CN103981122A - Bradyrhizobium ganzhouense sp.nov. and application thereof - Google Patents
Bradyrhizobium ganzhouense sp.nov. and application thereof Download PDFInfo
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Abstract
The invention discloses bradyrhizobium ganzhouense sp.nov. and application thereof. Bradyrhizobium ganzhouense sp.nov. RITF806 is preserved in China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China since 26th Feburary, 2014, and the preservation number is CCTCC NO: M2014044. Bradyrhizobium ganzhouense sp.nov. is separated and screened out from acacia melanoxylon rootnodule, is capable of realizing nodulation symbiosis with acacia melanoxylon, acacia aneura, acacia implexa and the like, has high stress resistance, and has wide application prospect for azotification microbe inocula and bio-organic fertilizers.
Description
Technical field:
The invention belongs to microorganism field, be specifically related to a kind of Ganzhou raw root nodule bacterium and uses thereof slowly.
Background technology:
In atmosphere, contain 78% nitrogen, animals and plants can not directly utilize, and nature only has part prokaryotic micro-organisms to have the ability of directly utilizing nitrogen in atmosphere, are reduced ammonification, nitrogen fixation that Here it is.Population in the world, resource, environment have become current global problem, and the biological nitrogen fixation that is called as " Green Revolution " comes into one's own day by day, especially the application in forestry.Due to forestry, to have the production cycle long, and the features such as land occupation condition complexity make the fertilising of forestry the convenience unlike agricultural, effectively.Therefore, biological nitrogen fixation becomes the important sources of the required nitrogen of forest.
In the production of forestry of some developed countries, on certain degree, rely on the contribution of biological nitrogen fixation, pay attention to the development and utilization of nitrogen-fixing microorganism resource.Due to the raising of the rise of price, protection of the environment consciousness, biotechnological formulation is inexpensive with it, environmental protection and solely being favored.Many countries are all using researching and developing microbial fertilizer and goods as a far-reaching plan in the world.
Acacia Species (Acacia) is the important quick growing species of trees that China introduces a fine variety; it is fast-growing but also wide adaptability not only; drought-resistant hemorrhoid are thin; fast growing; can produce root nodule, have nitrogen fixation, there is good improvement soil function; plant experimentally large-scale planting up till now from its introduction, in forestry industry and improvement of the ecological environment, brought into play significant role.Need for progress of research and production of forestry application, select suitable root nodule bacterium microbial fertilizer, reduce discharging of chemical fertilizer, can realize the doulbe-sides' victory of economic benefit and ecological Efficiency, and can drive forestry technology progress.
Bacterial classification is the basis of microbial fertilizer production application.The bottleneck of the China of restriction at present microbial fertilizer industry development is the seed selection problem of strain excellent.Production of forestry active demand nitrogen fixing capacity is strong, the microbial fertilizer production bacterial strain of strong stress resistance.
Summary of the invention:
The object of this invention is to provide a kind of high-efficiency nitrogen-fixing dross, can think with black wooden yearning between lovers, net pulse-phase and novel bacterial-Ganzhou raw root nodule bacterium (Bradyrhizobium ganzhouense sp.nov.) RITF806 slowly of grey wooden yearning between lovers symbiosis dross, this bacterium is preserved in Chinese Typical Representative culture collection center (CCTCC) on February 26th, 2014, address: Wuhan, China Wuhan University, deposit number is CCTCC NO:M2014044.
The present invention also provide Ganzhou slowly raw root nodule bacterium (Bradyrhizobium ganzhouense sp.nov.) RITF806 in the application of preparing in nitrogen-fixing microorganism microbial inoculum and biological organic fertilizer.
The present invention is Ganzhou raw root nodule bacterium (Bradyrhizobium ganzhouense sp.nov.) slowly that separate from black wooden yearning between lovers root nodule, such bacterial strain can be thought the dross symbiosis such as (Acacia aneura) and grey wooden yearning between lovers (Acacia implexa) with black wooden yearning between lovers (Acacia melanoxylon), net pulse-phase, there is strong resistance, in nitrogen-fixing microorganism microbial inoculum and biological organic fertilizer production, have broad application prospects.
