CN107904192A - Rhizobium V9 2 and its application - Google Patents
Rhizobium V9 2 and its application Download PDFInfo
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Abstract
The invention discloses rhizobium (Rhizobium anhuiense) V9 2 and its application, China typical culture collection center is preserved in, deposit number is CCTCC NO：M 2017610.The rhizobium V9 2 of the present invention is that one plant of symbiotic nitrogen fixation ability is strong, obvious to the son volume increase of Sichuan Hairy vetch, with producing IAA ability, has the strong excellent pale reddish brown sweet potato rhizobium strains of molten Phos, molten potassium capability, resistance.In not nitrogen fertilizer application, Rhizobium Inoculation V9 2 green forage yield of pale reddish brown sweet potato can be made to increase production 22.7%, fresh quench yield volume increase 79.8%.
Description
Technical field
The invention belongs to microorganism field, and in particular to rhizobium V9-2 and its application.
Background technology
Pale reddish brown sweet potato (Vicia villosaL.) is the main leguminous greenmanuring crop of China Water rice region, and it is simultaneous to be used as green manure
Forage grass cultivation main sweet potato kind, be good organic fertilizer (Fu Yunzhang, 2017；Cao Weidong etc., 2010).It is soft with stem juice
Tender, the features such as leaf amount is more, yield is high, palatability is good, full of nutrition, be good green forage, and the good of concentrated feed is replaced
Dai Pin and mixed feed raw material, it is deep liked by the aquaculture peasant such as Sichuan, Yunnan, Guizhou (Guo Tailei, 2013；Shi Wanqing,
2016).Pale reddish brown sweet potato is general in planting process to be achieveed the purpose that to increase yield using chemical fertilizer, and research finds that Yuexi County peasant exists
Plant pale reddish brown sweet potato per acre apply farmers' farmyard manure 300kg, phosphate fertilizer 10kg can achieve the purpose that stable yields volume increase (Wang Demeng,
2006).But there are the problems such as production cost is high, utilization rate is low, pollution of ecological environment for chemical fertilizer application.And legume because with root nodule
Bacterium characteristics of Symbiotic Nitrogen can reach crop yield without applying chemical fertilizer and being only inoculated with high-efficiency root-nodule bacteria in process of production.Example
Such as Brazil plantation soybean, nitrogen fertilizer application is not only inoculated with effective rhizobium, and yield applies the suitable of 400kg chemical fertilizer with per hectare, annual to save
Nitrogenous fertilizer be worth up to 2,500,000,000 dollars.By Rhizobium Inoculation, annual biological nitrogen fixation amount is about 6,200,000 t, accounts for the U.S. and disappears then in the U.S.
Consume more than the 55% of nitrogenous fertilizer total amount.Therefore, artificial infection rhizobium are to promote legume growth, improve legume yield,
The Important Agricultural measure improved the ecological environment.
Focus mostly at present on high-efficiency root-nodule bacteria screening application study in the higher legume of the economic values such as soybean,
Research report applied to pale reddish brown sweet potato high-efficiency root-nodule bacteria is seldom.Nanjing Soil Inst., Chinese Academy of Sciences's rhizobium group (1974)
The screening of sweet potato rhizobium strain excellent is carried out between 1971 and 1973 in Jiangsu Province, from the 162 plants of rhizobium isolated
In filter out 7 plants of Rhizobium strains that 7 plants of growths are fast, dross is early, nitrogen fixing capacity is strong；In September, 1972 is in yangtze river county Zhang Jigong
Society has carried out the experiment of sweet potato Rhizobium Inoculation, it is found that sweet potato rhizobium are inoculated with equal fertilizer application condition can improve the first flower bud phase of sweet potato
Green forage yield, increases production 6.2%-39.1%.But the population distribution of rhizobium has geographic limitations, in the screening of high-efficiency root-nodule bacteria
In, it is necessary to pay attention to the adaptability (Chen Wen new etc., 2004) of rhizobium correspondence area surroundings.In general, in some region
Interior maximally efficient rhizobium often from this area or regional bacterial strain similar to this area condition (Jiaetal, 2008；
Chen Wen is new etc., and 2011).The research of the efficiently pale reddish brown sweet potato rhizobium of suitable Sichuan ecological environment is almost blank at present.
