CN103642730A - ACC (1-aminocyclo-propane-1-carboxylic acid) enzyme generating active growth-promoting bacteria SC-12 and application thereof in promoting plant growth - Google Patents
ACC (1-aminocyclo-propane-1-carboxylic acid) enzyme generating active growth-promoting bacteria SC-12 and application thereof in promoting plant growth Download PDFInfo
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Abstract
The invention relates to ACC (1-aminocyclo-propane-1-carboxylic acid) enzyme generating active growth-promoting bacteria SC-12 and application thereof in promoting plant growth. The strain SC-12 is separated from the rhizosphere soil of plants in the coastal beach of the Rudong country of Jiangsu province and identified as the bacillus stratosphericus with collection number CGMCC No.7622; in the condition of taking ACC as the unique nitrogen source, the ACC deaminase activity of the strain is 23.57umol<alpha->ketobutyric acid.mg<-1>pro.h<-1>. Moreover, the strain SC-12 also has the features of generating indoleacetic acid (IAA), resisting salt and dissolving phosphorus; meanwhile, the strain has good adaptability to acid and base. In a salt-resistant condition, the strain SC-12 can be used for remarkably promoting the germination rate and germinal length of pepper and tomato. The field trail result indicates that the growth-promoting bacterial fertilizer developed by use of the strain SC-12 can be used for remarkably increasing the biomass and output of tomatoes.
Description
Technical field
The invention belongs to intensive agriculture production technical field, relate in particular to the growth of a kind of SC-12 bacterial strain solanaceous vegetables such as capsicum, tomato in promoting booth adverse circumstance environment.
Background technology
Due to facility cultivation (being mainly plastic greenhouse, heliogreenhouse and film-mulching technique) the anti-season of China vegetables and other Important Economic crops supply with and trans-regional plantation in the vital role that risen, industrialized agriculture has obtained large-area applying throughout the country.
Greenhouse, soil under the cultivation conditions such as booth lacks rainwater drip washing, and temperature, humidity, aeration status and water and fertilizer management etc. all have bigger difference (Yin Yongxian with outdoor cropping, Liu Hongyan. nitrated in soil under facility cultivation, the research of denitrification. Acta Ecologica Sinica, 1996, 16 (3): 246-250.), facility cultivation is intensive in height for a long time again, high multiple crop index, under the production status of high rate of fertilizer, the ecotope that it is special and irrational water and fertilizer management measure have caused soil secondary salinization, nutrient is uneven, many production problems such as soil acidification, wherein the most outstanding is soil secondary salinization, become the critical limitation factor (Yu Haiying in agriculture production, Li Tingxuan, Zhou Jianmin. facility soil secondary salinization and the impact on soil property thereof. soil, 2005, 37 (6): 581-586.).The salinification of soil not only affects plant metabolism and photosynthesis, also can reduce the absorption of plant to soil nutrient and trace element, because the iron in soil mainly exists with the forms such as high oxide of insoluble, and saline-alkali soil can further reduce the content of soil soluble iron, thereby cause the chlorosis of plant.Therefore, be grown in harm and the sideropenic threat that plant in salinization soil is often subject to salt stress simultaneously.
