinoma recurrence rate is only 50%, and preventive effect is up to
88.69%.The peach carcinoma average diameter for not cutting off carcinoma then reaches 68.14mm.Coordinate the above result shows that being cut off using carcinoma
Lime sulfur sterilizing, then root irrigation is smeared to Peach crown gall with good therapeutic effect, this side using PAR microbial inoculums
Method can be applied to the tone fruit trees that root knot has occurred for field, be a set of effective and feasible therapeutic scheme.
The Optimization of preparation of embodiment 6, radiation rhizobium (Rhizobium radiobacter) PAR microbial inoculums
Compared with two with embodiment 2 the step of, nutrient broth therein is only replaced with into bulk fermentation culture solution, remaining step
All same.Concrete operations are as follows：
1, the lawn for the radiation rhizobium PAR CGMCC No.11639 that embodiment 1 obtains is transplanted to nutrient broth
In (formula is same as above), 28-30 DEG C of shaking table shaken cultivation 22h obtains seed liquor.
2, the seed liquor that step 1 obtains is inoculated into bulk fermentation culture solution in 0.8% ratio (volume ratio), culture temperature
28~30 DEG C of degree, rotating speed 150rpm, initial pH 6.0~7.0, incubation time is for 24 hours.Zymotic fluid, zymotic fluid are obtained after fermentation
The viable count of middle radiation rhizobium PAR CGMCC No.11639 is up to 50,000,000,000/mL.
Wherein, bulk fermentation culture formula of liquid (mass percentage) is：Corn flour 1.0%, peptone 0.3%, sucrose
1.2%, yeast powder 0.4%, NH4Cl 0.4%, MgSO4·7H2O 0.06%, remaining is water.
3, the radiation root nodule fermented liquid and absorption carrier obtained step 2 --- turf and vermiculite (volume ratio 1:1)
It is 1 according to volume mass ratio:2 (i.e. 1L：Ratio 2Kg) is mixed evenly to get microbial inoculum.Radiation rhizobium in the microbial inoculum
The viable count of PAR CGMCC No.11639 is up to 12,000,000,000/g, and water content is within 3%.
Bulk fermentation culture solution in the present invention it can be seen from the experimental result of the present embodiment is for radiation rhizobium
Preparing for (Rhizobium radiobacter) PAR microbial inoculums is most important, using identical method, by microbial inoculum in embodiment 2
Viable count improves more than ten times.
Claims (17)
- Radiation rhizobium 1. (Rhizobium radiobacter) PAR, it is in China Committee for Culture Collection of Microorganisms The deposit number of common micro-organisms center is CGMCC No.11639.
- 2. a kind of microbial inoculum, its active constituent is radiation rhizobium (Rhizobium described in claim 1 radiobacter)PAR。
- 3. microbial inoculum according to claim 2, it is characterised in that：In the microbial inoculum, the radiation rhizobium The viable count of (Rhizobium radiobacter) PAR is microbial inoculum described in 5~20,000,000,000/g.
- 4. microbial inoculum according to claim 2 or 3, it is characterised in that：The microbial inoculum is by the radiation rhizobium The zymotic fluid and absorption carrier of (Rhizobium radiobacter) PAR is according to 1L：2Kg to 1L：The proportioning of 4Kg mixes.
- 5. the preparation method of microbial inoculum described in claim 4, includes the following steps：(1) radiation rhizobium (Rhizobium radiobacter) PAR described in claim 1 is cultivated, the radiation is obtained The zymotic fluid of shape rhizobium (Rhizobium radiobacter) PAR；(2) by zymotic fluid obtained by step (1) and the absorption carrier described in claim 4 according to 1L：2Kg to 1L：The proportioning of 4Kg It mixes, obtains the microbial inoculum.
- 6. according to the method described in claim 5, it is characterized in that：In step (1), the radiation rhizobium are cultivated The culture medium of (Rhizobium radiobacter) PAR is bulk fermentation culture solution；The solvent of the bulk fermentation culture solution is Water, solute and concentration are as follows：Corn flour 0.8~1.5%, peptone 0.2~0.5%, sucrose 0.8~1.2%, yeast powder 0.2 ~0.6%, NH4Cl 0.3~0.8%, MgSO4·7H2O 0.03~0.08%；Wherein, % indicates mass percentage.