Now some researches show that only a few rhizobium also have the growth-promoting characteristics such as Soluble phosphorus, secretion growth hormone (IAA).Shi Shangli
(2007) etc. more than 730 lucerne rhizobiums study and be just sieved to 29 plants of rhizobium with preferable growth-promoting ability, only from this
29 plants of rhizobium screen 10 plants of strong rhizobium of producing IAA ability, and dissolved organic phosphorus and Phos energy are carried out to this 29 plants of bacterium
The measure of power, find all strains examined there is no the ability of dissolved metals, this 29 rhizobium meliotis can dissolved organic phosphorus, but
Phosphate solubilization differs greatly, and only 8 bacterial strains have the ability of stronger dissolved organic phosphorus, remaining bacterial strain has weak or faint dissolved with machine
The ability of phosphorus.
It can be seen from the above that the growth area surroundings of the practical effect of rhizobium and the species of legume and legume
Closely related, effect may not just very well, at certain on another legume for the good rhizobium of effect on a kind of legume
The good rhizobium of ground effect may not be good in other Regional Effect.
Therefore it is particularly important that screening the high-quality and efficient multi-functional growth-promoting Rhizobium strains for being adapted to specific region and specified plant.
The content of the invention
The technical problems to be solved by the invention are：How a kind of the high-quality of the pale reddish brown sweet potato plantation in adaptation Sichuan province is provided
Efficient rhizobium.
The technical scheme is that：Rhizobium (Rhizobium anhuiense) V9-2, was protected on October 23rd, 2017
The China typical culture collection center being hidden in Wuhan University of Wuhan City of Hubei China province, deposit number are：CCTCC NO：
M2017610。
Applications of the rhizobium V9-2 in the son plantation of Sichuan province Hairy vetch.
The present invention boorishly gives birth to from the double lions in Liangshan State of Sichuan Province Yanyuan County plum rains town and root is separated and purified in pale reddish brown sweet potato root nodule
Knurl bacterium, selection have the characteristics that typical rhizobium bacterium colony, and Gram's staining result is in small rod-shaped, gram-negative bacterium for cell
Strain.
Water planting tieback experiment is carried out with Hairy vetch kind to primary election bacterial strain, filtering out can dross and Noduling ability
By force, the good bacterial strain of symbiotic nitrogen fixation effect is aimed strain rhizobium V9-2.Compared to not Rhizobium Inoculation, pale reddish brown sweet potato plant does
Bring up again high by 25.8%.
STb gene is extracted to rhizobium V9-2, multiple housekeeping genes are sequenced, phylogenetic tree construction, hard objectives
The classification position of bacterial strain rhizobium V9-2.Show that rhizobium V9-2 belongs to Rhizobium anhuiense.
Rhizobium V9-2 carries out acid and alkali-resistance, salt tolerant, the experiment of the resistance such as growth temperature range, and producing IAA ability, molten
The growth-promoting experiment of phosphorus ability, and Field inoculation experiment.The result shows that：The acidproof alkali abilities of rhizobium V9-2 are stronger, can pH4~
Grown on 11 tablet, but peracid or positive control of the colony diameter less than pH7 excessively on the tablet of alkali, illustrate that peracid crosses alkali to it
Growth has certain inhibitory action；Salt resistance ability is also relatively strong, can be grown in the YMA tablets of NaCl 0.2%~4%；Growth temperature
It is wider to spend scope, can be grown in 20~28 DEG C of temperature ranges.Rhizobium V9-2 producing IAA 0.29mg/L, show rhizobium V9-
2 have the ability of producing IAA concurrently.Rhizobium V9-2 has calcium phosphate certain solvability, is measured on calcium phosphate tablet molten
Phosphorus loop diameter and colony diameter ratio are respectively 1.02, are not grown on organophosphor culture medium.Rhizobium V9-2 dissolves potassium
1.48mg/L, shows the ability that rhizobium V9-2 has molten potassium.Field inoculation shows Rhizobium Inoculation V9-2 compared to not being inoculated with
Rhizobium, can make the green forage yield of pale reddish brown sweet potato increase production 22.7%, fresh quench yield volume increase 79.8%.