Ganzhou of the present invention slowly raw root nodule bacterium (Bradyrhizobium ganzhouense) RITF806 is preserved in Chinese Typical Representative culture collection center (CCTCC) on February 26th, 2014, address: Wuhan, China Wuhan University, deposit number is CCTCC NO:M2014044.
brief description of the drawings:
Fig. 1 is Ganzhou 16S rRNA of raw root nodule bacterium and phylogenetic tree of reference bacterial strain slowly
Fig. 2 is the phylogenetic tree that adopts maximum likelihood method to build, it is that the sequence assembly of glnII (497nt) and atpD (391nt) builds with Ganzhou housekeeping gene recA (355nt) of more close population in raw root nodule bacterium and Bradyrhizobium slowly.
Fig. 3 is the composition of Bradyrhizobium representative strain polarity ester, 1: Ganzhou is raw root nodule bacterium RITF806 bacterial strain slowly; 4:Bradyrhizobium huanghuaihaiense CCBAU23303
tbacterial strain
Fig. 4 is black wooden yearning between lovers seedling inoculation Ganzhou raw root nodule bacterium RITF806 slowly;
Fig. 5 is Ganzhou transmission electron microscope photo of raw root nodule bacterium RITF806 slowly.
Embodiment:
Following examples are to further illustrate of the present invention, instead of limitation of the present invention.
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
YMA medium: N.F,USP MANNITOL 10.0g, Sodium Glutamate 0.5g, K
2hPO
40.5g, MgSO
47H
2o0.2g, NaCl0.01g, yeast powder 0.5g, agar powder 17g, distilled water 1000mL, pH7.0.Sterilizing is for subsequent use.
Jensen ' s is without nitrogen nutrition liquid: K
2hPO
40.05g, NaCl0.05g, MgSO
47H
2o0.05g, CaHPO
40.25g, FeCl
30.05g, 1mL trace element solution (H
3bO
32.86g/L, MnSO
4h
2o2.08g/L, ZnSO
47H
2o0.022g/L, CuSO
47H
2o0.08g/L, NaMoO
40.11g/L), distilled water 1000mL.Sterilizing is for subsequent use.
Embodiment 1: Ganzhou is separation and the qualification of raw root nodule bacterium RITF806, RITF807, RITF211 slowly
One, the separation of root nodule bacterium
The root nodule of strains separation 12 years raw black wooden yearning between lovers from Ganzhou, Jiangxi Province.Root nodule is rinsed surperficial silt well with tap water, after 95% alcohol-pickled 15-30 second, puts into 0.1% mercuric chloride solution surface sterilization 3-5 minute, aseptic water washing 7-8 time.Root nodule after surface sterilization is placed between two aseptic slide glasss, and firmly extruding makes root nodule fragmentation, picks root nodule liquid at the flat lining out of YMA with aseptic inoculation pin, and 28～30 DEG C of constant incubators are cultured to bacterium colony appearance, picking colonies typical purifying and qualification.Purifying obtains root nodule bacterium RITF806, RITF807 and RITF211 thus.
Two, the qualification of root nodule bacterium
Identify root nodule bacterium RITF806, RITF807 and RITF211 from the following aspects:
1, Morphological Identification
Above-mentioned steps one is separated to also root nodule bacterium RITF806, RITF807 and the RITF211 of purifying and be cultured to logarithmic phase, and when bacterium colony size is stablized, carry out single bacterium colony state description, mainly comprise size, color, transparency, wettability, bacterium colony condition of surface, the colony edge state of bacterium colony.
For the described root nodule bacterium in logarithmic phase, after smear staining, adopt the form of observation by light microscope thalline.
Result shows, 3 strain root nodule bacterium RITF806, RITF807 and the RITF211 of above-mentioned steps one separation and purification are Gram-negative bacteria, bacterium colony circular protrusions, and oyster white, translucent, smooth surface, neat in edge; Thalline is shaft-like, and 1.29～3.32 × 0.50～0.60 μ m, without gemma.Its transmission electron microscope photo as shown in Figure 5.