- 7. according to the method described in claim 6, it is characterized in that：Cultivate the radiation rhizobium (Rhizobium Radiobacter) condition of culture of PAR is：By the kind of the radiation rhizobium (Rhizobium radiobacter) PAR Sub- liquid is inoculated into the bulk fermentation culture solution, and inoculum concentration is 0.5~5% (volume ratio), 28~30 DEG C of cultivation temperature, rotating speed 150rpm, initial pH 6.0~7.2, incubation time 18~for 24 hours.
- 8. the zymotic fluid of radiation rhizobium (Rhizobium radiobacter) PAR described in claim 1.
- 9. for preventing and/or treating the product of plant root cancer, including in the claim 2-4 of independent packaging it is any described Microbial inoculum and fungicide and/or disinfectant；The fungicide and/or disinfectant be it is following in it is any：Lime sulfur, hydrogen peroxide, Peracetic acid, agricultural streptomycin, Ferrous sulfate, copper sulphate, potassium permanganate.
- 10. appointing in radiation rhizobium (Rhizobium radiobacter) PAR or claim 2-4 described in claim 1 The product described in microbial inoculum or zymotic fluid according to any one of claims 8 or claim 9 described in one is preventing and/or is treating plant roots Application in carninomatosis.
- 11. appointing in radiation rhizobium (Rhizobium radiobacter) PAR or claim 2-4 described in claim 1 The product described in microbial inoculum or zymotic fluid according to any one of claims 8 or claim 9 described in one is being prepared for preventing and/or controlling Treat the application in the product of plant root cancer.
- 12. according to the application of claim 10 or 11, it is characterised in that：The root knot is by Agrobacterium tumefaciens Root knot caused by (Agrobacterium tumefaciens).
- 13. a kind of method for preventing plant root cancer, includes the following steps：It is added in claim 2-4 and appoints into vegetable seeds One microbial inoculum, is sowed after being dressed seed.
- 14. a kind of method for preventing plant root cancer, includes the following steps：The root system of plant is placed in claim 2-4 and is appointed It impregnates in the dilution of one microbial inoculum, is planted after immersion.
- 15. a kind of method for treating plant root cancer, includes the following steps：The carcinoma for cutting off root knot morbidity plant, then exists Incision smears fungicide and/or disinfectant, after drying, the microbial inoculum of claim 3 or 4 is smeared, then again with the microbial inoculum Dilution carry out root irrigation；The fungicide and/or disinfectant be it is following in it is any：Lime sulfur, hydrogen peroxide, peroxide Acetic acid, agricultural streptomycin, ferrous sulfate, copper sulphate, potassium permanganate.
- 16. according to the method for claim 15, it is characterised in that：Described method includes following steps：Cut off root knot hair The carcinoma of sick plant, then incision smear mass percentage be 1% hydrogen peroxide, after drying, smear claim 3 or 4 microbial inoculums, then carry out root irrigation with the dilution of the microbial inoculum again.
- 17. according to the method for claim 15, it is characterised in that：Described method includes following steps：Cut off root knot hair Then the carcinoma of sick plant smears the dilution of lime sulfur in incision, after drying, smear the bacterium of claim 3 or 4 Then agent carries out root irrigation with the dilution of the microbial inoculum again.
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| Bacillus amyloliquefaciens strain 32a as a source of lipopeptides for biocontrol of Agrobacterium tumefaciens strains;Ben Abdallah D.等;《Journal of Applied Microbiology》;20150731;第119卷(第1期);196-207 * |
| Potential effect of rhizobacteria in the management of crown gall disease caused by Agrobacterium tumefaciens biovar 1;Rhouma A.等;《Journal of Plant Pathology》;20081130;第90卷(第3期);517-526 * |
| 放射形土壤杆菌（Agrobacterium radiobacter）K84菌株发酵条件及其生物制剂;陈龙英 等;《上海农业学报》;19960225;第12卷(第1期);50-55 * |
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