Compared with prior art, the invention has the advantages that：
The rhizobium V9-2 of the present invention is that one plant of symbiotic nitrogen fixation ability is strong, obvious to the volume increase of the pale reddish brown sweet potato in Sichuan, with
Producing IAA ability, have the strong excellent pale reddish brown sweet potato rhizobium strains of molten Phos, molten potassium capability, resistance.Do not applying nitrogen
Fertilizer, Rhizobium Inoculation V9-2 can make the green forage yield of pale reddish brown sweet potato increase production 22.7%, fresh quench yield volume increase 79.8%.
Preservation explanation：
Rhizobium (Rhizobium anhuiense) V9-2, Hubei China province Wuhan is preserved on October 23rd, 2017
China typical culture collection center in Wuhan University of city, deposit number are：CCTCC NO：M 2017610.
Brief description of the drawings
Fig. 1 is colonial morphologies of the rhizobium V9-2 on YMA medium；
Fig. 2 is the systematic growth figure of the 16rRNA gene orders of rhizobium V9-2；
Fig. 3 is the systematic growth figure of tri- housekeeping gene joint mappings of recA, atpD, glnII of rhizobium V9-2.
Embodiment
Separation, purifying and the preservation of 1 rhizobium V9-2 of embodiment
From the wild pale reddish brown sweet potato of Liangshan State of Sichuan Province Yanyuan County plum rains town Shuan Shi villages collection, healthy and strong plant main root is selected
Upper big and full red root nodule, root nodule are cleaned, and band part root skin is adopted down, carries out surface sterilization：5min is soaked with ethanol (95%),
Outwell, then 5min is soaked with mercuric chloride (0.1%), finally with aseptic water washing 6-8 times.Root nodule is put into superclean bench and is gone out
On white plaque after bacterium, clip broken root nodule with bamboo stick and be applied on the tablet of YMA medium, carry out line purifying, in 28 DEG C of perseverance
Cultivated in incubator.YMA medium formula：KH2PO40.25g, MgSO4`7H2O 0.2g, CaCl2`6H2O 0.1g, NaCl 0.1g,
Ammonium molybdate (1%) 2mL, boric acid (1%) 2mL, Congo red (1%) 2.5mL, mannitol 10g, dusty yeast 0.8g, agar 18~
20g, water 1000mL, pH6.8-7.0.
After tablet grows bacterium colony, the clearance permit bacterium colony of not inhaling in form as rhizobium is selected from tablet and is carried out on tablet
Dilution line culture.3d or so observes colonial morphology, observes 7d or so always, because slow raw rhizobium need 6~10d bacterium occur
Fall.Dilution streak plate partition method is purified.Whether preliminary judgement bacterium colony is that rhizobium then carry out according to following two aspects：
(1) colonial morphology on YMA medium：Milky, do not inhale red, bacterium colony is circular, protuberance, neat in edge is not spread, surface is smooth,
Compared with moistening, it is more sticky.What culture 3d~5d grew bacterium colony is fast raw rhizobium, and what 6~10d of culture grew bacterium colony is slow knurl of taking root
Bacterium.(2) cellular morphology：Mark to the rhizobium bacterium colony of confirmation, film-making carries out Gram's staining, and the microscopy result of rhizobium is
Cell is consistent in small rod-shaped and form, no gemma, intracellular often in the form of link, the Gram-negative (G containing beta-hydroxy-butanoic acid-)。
If above-mentioned mark bacterium colony has above-mentioned two aspects feature, the test tube slant culture of bacterium colony access YMA medium is preserved.