Alkaline land improving is a complexity and large order, and advocates under the prerequisite that ecological benefits attach most importance to current, and biological modification measure has become the focus of research.Plant growth-promoting bacteria (plant growth-promotingrhizobacteria, PGPR) can suppress or alleviate the detrimentally affect that Plant diseases produces plant by indirect generation microbiotic and mode such as secretion siderophore etc., and by phosphorus decomposing, fixed nitrogen, produce plant hormone, enzymolysis reduces the modes such as ethylene levels and directly stimulates and coordinate plant growth (Kloepper J W, Schroth M N.Plant growth-promoting rhizobacteria on radishes//Proceedings of the Fourth International Conference on Plant Pathogen Bacteria[C] .vol.2.INRA, 1978:879 – 882).Wherein, a kind of PGPR containing 1-amino-cyclopropane-1-carboxylic acid (1-aminocyclo-propane-1-carboxylic acid, ACC) desaminase has received the concern of various countries' researchers.Acc deaminase is a kind of external source growth-promoting material of effective reduction stress ethylene content, this enzyme can significantly improve the resistance of farm crop and increase output (Glick BR under the adverse environmental factors such as arid, salt stress and heavy metal contamination, Cheng Z, Czarny J, Duan J.Promotion of plant growth by ACC deaminase-producing soil bacteria.European Journal of Plant Pathology, 2007,119:329 – 339).Some microorganism can secrete 1-amino-cyclopropane-1-carboxylic acid (being called for short ACC) thereby the remarkable Promoting plant growth of desaminase, as food crop such as the vegetable melon and fruit classes such as tomato, cucumber, capsicum and wheat, pulse families.In its energy hydrolyzing plant body, the direct precursor ACC of biosynthesizing ethene, produces hydroxybutyric acid and ammonia, stops the release of ethene.When this bacterium is attached to seed epidermis, it can be hydrolyzed ACC, reduce seed a large amount of plant growth inhibitor and plant senescence promotor---concentration of ethene producing under adverse environmental factor, promote root to extend and plant-growth (Xu Yuquan, Shi Rong, Lin Zhixin. there is the pseudomonas strains B*8. Shanghai Communications University journal of acc deaminase activity and anti-blight bacterium, 1999,33 (2): 206-209.).
In actual production, the growth-promoting bacterium of can growth-promoting if can screen, can strengthen again crop anti-adversity ability, and disclose in theory its mechanism of action, not only can effectively reduce the excessive harm that chemical fertilizer, agricultural chemicals bring to environment and human health of using, can also extensively promote the growth of plant in various adverse circumstance environment, for the development of current green sustainable agriculture, there is important theory and practical significance.
Summary of the invention
The invention provides the new growth-promoting bacterium of a strain, can be in promoting booth adverse circumstance environment the growths such as vegetable melon and fruit class such as capsicum, tomato.
Technical solution of the present invention is: the bacterial strain that the present invention adopts is stratospheric genus bacillus SC-12, be preserved in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center, Institute of Microorganism, Academia Sinica), preservation date is on May 15th, 2013, and preserving number is: CGMCC NO..7622.
In the coastal beach plant rhizosphere soil in Rudong County, bacterial strain SC-12 Xi Cong Jiangsu Province of the present invention, separation obtains, observation analysis this bacterium morphological specificity, cultural characteristic, physio-biochemical characteristics and 16SrDNA sequence:
(1) bacterium colony and morphological features and physiological and biochemical property
Bacterial strain SC-12 is rod-short, and bacterium colony is sticky translucent, and surface wettability is smooth, neat in edge, and Gram-negative, its Electronic Speculum picture is Fig. 1.Physiology and biochemistry qualification result shows: bacterial strain SC-12 can utilize starch, lactose, glucose, sucrose, seminose fully, but can not liquefy gelatin; Methyl red test, Fu-Pu test, oxidase test, nitrate reduction test are all negative; Indole test, citrate test, catalase test result are all positive.
(2) 16SrDNA sequence
The 16Sr DNA nucleotide sequence of bacterial strain SC-12 contains 1422bp, sequence is as shown in SEQ ID NO.1, to in the databases such as itself and GeneBank, carry out maximum homology comparison, find that 16S rDNA sequence and the stratospheric bacillus stratosphericus KC172060 sequence homology of SC-12 reaches 100%.
The present invention also provides a kind of growth-promoting bacterial manure, is described bacterial strain SC-12 is inoculated in the pig manure straw compost becoming thoroughly decomposed and ferments and obtain, and in gained growth-promoting bacterial manure, contains SC-12 bacterial strain 1 * 10
8more than/g.
Concrete method is: bacterial strain SC-12 described in the claim 1 of activation is inoculated in LB liquid nutrient medium, 30 ℃, shaking table is cultivated after 20h, by fermented liquid by 20%(kg/liter) amount be inoculated in the pig manure straw compost becoming thoroughly decomposed, and fully mix, regulate heap body humidity, carry out solid fermentation, in fermenting process, stir fertilizer every day, controls heap temperature, ferment 20 days, in fertilizer, contain SC-12 bacterial strain 1 * 10
8more than/g, be growth-promoting bacterial manure.