2, analysis of physio biochemical characteristics
With reference to " common bacteria system identification handbook " (eastern elegant pearl, Cai Miaoying. common bacteria system identification handbook. Beijing: Science Press, 2011.) and " Microbiology Experiment " (Shen Ping, Fan Xiurong, Li Guangwu. Microbiology Experiment (third edition). Beijing: Higher Education Publishing House, 1999) measure the physiological and biochemical properties of above-mentioned root nodule bacterium.
The physiological and biochemical property measurement result of described root nodule bacterium is as follows:
The bacterium colony of root nodule bacterium RITF211, RITF806, RITF807 is sticky, neat in edge; On BTB substratum, produce alkali; React with litmus milk and produce alkali; Bacterial strain can utilize tween 80, D-Fructose, alpha-D-glucose, PEARLITOL 25C, Beta-methyl-D-Glucose glycosides, turanose, methyl-prop ketone acid, citric acid, D-galacturonic acid, D, and Pfansteihl is as sole carbon source; Can on glucuronamide, the substratum of hydroxyl-L-PROLINE as only nitrogen source, grow; Can on the substratum of following antibiotic concentration (μ g/ml), grow: penbritin 5～300, paraxin 5～300, erythromycin 5～100, neomycinsulphate 5～300, tetracycline hydrochloride 5; Can in the YMA medium that contains dyestuff below 1%, grow: hydrochloric acid acridine, Sodium desoxycholate, Congo red, algae is red, dibromothymolsulfonphthalein, Viola crystallina, toluylene red, Sodium Nitrite; Catalase experiment, urease test, oxidase test, nitrate reduction reacting positive; The test of L-Phe desaminase, 3-ketone group lactose test, Nile blue reduction reaction, methylenum coeruleum reduction reaction feminine gender; Can not on broth culture, grow.
3,16S rRNA sequencing and Phylogenetic Analysis
Ordinary method is cultivated root nodule bacterium RITF806, RITF807 and RITF211, extract total DNA of bacterial strain as gene amplification template, with bacterial 16 S rRNA universal primer, 27F:5 '-AGAGTTTGATCCTGGCTCAG-3 ', 1492R:5 '-TACGGTTACCTTGTTACGACTT-3 ' carries out PCR reaction.Reaction system adopts Shanghai biotechnology company limited pcr amplification test kit.Response procedures is: 95 DEG C of sex change 1min, 56.7 DEG C of 1min, 72 DEG C of extensions 1.5min, totally 30 circulations.DNA sequencing is completed by the English Weihe River prompt base (Shanghai) biotech company, and sequence assembly and similarity analysis use DNAMAN software to complete, and sequence alignment completes online by American National biotechnology information center ncbi database.
Root nodule bacterium RITF806(SEQ ID NO.1), RITF807(SEQ ID NO.2) and RITF211(SEQ ID NO.3) 16S rRNA sequence respectively as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.With the contribute phylogenetic tree (Fig. 1) of (1250nt) of the adjacent method of 16S rRNA gene, Bradyrhizobium ganzhouense sp.nov.RITF806 and other kinships of taking root slowly between knurl bacterial classification group are shown.3 protein coding housekeeping gene recA, glnII, atpD phylogenetic trees of root nodule bacterium RITF806, RITF807, RITF211 bacterial strain are shown in Fig. 2.16S rRNA between root nodule bacterium RITF806, RITF807, RITF211 bacterial strain and other slow raw root nodule bacterium and the similarity scope of 3 protein coding housekeeping genes are in table 1.
In watch 1Bradyrhizobium ganzhouense kind bacterial strain and and other slow raw root nodule bacterium between 16S RNA and the similarity of 3 protein coding housekeeping genes
*aligned region length (bp): 16S rRNA gene (1250), recA (355), glnII (497) and atpD (391)
three bacterial strain: RITF806
t, RITF807and RITF211.
4, DNA homology is analyzed
Utilize a large amount of 3 strain root nodule bacterium RITF806, RITF807, RITF211 and the reference bacterial strain DNA of extracting of ordinary method.The reference bacterial strain of choosing to comparatively similar for examination root nodule bacterium, is respectively Bradyrhizobium cytisi CTAW11 aspect genotype and phenotype
t, Bradyrhizobium huanghuaihaiense CCBAU23303
t, Bradyrhizobium diazoefficiens USDA110
t, Bradyrhizobium rifense CTAW71
t, Bradyrhizobium canariense LMG22265
t, Bradyrhizobium japonicum USDA6
t, Bradyrhizobium betae PL7HG1
t.Adopt renaturation rates to measure the DNA homology of bacterial strain.The results are shown in Table 2.