The rhizobium V9-2 isolated and purified of the present embodiment is fast raw rhizobium, is adding Congo red YMA medium
Upper culture, thalline are not inhaled red, and milky, bacterium colony are smaller, circular, sticky, Rana guadranus is higher, slightly transparent, and 2-3d grows bacterium colony.Through
Gram stain microscopy is G-, in small rod-shaped.
The tieback experiment of 2 rhizobium of embodiment
The tieback experiment sandy culture of rhizobium, the pale reddish brown sweet potato kind in experiment are Hairy vetch, are purchased from into
All greenweed Yuan Zhong industry Co., Ltds.In illumination room (22~24 DEG C of temperature control, 2800 lux of intensity of illumination or so, during sunshine
Between 14h) carry out, plantation 46d harvest.Regular replenishment sterilizing nitrogen-free nutrient solution, rhizobium V9-2 is inoculated into sand culture device, sand culture
Device uses the plastic cup of 300mL, and matrix selects vermiculite, using the same kind plant of not Rhizobium Inoculation V9-2 as control CK, everywhere
Reason is repeated three times.Imitated after harvest with the root nodule number and plant weights of pale reddish brown sweet potato plant to evaluate the inoculation of rhizobium V9-2
Fruit.
(1) bacterium solution culture：Rhizobium V9-2 is inoculated in YMA fluid nutrient mediums, places and uses 120rpm/min on shaking table
Rotating speed, 28 DEG C of cultures to exponential phase (about 3d or so).
(2) vernalization of seed：Big, the full undamaged pale reddish brown sweet potato seed of grain is selected, 5min is soaked with 95% alcohol, falls
Alcohol is removed, adds mercuric chloride (0.1%) immersion surface sterilization 5min.Finally use sterile water wash 6~8 times, each 5min.Finally will
Seed spreads and vernalization is carried out in sawdust after sterilization, and 28 DEG C of germination temperature, treats that main root grows to 2~3cm or so, when fibrous root is not grown
Sowing.
(3) making of sand culture device：Select the plastic cup of 300mL to make sand culture device, first the vermiculite after sterilizing is added and was sterilized
Plastic cup in volume account for the 3/4 of whole cup, add sterile nitrogen-free nutrient solution and keep vermiculite moistening, plastic cup and leech
Stone is sterilized separately, and selects particle diameter to be used for separating mould contamination for the quartz sand paving of 2-4mm, quartz sand also needs to sterilize.
(4) nitrogen-free nutrient solution prescription：0.46g calcium sulfate, 0.136g dipotassium hydrogen phosphates, 0.075g potassium chloride, 0.075g lemons
Lemon acid iron, 0.06g magnesium sulfate, 0.03g calcium nitrate, 1000mL distilled water, 1mL trace element solutions.
(5) trace element solution formula：2.86g boric acid, 1.81g manganese sulfates, 0.8g copper sulphate, 0.22g zinc sulfate,
0.02g molybdic acids add distilled water to 1000mL.
(6) plantation and testing index：Vernalization seed is planted in the plastic cup for installing vermiculite, bacterium solution is drawn with liquid-transfering gun
2mL is gently beaten near pale reddish brown sweet potato root.It is another to set the same kind plant for not being inoculated with processing as control (CK).CK is first planted during plantation
Avoid connecing bacterium pollution.Each processing is repeated 3 times.The results are shown in table 1 for water culture experiment.
Table 1 the result shows that, the rhizobium V9-2 with for examination Hairy vetch sublist reveal preferable Noduling ability and be total to
Growing nitrogen-fixing ability；Compared with the not control of Rhizobium Inoculation V9-2, V9-2 can significantly improve the plant weights of Hairy vetch,
Control than not Rhizobium Inoculation improves 25.8%.As it can be seen that the rhizobium V9-2 be matched with Hairy vetch it is excellent
Good high efficient strain.