The present invention be take ACC as only nitrogen source adopts enrichment directed screening method, is separated to the plant growth-promoting bacteria that acc deaminase is produced in 4 strains from the coastal beach plant rhizosphere soil in Rudong County, Jiangsu Province.The active size of its acc deaminase, product IAA, product siderophore and molten phosphorus ability are measured.Result demonstration, the ability that bacterial strain SC-12 produces desaminase is the strongest, also has stronger product IAA and molten phosphorus ability simultaneously.Salt tolerant and resistance to acids and bases test also show that bacterial strain SC-12 has certain patience to salt, particularly evident to the patience of soda acid, normal growth in the scope of pH4～10.By it being carried out to physio-biochemical characteristics and 16SrDNA sequential analysis, this strain bacterial strain is initially identified as stratospheric genus bacillus.On this basis, under condition of salt stress, bacterial strain SC-12 has obvious growth-promoting functions to capsicum, tomato seeds percentage of germination and bud length.Because bacterial strain SC-12 strain has stronger product ACC enzyme activity and very strong adaptive capacity to environment, to also thering is obvious growth-promoting functions.The PGPR filtering out from plant rhizosphere saline-alkali soil more can adapt to saline-alkali environment with respect to the PGPR sifting out in rich soil, and institute, for alkaline land improving, saline land greening, has more practicality.Therefore, this bacterium is expected to be applied to the solanaceous vegetableses such as shed for pepper, tomato, improves the adaptive faculty of plant to adverse circumstance, increases output.
Accompanying drawing explanation
The transmission electron microscope picture of Fig. 1 SC-12 bacterial strain;
The phylogenetic tree of the 16S rDNA complete sequence of Fig. 2 SC-12 bacterial strain;
Under the different nitrate concentrations of Fig. 3, bacterial strain SC-12 is on capsicum and tomato seeds percentage of germination and the long impact of bud.
Embodiment
Below in conjunction with example, the invention will be further described:
Embodiment 1:
1 materials and methods
1.1 bacterial classification
Separated from the coastal beach plant rhizosphere soil in Rudong County, Jiangsu Province.
1.2 substratum
LB substratum: Tryptones 10g, yeast extract 5g, NaCl10g, pH7.0, deionized water 1L;
SM substratum: glucose 1.0g, sucrose 1.0g, Trisodium Citrate 1.0g, oxysuccinic acid 1.0g, N.F,USP MANNITOL 1.0g, sodium-acetate 1.0g, KH
2pO
40.4g, K
2hPO
42.0g, MgSO
40.2g, CaCl
20.1g, CuSO
41mg, NiSO
41mg, ZnSO
45mg, FeSO
45mg, MnSO
43mg, CoSO
41mg, Na
2moO
41mg, H
3bO
32mg, vitamin H 2 (VB
6) 10mg, thiamines (VB
1) 2mg, cyanocobalamin (VB
12) 0.1mg, pantothenic acid (VB
3) 5mg, folic acid 2mg, riboflavin 5mg, nicotinic acid 5mg, distilled water 1000mL, pH6.4.After VITAMIN filtration sterilization, add.
SMA substratum: add the ACC of filtration sterilization in SM substratum, making its concentration is 0.5g.L
-1.
SMN substratum: add (NH in SM substratum
4)
2sO
4, making its concentration is 0.5g.L
-1.
SRSM substratum: glucose 10g/l; Ca
3(PO
4)
25g/l; (NH
4) SO
40.5g/l; KCl0.2g/l; MgSO
4.7H
2o0.3g/l; MnSO
40.004g/l; FeSO
4, 0.002g/l; NaCl20g/l; Yeast extract, 0.5g/l; Dibromo-ocresolsulfonphthalein 0.1g/l.
1.3 produce the screening of 1-amino-cyclopropane-1-carboxylic acid (ACC) desaminase bacterium
Bacterial strain is accessed to 28 ℃ of shaking tables in LB liquid nutrient medium and cultivate 18h, centrifugal collection thalline, accesses respectively SM liquid nutrient medium, SMA liquid nutrient medium, SMN liquid nutrient medium (3mL) by twice rear equivalent of aseptic washing (100 μ L) for thalline.28 ℃ of shaking culture 48h, with the OD of 722 spectrophotometer colorimetric method for determining different treatment bacteria suspensions
600value, and compare.Not long in SM substratum, in SMA and SMN substratum, the bacterium of good growth has acc deaminase activity.