Table 2. is for the DNA homology of examination root nodule bacterium and close reference bacterial strain
Result shows, the DNA homology between described 3 strain root nodule bacterium RITF806, RITF807, RITF211 is higher than 78%, and with reference bacterial strain homology lower than 70%, belong to the novel species of root nodule bacterium.(G+C) mol% content of bacterial strain RITF211, RITF806, RITF807 is respectively 62.8,64.6,65.2, and result is consistent with (G+C) mol% content value (62.7-65.1mol%) of close reference bacterial strain.
5, bacterial strain determination of fatty acid
Lipid acid contamination is measured and is adopted MIDI Sherlock microbial identification system, contains 9 kinds of lipid acid, wherein 16:0 and Summed Feature8 content the highest (table 3) in bacterial strain RITF806 cell.
Table 3.Bradyrhizobium ganzhouense RITF806
tfatty acid type with close slow raw root nodule bacterium
*Summed?feature3=16:1ω6c/16:1ω7c.
Summed?feature8=18:1ω6c/18:1ω7c.
These data are to detect gained by MIDI system, and database is TSBA6.Bacterial strain: 1, B.ganzhouense RITF806
t; 2, B.cytisi CTAW11
t; 3, B.diazoefficiens USDA110
t; 4, B.rifense CTAW71
t; 5, B.huanghuaihaiense CCBAU23303
t.-, do not detect.
6, polarity ester measurement result
The composition of Bradyrhizobium representative strain polarity ester is shown in Fig. 3.Known according to figure, the polarity ester that representative strain RITF806 contains is mainly diphosphatidylglycerol (DPG), aminolipid (AL), phosphatidyl glycerol (PG), phosphatidylethanolamine (PE), Yelkin TTS (PC) and unknown polar lipid (L).Reference bacterial strain Bradyrhizobium huanghuaihaiense CCBAU23303
tin also detect phosphatidylmonomethylethanolamine (PL), and PL does not exist in representative strain RITF806, and the two aminolipid containing (AL) kind difference.
5, growth characteristics analysis
Bacterial strain optimum temperuture and optimal pH and salt tolerant growth experiment are carried out.Adopt YMA medium, cultivate the thermal adaptability of observing, recording bacterial strain respectively at 4 DEG C, 10 DEG C, 28 DEG C, 37 DEG C, 60 DEG C, each processing repeats for 3 times.Adjust YMA medium acidity respectively at pH4, pH5, pH7, pH9, pH10, pH11 and pH12, each processing repeats for 3 times, cultivates, observes, records the optimal pH of bacterial strain.In YMA medium, add NaCl, make its final concentration be respectively 1%, 2%, 3%, 4% and 5%, each processing repeats for 3 times, the growth of cultivating, observe, recording bacterial strain.
Result shows, the optimum growth temperature of 3 described strain root nodule bacterium is 28 DEG C, and the most suitable growth pH is 7.Can under 4 DEG C, 10 DEG C, 28 DEG C, 37 DEG C conditions, grow, cultivate and can not grow after 10 minutes through 60 DEG C and 70 DEG C of heat shocks, optimum growth temperature is 28 DEG C; PH tolerance range is 5-12, and optimal pH is 7.0; NaCl tolerance range is 0-5%, and more than 3% growth is poor.
In view of results such as above-mentioned form, physiological and biochemical property, 16S rRNA sequencing and Phylogenetic Analysis, DNA homology analysis and lipid acid, polar lipid content, the 3 strain Rhizobium Identifications that step 1 separation and purification is obtained are Ganzhou raw root nodule bacterium (Bradyrhizobium ganzhouense sp.nov.) slowly.Type strain-Ganzhou of this kind slowly raw root nodule bacterium (Bradyrhizobium ganzhouense sp.nov.) RITF806 has been deposited in Chinese Typical Representative culture collection center (CCTCC) on February 26th, 2014, depositary institution address is: Wuhan, China Wuhan University, deposit number is CCTCC NO:M2014044.