The water culture experiment result of 1 rhizobium V9-2 of table
Note：Data are average value three times repeatedly；
The anti-adversity ability of 3 rhizobium V9-2 of embodiment
The measure of salt tolerant, acid and alkali-resistance has been substantially carried out to the anti-adversity ability of rhizobium V9-2.Trained based on YMA medium
Base is supported, using pH7,28 DEG C of YMA tablets for cultivating 7d as positive control (CK).The YMA inclined-planes of above-mentioned rhizobium V9-2 are trained
Support thing and scrape that to be made as bacteria suspension stand-by with sterile water.Using an inoculation method, each processing is repeated 3 times.Acid and alkali-resistance, salt tolerant examination
Test, the tablet of suitable growth temperature scope measure observes record result after cultivating 4d at 28 DEG C.
(1) acid and alkali-resistance measure culture medium prescription：Using YMA medium as basic culture medium, pH is adjusted with HC1 and NaOH
Value, makes the pH value of culture medium be followed successively by 4.0,5.0,6.0,8.0,9.0,10.0,11.0.
(2) Salt resistant test culture medium prescription：Using the YMA medium for removing NaCl as basic culture medium, NaCl is set
Concentration, make NaCl0.2%, 0.5%, 1.0%, 1.5%, 2%, 2.5%, 3%, 3.5% and 4% of culture medium.
Result of the test shows that the acidproof alkali abilities of rhizobium V9-2 are stronger, can be grown on the tablet of pH4~11, but peracid or
The positive control that colony diameter on the tablet of alkali is less than pH7 is crossed, illustrates that peracid crosses alkali and has certain inhibitory action to its growth；It is resistance to
Salt ability is also relatively strong, can be given birth in the YMA tablets of NaCl 0.2%-4%.
The growth-promoting ability of 4 rhizobium V9-2 of embodiment
The growth-promoting ability of rhizobium V9-2 has mainly investigated plant auxin secretion (IAA), Soluble phosphorus and molten potassium capability.
(1) measure of plant auxin secretion ability
It is firm using improvement using the ability of colorimetric method for determining rhizobium plant auxin secretion (IAA), measure culture medium
Arnotto fluid nutrient medium, culture medium composition：10g mannitol, 1g yeast extracts, 1g NH4NO3、0.5g K2HPO4·3H2O、0.2g
MgSO4·7H2O, 0.1g NaCl, 10mL Congo red (0.25%), 100mg L-Trps, 1000mL distilled water, pH=7.0.
Colorimetric formula of liquid：0.5M FeCl31mL, dense H2SO430mL, distilled water 50mL.
By inoculation in the triangular flask for filling 50mL culture mediums, rotating speed 160rpm/min is placed in, 28 DEG C of temperature is shaken
Bed culture, 3 repetitions, after cultivating 7d, 8000rpm/min centrifugation 10min, take bacterium solution 10mL, add 10mL color solutions, lucifuge
Wavelength 530nm measure OD values are selected after 30min on ultraviolet specrophotometer.According to mark song conversion IAA contents.The system of mark song
Make：Be made into 0 with IAA storing solutions, 5,10,20, the series standard liquid of 40mg/L make working solution.Colorimetric results measure, rhizobium V9-
2 producing IAA 0.29mg/L, show the ability that rhizobium V9-2 has producing IAA concurrently.
(2) ability of molten organophosphor and Phos
Select the ability of the molten organophosphor of Soluble phosphorus circle method measure bacterial strain and Phos.Organic phosphorus sources are lecithin, inorganic phosphorous sources
For Ca3(PO4)2、AlPO4.2H2O、FePO4.2H2O, above reagent are ommercially available AR.By Meng Jinna culture mediums and
PKO culture mediums make tablet, and prepared by strain and point inoculation method is tested with 3 resistance of embodiment, and each processing is repeated 3 times.28
Cultivate whether observation bacterial strain after 7d grows and whether to have Soluble phosphorus to iris out existing in DEG C incubator.