1.4ACC deaminase activity determination
Isolated strains is inoculated in shaking table shaking culture 20h in SMN nutrient solution, and 4 ℃ of centrifugal collection thalline, wash centrifugal twice with SM nutrient solution, thalline is suspended in SM nutrient solution, and be inoculated in SMA nutrient solution by 5% inoculum size, under the condition of 28 ℃ of 150rpm, cultivate 48h, to induce generation acc deaminase.4 ℃ of centrifugal collection thalline, use 0.1mol.L
-1it is centrifugal twice that Tris-HCl damping fluid (pH value is 7.6) washs, and is resuspended to 600 μ L0.1mol.L
-1in Tris-HCl damping fluid (pH value is 8.5), add 30 μ L toluene rapid vibration 30s with smudge cells.
Get 100 μ L containing the cell extract of toluene in 1.5mL centrifuge tube, add 10 μ L0.5mol.L
-1aCC mixes, 30 ℃ of reaction 30min.Add 1.0mL0.56N HCl termination reaction, the centrifugal 5min of 14000rpm removes cell debris, draws 1mL supernatant liquor in 7mL centrifuge tube, adds the 2,4 dinitrophenyl hydrazine (being dissolved in 2M HCl) of 0.15mL0.1%, 30 ℃ of reaction 15-30min.Taking-up adds 1mL2N NaOH.Take and add 1mL Tris-HCl damping fluid (pH value is 8.5) and carry out same reaction for blank tube zeroing, take 1mL concentration as 0,0.1,0.5,1.0,2.0, α-batanone acid of 3.0mM is standardized solution, light absorption value under mensuration 540nm wavelength.
Bacterial protein content adopts Xylene Brilliant Cyanine G method to measure.Take 1mL concentration as 0,20,40,60,80 and 100mg.L
-1bovine serum albumin be standardized solution, add 5mL Coomassie brilliant blue G250 staining fluid, reaction is measured light absorption value under 595nm wavelength after 5min.The scale of α-batanone acid that the activity of acc deaminase forms with every milligram of albumen per minute in surveying enzyme system shows, unit is μ mol α-batanone acid .mg
-1protein.min
-1.
1.5 bacterial strains produce the mensuration of siderophore, 3-indolyl acetic acid (IAA), molten phosphorus ability
The measuring method of siderophore: isolated strains is inoculated in MSA liquid nutrient medium, after 30 ℃ of 150r/min shaking culture 48h of shaking table, the centrifugal 10min of fermented liquid 6000r/min, get 1.5ml supernatant liquor and add 1.5mlCAS detection liquid, fully mix, the light absorption value (A) of measuring 630nm wavelength place after standing 1h, returns to zero in contrast with deionized water.Separately get 1.5ml CAS and detect the MSA liquid nutrient medium supernatant liquor that liquid and 1.5ml do not connect bacterium and fully mix, be measured in the same method light absorption value and be reference value (Ar).With A/Ar, make quantitative target.Ratio is less, and the output of reflection siderophore is larger.General reference standard is: A/Ar0～0.2, +++ ++; 0.2～0.4, ++++; 0.4～0.6, +++; 0.6～0.8, ++; 0.8～1.0 ,+.
IAA measuring method: the solution that tryptophane is configured to 2.5mg/ml, LB liquid nutrient medium is sub-packed in test tube, every pipe 4ml, the tryptophane solution 1ml that adds filtration sterilization after 121 ℃ of autoclavings, the concentration that makes tryptophane in substratum is 0.5mg/ml, strains tested is inoculated in above-mentioned substratum, shaking table shaking culture 3 days, centrifuging and taking 1ml supernatant, the ortho-phosphoric acid that adds 50 μ l10mmol/L, add 2ml Salkowski ' s developer, dark lower 25 ℃ of colour developing 30min, measure the light absorption value under 530nm wavelength again.With distilled water, replace the same reaction of nutrient solution zeroing in contrast, with the IAA reference liquid of different concns, with method, make typical curve, calculate the concentration of IAA in fermented liquid.
The measuring method of molten phosphorus ability: bacterial strain to be measured is first activated on LB solid medium, then improve on SRSM substratum with sterilizing toothpick point inoculating strain to solid.Every bacterial strain connects 4 repetitions on a ware.The size of observing bacterial strain transparent circle cultivate 5d in 28 ℃ of incubators after.