Embodiment 2, Ganzhou is the black wooden yearning between lovers effect of raw root nodule bacterium (Bradyrhizobium ganzhouense) RITF806 inoculation slowly
One, bacterium liquid is cultivated
By Ganzhou slowly raw root nodule bacterium RITF806 bacterial strain after activating, proceed to YMA liquid nutrient medium on YMA inclined-plane, 28-30 DEG C of shaking table shaking culture (150 revs/min of rotating speeds) is to the logarithmic growth later stage.
Two, inoculation
Nursery stock adopts black wooden Tissue Cultured Acacia seedling, the yellow soil of matrix without sterilizing, and selecting diameter is the plastic tub of 12cm, basin bottom part is put double-deck filter paper, adds yellow soil to pelvic surface of sacrum 1.5cm place.Get the approaching Tissue Cultured Acacia bottle seedling of growing way, root agar cleaned up to rear plantation in filling the plastic tub of yellow soil with distilled water, every pot transplanting 1 young plant, between nursery stage according to conventional seedbed system management by methods.Transplant and inoculate after 1 month, (cell concentration is 10 in each basin, to inject above-mentioned bacterium liquid
8cUF/mL) 5mL, contrast (CK) is not inoculated.Test adopts single factors randomized blocks design, and each processing repeats for 30 times.Inoculate and after 5 months, observe dross and growing state.
Three, mensuration and method of calculation
1, statistics plant height of seedling, over-ground part and underground part dry weight, root nodule numbers, root nodule dry weight.
2, biomass estimation adopts plant sample at 80 DEG C of title dry weights that dry to constant weight, sample nitrogen content Kjeldahl nitrogen determination.
3, the calculating of total nitrogen, amount of nitrogen fixation and fixed nitrogen benefit: total nitrogen=biomass × nitrogen content; Amount of nitrogen fixation=the connect total nitrogen of total nitrogen-adjoining tree of bacterium plant; Fixed nitrogen benefit=amount of nitrogen fixation/total nitrogen × 100%.
Result is as shown in Fig. 4, table 4 and table 5, and black wooden yearning between lovers inoculation Ganzhou after raw root nodule bacterium RITF806, can promote the growth of nursery stock slowly, improves its dross quantity, height of seedling, over-ground part and over-ground part dry weight, respectively than contrast raising 81.09-152.9%(table 4); Fixed nitrogen benefit reaches 61.47%(table 5).
Watch 4 black wooden yearning between lovers inoculation Ganzhou growth indexes of raw root nodule bacterium RITF806 slowly
Watch 5 black wooden yearning between lovers inoculation Ganzhou fixed nitrogen effect of raw root nodule bacterium RITF806 slowly
Embodiment 3, Ganzhou is raw root nodule bacterium (Bradyrhizobium ganzhouense) RITF806, RITF211 and the black wooden yearning between lovers of RITF807 inoculation, net pulse-phase think of and grey wooden yearning between lovers slowly.
One, bacterium liquid is cultivated
By Ganzhou slowly raw root nodule bacterium RITF806, RITF211 and RITF807 bacterial strain on YMA inclined-plane, after activation, proceed to YMA liquid nutrient medium respectively, 28-30 DEG C of shaking table shaking culture (150 revs/min of rotating speeds) is to the logarithmic growth later stage.
Two, seed treatment and sprouting
First soak seed half an hour with the vitriol oil, then the vitriol oil is poured out, then with after sterilized water washing 7～8 times, be dipped into seed with sterilized water and expand.The seed of processing is placed on to (interior pad aseptic filter paper adds sterilized water moisturizing) in sterile petri dish, is placed in 30 DEG C of incubators and germinates.
Three, inoculation and nursery stock are cultivated
After seed germination, seed is entered to be equipped with in the test tube of aseptic vermiculite (mix without nitrogen water planting liquid with Jensen ' s wet, aluminium foil sealing, 121 DEG C of sterilizing 1h), every pipe 1 strain, cultivate 2～3 days in 28 DEG C of growth cabinets after, bacterium liquid is poured in test tube, every pipe 2ml, every strain bacterium connects 10 young plants, then putting into growth cabinet cultivates, intensity of illumination 7000lux, temperature maintains 26 DEG C of left and right, light application time 12h.In process of growth, within every 5 days, water one time sterilized water.Cultivate after 3 months, observe dross and growing state.