1. the culture medium of dissolved organic phosphorus ability is with Meng Jinna culture medium prescriptions (g/L)：10g glucose, 5g CaCO3,
0.5g(NH4)2SO4, 0.4g dusty yeasts, 0.3g KCl, 0.2g lecithin, 0.3g NaCl, 0.03g MnSO4·4H2O, 0.03g
FeSO4·7H2O, 20g agar, 1000mL distilled water, pH value 6.8~7.0.Wherein lecithin is dissolved by heating with 75% alcohol, single
Solely sterilizing, is cooled to after 60 DEG C or so of culture medium mixes with sterilizing and is down flat plate.
2. the culture medium of dissolved metals ability is with PKO culture medium prescriptions (g/L)：10g glucose, the above-mentioned Phos of 3.0g
Source material (Ca3(PO4)2、AlPO4.2H2O、FePO4.2H2O), 0.5g (NH4)2SO4, 0.2g KCl, 0.2g NaCl, 0.03g
MnSO4, 0.03g MgSO4.7H2O, 0.003g FeSO4.7H2O, 0.5g dusty yeast, 20g agar, 1000mL distilled water, pH value
6.8~7.0.After wherein calcium phosphate, aluminum phosphate, ferric phosphate pulverized 300 mesh sieves and individually hot air sterilization with mortar, with sterilizing temperature
The culture medium mixing that degree is down to 60 DEG C or so is down flat plate.
The result shows that rhizobium V9-2 has calcium phosphate on certain solvability, the Soluble phosphorus circle measured on calcium phosphate tablet
Diameter and colony diameter ratio are respectively 1.02, are not grown on organophosphor culture medium.
(3) ability of molten potassium
With the molten potassium capability of flame spectrophotometric determination.Potassium resource selection washing potassium feldspar miberal powder (800 mesh).Without potassium nutrient solution
(1L)：Sucrose 10g, MgSO4·7H2O 0.5g, NaCl 0.1g, yeast extract 0.5g, pH7.2-7.4.
Shaking flask is done using 250mL triangular flasks, loads no potassium basal liquid medium 100mL, addition potassium feldspar miberal powder (800
Mesh) 0.5g, the bacteria suspension of 5mLV9-2 is inoculated with, compares and connects equivalent sterile water, 3 repetitions, 28 DEG C of 160rpm/min cultures are set
7d, takes 10mL nutrient solutions, 8000r/min centrifugation 10min, take supernatant, soluble potassium in supernatant is surveyed with flare photometer
Content.Rhizobium V9-2 dissolves potassium 1.48mg/L, shows the ability that rhizobium V9-2 has molten potassium.
The amplification of the 16S rRNA genes and other housekeeping genes recA, glnII, atpD of 5 rhizobium V9-2 of embodiment and
Phylogenetic Analysis
Bacterial strain STb gene is extracted, primer carries out PCR amplification, PCR reactions Bio- to above-mentioned 4 genes respectively shown in table 2
RAD MyCyclerTM instruments, after pcr amplification product detects on 1.0% agarose gel electrophoresis, are sent to Chengdu Qing Ke Chinese catalpas
Prosperous Bioisystech Co., Ltd carries out the measure of sequence.The calculating of gene order similarity is carried out with software DNAman 6.0.
PCR primer used in 2 experiments of table
Note：Y=C or T, H=A, C or T, R=A or G, S=C or G, K=G or T, N=A, C, G or T,
I=inosine, M=A or C, N=any base.