1.6 strain identification
1.6.1 bacterium colony and thalli morphology are observed
On LB substratum, line obtains single bacterium colony, observes single colony edge shape, size, protuberance situation, configuration of surface, colony colour, transparency, pigment formation situation etc.By after strain culturing 16-18h, carry out gramstaining, and observe the form of Gram-stained yin and yang attribute and thalline.
1.6.2 Physiology and biochemistry is identified:
Physiological and biochemical property is measured with reference to < < common bacteria system identification handbook > > (eastern elegant pearl etc., 2001).
1.6.3 molecular biology identification
With 16S rDNA universal primer 27F(forward primer): 5 '-AGAGTTTGATCCTGGCTCAG-3 ' (SEQ ID NO.2); 1492R(reverse primer): 5 '-TACGGCTACCTTGTTACGACTT-3 ' (SEQ ID NO.3), the total DNA of isolated strains of take carries out pcr amplification as template.Amplified reaction volume 50 μ l, reaction conditions is 94 ℃ of denaturation 5min; 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 1.5min, circulate 30 times; Last 72 ℃ are extended 10min.Get PCR product 2.5 μ L in 0.8% agarose gel electrophoresis detection PCR product.PCR product is delivered to biotech firm and is carried out purifying and order-checking.Submit sequencing result to GenBank database, with BLAST software, compare.Sequencing result is inputted to MEGA4.0 together by BLAST software and the sequence of the relevant bacterial strain of 16S rDNA obtaining in GenBank and carry out DNA homology relatively and arrange, constructing system is grown pedigree chart.
The impact on strain growth of 1.7 temperature, initial pH value and salt concn
Strains tested point is connected to solid plate, at the temperature of 15 ℃, 25 ℃, 35 ℃, 45 ℃, cultivate 72h respectively, under 600nm wavelength, measure the OD value of respectively processing bacterium liquid, by OD value, represent the biomass that it cultivates in gradient salt concn substratum, OD value is larger, shows that biomass is just larger.
Use 1mol.L
-1naOH and 1mol.L
-1hCl the pH value of LB substratum is adjusted to respectively to 4.0,5.0,6.0,7.0,8.0,9.0,10.0.Can the strains tested of activation be put respectively on the flat board that is connected to above-mentioned different pH, cultivated 72h for 28 ℃, observe it and grow and growing state, method be the same.
The bacteria suspension of the strains tested of incubated overnight is linked into Ca (NO
3)
2concentration is respectively 0,50,100,150,200,250gkg
-1liquid LB substratum in (tryptophane concentration is 0.5mg ml
-1), can 24h be cultivated in 28 ℃ of concussions, observe it and grow and growing state, and method is the same.
The impact of bacterial strain SC-12 on capsicum, tomato salt-tolerant under 1.8 different salinity conditions
By deionized water (CK), 8,16,24g/kg Ca (NO
3)
2after solution sterilization, add in the culture dish of sterilizing (built-in 3～5 metafiltration paper) every plate 10ml.By the capsicum of sterilizing, tomato (70% alcohol 1min, 1% clorox 10min), be dipped in 0.03mol/L MgSO
4bacteria suspension (OD
600=0.5) or 0.03mol/L MgSO
4in inactivated bacteria suspension (CK), after 1h, be placed in above-mentioned sterilized culture dish.Add 3mL sterilized water moisturizing vernalization (25 ℃-28 ℃) every day in plate, and 4 days " Invest, Then Investigate " percentage of germination and bud are long.20 seeds of every culture dish, arrange 5 repetitions.
The growth-promoting test of 1.9SC-12 bacterial manure field
1.9.1 the preparation of growth-promoting bacterial manure is inoculated into the bacterial strain SC-12 of activation in 100mL (500mL shaking flask) LB liquid nutrient medium, and 30 ℃, 200rmin
-1shaking table is cultivated after 20h, by fermented liquid by 20%(v/m) amount of (kg/liter) is inoculated in the pig manure straw compost becoming thoroughly decomposed, and fully mix, regulate heap body humidity, carry out solid fermentation, in fermenting process, stir fertilizer every day, controls heap temperature, ferment 20 days, in fertilizer, contain SC-12 bacterial strain 1 * 10
8more than/g, be growth-promoting bacterial manure.