Four, mensuration and method of calculation
1, statistics plant knurl number, knurl weight, fresh weight etc.
2, biomass estimation adopts plant sample at 80 DEG C of title dry weights that dry to constant weight, sample nitrogen content Kjeldahl nitrogen determination.
3, the calculating of total nitrogen, amount of nitrogen fixation and fixed nitrogen benefit: total nitrogen=biomass × nitrogen content; Amount of nitrogen fixation=the connect total nitrogen of total nitrogen-adjoining tree of bacterium plant; Fixed nitrogen benefit=amount of nitrogen fixation/total nitrogen × 100%.
Result shows, black wooden yearning between lovers, grey wooden yearning between lovers and net pulse-phase are thought inoculation Ganzhou slowly after raw root nodule bacterium RITF806, RITF211 and RITF807, can promote the dross quantity of seedling, promote the growth (showing 6-9) of seedling.Nursery stock fresh weight 14.40-55.18% is improved in black wooden yearning between lovers inoculation Ganzhou slowly raw root nodule bacterium; Nursery stock fresh weight 27.18-36.91% is improved in the wooden yearning between lovers inoculation of ash Ganzhou slowly raw root nodule bacterium; Net pulse-phase is thought inoculation Ganzhou raw root nodule bacterium raising nursery stock fresh weight 14.06-49.40% slowly.
Watch 6 black wooden yearning between lovers inoculation Ganzhou growth indexes of raw root nodule bacterium slowly
Watch 7 black wooden yearning between lovers inoculation Ganzhou fixed nitrogen effect of raw root nodule bacterium slowly
Watch 8 grey wooden yearning between lovers inoculation Ganzhou growth indexes of raw root nodule bacterium slowly
Watch 9 net arteries and veins inoculation Ganzhou growth indexes of raw root nodule bacterium slowly
Claims (2)
1. Ganzhou raw root nodule bacterium (Bradyrhizobium ganzhouense sp.nov.) RITF806 slowly, its deposit number is CCTCC NO:M2014044.
Ganzhou claimed in claim 1 slowly raw root nodule bacterium (Bradyrhizobium ganzhouense sp.nov.) RITF806 in the application of preparing in nitrogen-fixing microorganism microbial inoculum or biological organic fertilizer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567615A (en) * | 2016-03-14 | 2016-05-11 | 中国科学院烟台海岸带研究所 | Bradyrhizobium sp. and application thereof |
| CN113943674A (en) * | 2021-10-29 | 2022-01-18 | 中国农业科学院农业资源与农业区划研究所 | In-situ efficient composite rhizobium flora |
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| CN102174436A (en) * | 2011-01-24 | 2011-09-07 | 领先生物农业股份有限公司 | Bradyrhizobium japonicum capable of effectively fixing nitrogen and culture method and application thereof |
| CN102181391A (en) * | 2011-03-23 | 2011-09-14 | 西南林业大学 | T49MA strain for improving dry resistance and heat resistance of leguminous trees and culture method for root nodule seedling of T49MA strain |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102174436A (en) * | 2011-01-24 | 2011-09-07 | 领先生物农业股份有限公司 | Bradyrhizobium japonicum capable of effectively fixing nitrogen and culture method and application thereof |
| CN102181391A (en) * | 2011-03-23 | 2011-09-14 | 西南林业大学 | T49MA strain for improving dry resistance and heat resistance of leguminous trees and culture method for root nodule seedling of T49MA strain |
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| JUN KUN LU等: "Bradyrhizobium ganzhouense sp. nov., an effective symbiotic bacterium isolated from Acacia melanoxylon R. Br. nodules", 《INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567615A (en) * | 2016-03-14 | 2016-05-11 | 中国科学院烟台海岸带研究所 | Bradyrhizobium sp. and application thereof |
| CN113943674A (en) * | 2021-10-29 | 2022-01-18 | 中国农业科学院农业资源与农业区划研究所 | In-situ efficient composite rhizobium flora |
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