(1) structure of the amplification of 16S rRNA genes and phylogenetic tree
Using STb gene as template, 16S rRNA genes are expanded with table 2 universal primer P1 and P6.PCR reaction systems (50 μ l)：2
× PCR Mix 25 μ l, primer P1 and P6 (10 μM) each 1 μ l, 1 μ l of DNA profiling, add ultra-pure water to complement to 50 μ l.PCR reacts bar
Part：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 1min, 56 DEG C of annealing 30s, 72 DEG C of extension 1min, are circulated 30 times；72 DEG C of final extensions
10min.The result such as SEQID that Chengdu Qing Ke Zi Xi Bioisystech Co., Ltd is sequenced after amplified production detects as stated above
No.1。
By the sequence results of gained in EzTaxon (http://www.ezbiocloud.net/eztaxon) it is compared,
It was found that it is Rhizobium anhuiense with the highest type strain of 16S rRNA gene order similarities of rhizobium V14-2
CCBAU23252T, similarity 99.74%.With sequence compared on EzTaxon as a result, the high pattern of selection similarity
Bacterial strain is as reference strains, phylogenetic tree construction.Carried out with the adjacent method (Neighbor-joining) in Mega7 softwares
The structure of 16SrRNA Phylogenetic Trees, value of bootstrapping (bootstrap) 1000, its phylogenetic tree is shown in Fig. 1.
(2) structure of the association system development tree of more locus gene sequences
Further more accurately to determine the classification position of rhizobium V9-2, the alternative housekeeping gene atpD for selecting 3 sites,
RecA and glnII sequences carry out the structure of association system development tree.
Expand recA primer recAF2, recAR2, glnII primer GSII-5 and GSII-6, atpD primer
AtpDF1 and atpDR, primer sequence are as shown in table 2.Reaction system is 30 μ l, and reaction solution composition is as follows：Reaction system (30 μ l)
For：2×PCR Mix 15μl；Each 0.5 μ l of forward primer and reverse primer of 10mM；1 μ l of DNA profiling；ddH2O13μl。(1)
RecA amplification conditions：95 DEG C of pre-degeneration 3min；95 DEG C of denaturation 1min, 60 DEG C of annealing 1min, 72 DEG C of extension 2min, are circulated 30 times；
72 DEG C of final extension 2min.(2) glnII amplification conditions：95 DEG C of pre-degeneration 3min；95 DEG C of denaturation 1min, 56 DEG C of 1min that anneal, 72
DEG C extension 2min, circulate 30 times；72 DEG C of final extension 7min.(3) the pcr amplification reaction program of atpD is identical：95 DEG C of pre-degenerations
5min；94 DEG C of denaturation 45s, 57.5 DEG C of annealing 1min, 74 DEG C of extension 1.5min, are circulated 30 times；74 DEG C of final extension 5min.Amplification
Product send Chengdu Qing Ke Zi Xi Bioisystech Co., Ltd to be sequenced after detecting as stated above, two are carried out to each gene to sequencing
(sequence of positive and negative primer), then positive and negative primer sequence 6.0 softwares of DNAman are spliced, remove the sequence of positive and negative primer
It is 496nt, sequence results such as SEQID No.2 to obtain atpD sequence sizes respectively afterwards, and glnII sequence sizes are 637nt, sequence
As a result if SEQID No.3, recA sequence size are 493nt, sequence results such as SEQID No.4.
The sequence results of gained are compared in US National Bioinformatics Institute (NCBI), are found and rhizobium V9-2
The highest type strains of sequence similarity of tri- site housekeeping genes of atpD, glnII and recA be Rhizobium
anhuiense CCBAU 23252T, the similarity with the type strain is respectively 100%, 96.35%, 98.83%.Using every
A gene order comparison result on NCBI, selection is with 6 plants of high type strains of 3 gene similarities as the reference bacterium contribute
Strain.
The structure of 3 gene (atpD, glnII and recA) association system development trees：First by atpD, glnII, recA 3
The sequence of housekeeping gene corresponding gene sequences with reference strains respectively, are compared with MEGA7, had one's hair trimmed by standard of minimum length,
Sequence after having one's hair trimmed is saved as into FASTA forms, three gene order length are respectively 350nt, 460nt, 342nt after having one's hair trimmed.