1.9.2 tomato seedling tomato variety is No. 9, Jinpeng, chooses healthy full tomato seeds, in 75% alcohol, soaks 1min, with 0.1% mercuric chloride sterilization 2min, soak imbibition after rinsing 3 times with aqua sterilisa, be then layered on the culture dish that aseptic moistening filter paper is housed, 30 ℃ of vernalization, until seed shows money or valuables one carries unintentionally.Seed is sown into the cave basin of growing seedlings, and cultivating soil is the wormcast seedling medium that Yangzhou University Environmental Science and Engineering institute earthworm production base produces, and grows seedlings and within 20 days, selects afterwards the consistent booth that is transplanted to of growing.
1.9.3 test design is tested in 2013 and is carried out in Yangzhou are raised sub-Vegetable Company booth.Plastic shed soil physicochemical character: organic 29.2g/kg, alkali-hydrolyzable nitrogen 240mg/kg, rapid available phosphorus 140mg/kg, available potassium 240mg/kg, pH7.95, total salt 1.65g/kg and EC value are 0.67mS/cm.Booth area 50 * 6m2, minute 3 furrow.Every furrow duplicate rows plant is apart from 40cm, line-spacing 70cm.Every row 15 strains, totally 30 strains.Field test is established 4 processing: 1. growth-promoting bacterial manure (10000kg ha
-1); 2. day auxiliary fertilizer (12000kg ha
-1); 3. Poly composite fertilizer (1125kg ha
-1); 4. contrast.After incorporation of fertilizerin the soil, the shallow 15cm of turning over mixes immediately, ridging, overlay film, the field planting of transplanting seedlings.Random district group is arranged, and repeats for 4 times.Community daily administration is consistent.Tomato collection period, in each community, choose at random strain and list and sample collection period, measure plant overground part, underground part and fruit fresh weight, dry weight and plant height.
2 results and analysis
2.1 produce the screening of 1-amino-cyclopropane-1-carboxylic acid (ACC) desaminase bacterium
Plant is under adverse circumstance (drought, waterlogging, salt, heavy metal etc.) condition, and the ethylene content in its body can increase sharply, and it is even dead that the accumulation of excessive ethene can make plant-growth be obstructed.Glick et al (2005) (Glick BR.Modulation of plant ethylene levels by the bacterial enzyme ACC deaminase.FEMS Microbiology Letter, 2005,251:1 – 7) research shows that microorganism can produce acc deaminase, by the precursor substance ACC of decomposing ethylene, reduce and coerce the accumulation of ethene in plant materials under environment, thereby Promoting plant growth is grown.This research by by the bacterial suspension of strains tested respectively equivalent be inoculated in SM, SMA, SMN nutrient solution, to detect bacterial strain, whether can take ACC and grow as only nitrogen source.To in SM substratum, can get rid of the error in judgement that bacterial strain nitrogen fixation causes for examination microbionation, and be inoculated in SMN substratum and can get rid of the error in judgement to bacterial strain product acc deaminase ability such as microbial culture and culture medium prescription.Test-results shows, have multi-strain bacteria the growth potential of SMA nutrient solution the growth potential significantly better than SM nutrient solution, show that bacterial strain can take ACC and grow as only nitrogen source, by these bacterial strain called afters SC-3, SC-12, D18, D25.
2.2 isolated strains acc deaminases are active, product siderophore, the mensuration of IAA and molten phosphorus ability
This research is its acc deaminase activity of the further quantitative assay of isolated strains of only nitrogen source growth to the ACC of take above, the results are shown in Table 1.Result shows, 4 strain bacterium all show acc deaminase activity, LSD the result of multiple comparisons shows, 4 strain bacterium exist significant difference in 0.05 conspicuous level, and its acc deaminase activity shows as SC-12>D18>D25> SC-3.
PGPR both can be by phosphorus decomposing, produce the growth of the direct effect promotion plants such as plant hormone, also can and produce the resistance (Ding Yanqin that the indirect actions such as microbiotic improve plant by secretion siderophore, Du Binghai. the screening of PGPR and preliminary evaluation in Rhizosphere of Crops bacterium. soil and fertilizer, 2001, (3): 41-43.), this laboratory is by producing siderophore to 4 strain bacterial strains, the mensuration of IAA and molten phosphorus ability, LSD the result of multiple comparisons shows, bacterial strain SC-3 and D25 produce IAA ability and are significantly greater than bacterial strain SC-12 and D18(P < 0.05), bacterial strain D18 product IAA ability is minimum, molten phosphorus ability SC-12 > D25 > SC-3 > D18,4 strain bacterial strains all can produce siderophore, it is stronger that bacterial strain SC-12 and D18 produce siderophore ability, and bacterial strain SC-3 and D25 produce siderophore ability a little less than.The comprehensive above bacterial strain SC-12 that analyzes may be better than all the other 3 strain bacterium to the potential growth-promoting effect of plant in booth adverse circumstance environment.Following SC-12 bacterial strain is selected to further research.
Table 1 strains tested acc deaminase activity and product indolylacetic acid, siderophore and arginine decarboxylase situation
Note: in table, same column data lowercase is different represents that between processing, difference reaches P < 0.05 conspicuous level.
The evaluation of 2.3 bacterial strain SC-12
2.3.1 bacterial strain SC-12 morphological feature
Bacterial strain SC-12 is rod-short, and bacterium colony is sticky translucent, and surface wettability is smooth, neat in edge, and Gram-positive, its Electronic Speculum picture is Fig. 1.
2.3.2 bacterial strain SC-12 physiological and biochemical property
The physiological and biochemical property of bacterial strain is important evidence and the means of division bacteria and evaluation, and some specific biological characteristicses are also for exploitation and the practical application of its function provide reference.Bacterial strain SC-12 Physiology and biochemistry qualification result is in Table 2.
The physiological and biochemical property of table 2 bacterial strain SC-12
Note: "+" represents growth, "-" represents not grow.
2.3.3 the structure of bacterial strain SC-12 systematic evolution tree and analysis thereof
The bacterial strain SC-1216S rDNA genome sequence recording is listed in ncbi database and is compared through row BLAST, and choose type strain and carry out Phylogenetic Analysis, with Mega4.0 software, carry out Multiple Sequence Alignment phylogenetic tree construction (Fig. 2).As seen from the figure, bacterial strain SC-12 is positioned in Bacillus branch, reaches 100% with stratospheric bacillus stratosphericus KC172060 sequence homology.In conjunction with its physiological and biochemical property, this identification of strains is stratospheric genus bacillus, and GenBank accession number is (KF012032).
The impact on strain growth of 2.4 temperature, pH and salt concn
Culture temperature, the initial pH of substratum and Ca (NO have been measured
3)
2impact (table 3) etc. envrionment conditions on strain growth, to investigate the adaptive faculty of bacterial strain SC-12 to environment.Result shows, bacterial strain SC-12 is mesophilic bacteria, at 15-45 ℃, can grow, and can tolerate low temperature to a certain degree, and optimum temperuture is 25 ℃～30 ℃; Biomass to bacterial strain SC-12 in the LB of different pH substratum is measured.Result shows, pH is 4～10 o'clock, and bacterial strain SC-12 all can normal growth, so bacterial strain SC-12 has stronger soda acid patience.Bacterial strain SC-12 is carried out to salt tolerance test to be shown, at 0～50g/kg Variation of Salinity Condition, to SC-12, growth has no significant effect, and under 50～150g/kg Variation of Salinity Condition, the growth of SC-12 is suppressed gradually with the increase of salts contg, when salts contg is 200g/kg, be almost suppressed completely.However, under the environment of 100g/kg salt concn, in nutrient solution, the OD value of bacterial strain SC-12 is 1.7, shows stronger salt tolerance.
The impact on bacterial strain SC-12 growth of table 3 culture temperature, pH and salt concn
The impact that under 2.5 nitrate stress, bacterial strain SC-12 germinates on capsicum, tomato seeds
Under nitrate stress, bacterial strain SC-12 is shown in Fig. 3 to capsicum, tomato seeds percentage of germination, the long impact of bud.Result shows, in the too high or too low situation of nitrate content, and the growth-promoting DeGrain of bacterial strain SC-12.When salts contg is 8g/kg and 16g/kg, the percentage of germination of capsicum, tomato seeds, bud length are after inoculating strain SC-12, remarkable with CK comparing difference, show that this bacterium has significant growth-promoting functions to capsicum, tomato under nitrate stress.