Opened 3 sequence assemblies together with WORD, combined with the adjacent method (Neighbor-joining) in MEGA7 softwares
The structure of phylogenetic tree, value of bootstrapping (bootstrap) 1000, atpD, glnII, recA association system development tree are such as Fig. 3 institutes
Show.
Fig. 2 and Fig. 3 show that the 16S rRNA genes of V9-2, and 3 housekeeping genes of atpD, glnII, recA combine sequences
Row and type strain Rhizobium anhuiense CCBAU 23252TOn same branch node.Analyze also as before, it is described
Bacterial strain and type strain Rhizobium anhuienseCCBAU 23252TThis 4 genes similarity it is very high, atpD,
The similarity of glnII, recA are respectively 100%, 96.35%, 98.83%, show that rhizobium V9-2 belongs to Rhizobium
anhuiense。
6 Field inoculation effect of embodiment
Field inoculation effect test selection Sichuan Chongzhou City Qi Quan towns Sichuan Agricultural University modern agriculture base of the bacterial strain
Ground carries out.
This experiment sets two processing altogether, Rhizobium Inoculation V9-2 and is not inoculated with control treatment (CK), and beans kind selection Sichuan is purple
Flower sweet potato main breed " Hairy vetch ", not using any chemical fertilizer and organic fertilizer.Using the random district's groups arrangement in field.
Test and carried out on April 22nd, 30 days 1 in September in 2016.By the rhizobium V9-2 microbial inoculums prepared (viable count 5.7 ×
108CFU/g microbial inoculums) dress seed with pale reddish brown sweet potato, broadcasted sowing after drying in the shade.Plot area 7m2, when sowing first broadcasts CK, to avoid CK processing by
The influence of Rhizobium Inoculation.Sampled in plant full-bloom stage (breeding time 204d), measure plant root nodule number, aerial part plant are fresh
Weight, under ground portion root fresh weight, green forage yield.The management of period is performed by the Routine Management of the pale reddish brown sweet potato of Cotton Varieties by Small Farming Households.Try in field
Test that the results are shown in table 3.
The Field inoculation effect of the pale reddish brown sweet potato Rhizobium Inoculation V9-2 of table 3
The processing of Rhizobium Inoculation V9-2, full-bloom stage effective knurl number, total root nodule number, green forage yield than not being inoculated with pair
According to height, green forage yield is than CK increases by 22.7%.Fresh quench yield is than CK increases, volume increase 79.8%.As it can be seen that the excellent rhizobium of inoculation
It is obvious to Hairy Vetch suboutput production-increasing function.The rhizobium V9-2 is the excellent root nodule for being adapted to the son production of Sichuan Hairy vetch
Bacterium.
Field inoculation experimental study in Sichuan Chongzhou City Qi Quan towns is found, after Rhizobium Inoculation V9-2, the effective knurl of plant,
Total root nodule number and green forage yield are above not being inoculated with control, and volume increase 22.7%, fresh quench yield increases also above control treatment is not inoculated with
Production 79.8%, the increase explanation rhizobium V9-2 of above and below ground part biological amount promote the growth of pale reddish brown sweet potato.As it can be seen that this hair
The rhizobium V9-2 that bright separation obtains can be used for the popularization and application of large area in the pale reddish brown sweet potato production in Sichuan.
Embodiment described above only expresses the embodiment of the application, its description is more specific and detailed, but simultaneously
Therefore the limitation to the application protection domain cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, on the premise of technical scheme design is not departed from, various modifications and improvements can be made, these belong to this
The protection domain of application.
Claims (2)
- Rhizobium 1. (Rhizobium anhuiense) V9-2, is preserved in China typical culture collection center, deposit number is CCTCC NO：M2017610.
- 2. rhizobium (Rhizobium anhuiense) V9-2 according to claim 1 is in Sichuan province Hairy vetch Application in son plantation.
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