The growth-promoting test of 2.6SC-12 bacterial manure field
Different Fertilization mode to the growth-promoting functions of greenhouse tomato in Table 4.Can find out, growth-promoting bacterial manure has promoter action significantly to the plant height of tomato, fresh weight and output.The researchs such as Liu Yanji think that the bacterial strain screening from saltings has more the feature of salt tolerant, and this research also finds, from the bacterial strain of coastal beach plant rhizosphere screening, nitrate is had to patience, can promote the growth of plant.
Table 4 different fertilization is to tomato growth-promoting functions
Note: 1. growth-promoting bacterial manure (10000kg ha
-1); 2. day auxiliary fertilizer (12000kg ha
-1); 3. Poly composite fertilizer (1125kg ha
-1); 4. contrast.With the different lowercases after column data, represent significant difference in 0.05 level.
Claims (4)
1. ACC enzymic activity growth-promoting bacterium SC-12 is produced in a strain, is stratospheric genus bacillus (Bacillus stratosphericus), and its preserving number is CGMCC No.7622.
2. SC-12 bacterial strain claimed in claim 1 application in solanaceous vegetables growth in promoting booth adverse circumstance environment.
3. application according to claim 2, is characterized in that described solanaceous vegetables is tomato, capsicum.
4. a growth-promoting bacterial manure, is that described bacterial strain SC-12 is inoculated in the pig manure straw compost becoming thoroughly decomposed and ferments and obtain, and in gained growth-promoting bacterial manure, contains SC-12 bacterial strain 1 * 10
8more than/g.
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| CN103952349A (en) * | 2014-04-24 | 2014-07-30 | 烟台地元生物科技有限公司 | Bacillus aryabhattai, microbial agent and applications of bacillus aryabhattai and microbial agent |
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| CN109136153A (en) * | 2018-09-29 | 2019-01-04 | 中国科学院成都生物研究所 | One plant of salt tolerant bacillus for having a Plant growth promotion |
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| CN110938581A (en) * | 2019-12-27 | 2020-03-31 | 沈兰兰 | Culture medium for cultivating high-activity plant growth-promoting bacteria and preparation method thereof |
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| CN103952349B (en) * | 2014-04-24 | 2016-04-27 | 烟台地元生物科技有限公司 | A Shi genus bacillus and microbiobacterial agent and their application |
| CN104818233A (en) * | 2015-05-11 | 2015-08-05 | 南京农业大学 | Bacillus vallismortis and functional vegetable seedling raising biological matrix prepared from bacillus vallismortis |
| CN105349454A (en) * | 2015-11-03 | 2016-02-24 | 湖南中烟工业有限责任公司 | Bacillus stratosphericus strain and application thereof |
| CN105349454B (en) * | 2015-11-03 | 2018-10-19 | 湖南中烟工业有限责任公司 | One plant of stratosphere bacillus and its application |
| CN106831019A (en) * | 2017-01-11 | 2017-06-13 | 扬州大学 | A kind of prevention and controls of solanaceous vegetables soil continuous cropping obstacle |
| CN109136153A (en) * | 2018-09-29 | 2019-01-04 | 中国科学院成都生物研究所 | One plant of salt tolerant bacillus for having a Plant growth promotion |
| CN110616169A (en) * | 2019-09-17 | 2019-12-27 | 山东农业大学 | Stratospheric bacillus with growth promoting effect and application thereof |
| CN110616169B (en) * | 2019-09-17 | 2021-07-09 | 山东农业大学 | Stratospheric bacillus with growth promoting effect and application thereof |
| CN110938581A (en) * | 2019-12-27 | 2020-03-31 | 沈兰兰 | Culture medium for cultivating high-activity plant growth-promoting bacteria and preparation method thereof |
| CN112375717A (en) * | 2020-12-02 | 2021-02-19 | 安徽农业大学 | Phosphorus-dissolving microbial inoculum for corn as well as preparation method and application thereof |
| CN112375717B (en) * | 2020-12-02 | 2022-03-11 | 安徽农业大学 | Phosphorus-dissolving microbial inoculum for corn as well as preparation method and application thereof |
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