CN103275895A - Saline-alkali-tolerant heteroauxin-producing Bacillus subtilis and application thereof - Google Patents
Saline-alkali-tolerant heteroauxin-producing Bacillus subtilis and application thereof Download PDFInfo
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Abstract
The invention discloses a saline-alkali-tolerant heteroauxin-producing Bacillus subtilis and application thereof, belonging to the field of microorganisms. The Bacillus subtilis ZZ18 was collected by China General Microbiological Culture Collection Center on March 15<th>, 2013; the class name is Bacillus subtilis; and the collection number is CGMCC No.7324. The strain can produce heteroauxin at a high yield, can realize saline-alkali-tolerant growth, can grow by using insoluble phosphate as a phosphorus source and can be used for preparation of a biofertilizer.
Description
Technical field
The present invention relates to a kind of microorganism, relate to subtilis and application thereof that a strain salt tolerant alkali produces indolylacetic acid.
Background technology
Many microorganisms can be dissolved indissoluble attitude phosphorus in soil, promote plant to the absorption of phosphorus, increase crop yield and improve crop quality, with phosphate solubilizing microorganism as bio-feritlizer, not only can improve the utilising efficiency of phosphorus in the soil, the fertile volume increase of joint, and can improve Soil structure, improve soil organic matter content.Plant growth-promoting bacteria (Plant Growth Promoting Bacteria, be called for short PGPB) is defined as the free living that is conducive to plant-growth under certain condition on the bacterium of soil, rhizosphere, root table, phyllosphere.These bacteriums can fixed nitrogen, molten phosphorus, molten iron, and produce plant hormone, as growth hormone, Plant hormones regulators,gibberellins, phytokinin and ethene.In addition, they can also improve the resistance of plant, comprise arid, high salinity, heavy metal and organic pollutant murder by poisoning.Therefore but present most of plant growth-promoting bacteria is not also tamed into the bacterial strain that can effectively improve the various resistance of plant, and separating the plant growth-promoting bacteria that obtains effectively improving the various resistance of plant from plant rhizosphere soil becomes focus.
Summary of the invention
The objective of the invention is the above-mentioned defective at prior art, a kind of subtilis is provided, can efficiently produce indolylacetic acid and can and can utilize insoluble phosphate to grow for the phosphorus source in the growth of high salinity condition.
Purpose of the present invention can be achieved through the following technical solutions:
A kind of subtilis of the present invention (Bacillus subtilis ZZ18), in on March 15th, 2013 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, classification subtilis (Bacillus subtilis) by name, preserving number CGMCC No.7324.
Described subtilis bacterium colony is less to be white, protuberance, neat in edge, and smooth surface is moistening, and is opaque, produces gemma.
The physio-biochemical characteristics of described subtilis are: Gram-positive, and facultative aerobic, chemoheterotrophy, the catalase positive, M.R and VP test are negative, starch hydrolysis feminine gender, gelatin liquefaction positive, the nitrate reduction positive, Citrate trianion utilizes negative.
The major nitrogen source of using when subtilis of the present invention is cultivated includes but not limited to peptone, yeast powder, saltpetre, ammonium nitrate, ammonium sulfate, L-glutamic acid; The main carbon source of using includes but not limited to sucrose, wood sugar, N.F,USP MANNITOL, maltose, lactose, fructose, glucose; The inorganic component that uses includes but not limited to Repone K, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, dipotassium hydrogen phosphate, tricalcium phosphate, Calcium dichloride dihydrate, bitter salt, ferrous sulfate.Fermentation of bacillus subtilis of the present invention can carry out under the environment of pH6～10 at 20～35 ℃.
The ability of bacillus subtilis secretion indolylacetic acid of the present invention (IAA) is strong, under 30 ℃, 180r/min shaking table culture condition 24h its transform the IAA ability and reach 53.47 μ gmL
?1Indolylacetic acid is a kind of of plant hormone, can promote the growth of root.Produce the bacterial classification of indolylacetic acid, often be attached to root system of plant or leaf surface, produce IAA and a small amount of GA when utilizing plant metabolism to produce secretory product
3Influence physiological process and the metamorphosis of plant Deng plant hormone.Show as the elongation of direct promotion root, thus increased with soil in the chance that contacts of nutritive substance; Can improve the content of plant materials Endogenous IAA; Inducing plant defence expression of gene, it is disease-resistant to improve plant materials, resistance such as drought resisting.As prioritization scheme of the present invention, the fermentation of described subtilis is that 28 ℃, pH6～10 time carry out in temperature, and it is the highest to produce the IAA amount under this environment.
As further optimization of the present invention, the carbon source that described subtilis is adopted is N.F,USP MANNITOL, and the nitrogenous source of employing is the combination of yeast powder.The substratum that utilizes above-mentioned carbon source and nitrogenous source to make, the amount of the producing bacillus subtilis IAA that cultivates is the highest.
Subtilis of the present invention can grow under high salinity degree condition and produce IAA.Shake under bottle condition in the laboratory, described subtilis can be growth and produce IAA under 10 the condition at pH, and can grow under LB substratum saltness is 7% condition, is can produce IAA at 3% o'clock in saltness.
Subtilis of the present invention grows for the phosphorus source with the insoluble phosphate, and is translated into soluble phosphate.Shake under bottle condition in the laboratory, described subtilis reaches 133.52mgL to the inversion quantity of tricalcium phosphate
?1With respect to the result of blank, described subtilis exceeds 96.08mgL to the inversion quantity of tricalcium phosphate than blank
?1
Beneficial effect: the invention provides the subtilis that a strain salt tolerant alkali produces indolylacetic acid, can high yield indolylacetic acid and the saline and alkaline growth of ability and can utilize insoluble phosphate to grow for the phosphorus source, can be used for preparing bio-feritlizer.
Description of drawings
Fig. 1 is the bacterium colony figure of bacterial strain ZZ18 of the present invention;
Fig. 2 represents that the ZZ18 bacterial strain is to the situation of utilizing of insoluble phosphorus;
Fig. 3 represents that different liquid amounts produce the influence of IAA to bacterial strain ZZ18;
Fig. 4 represents the influence that the ZZ18 bacterial strain of different initial pH produces IAA;
Fig. 5 represents that different carbon sources are to the influence of bacterial strain ZZ18 product IAA;
Fig. 6 represents that different nitrogen sources is to the influence of ZZ18 bacterial strain product IAA;
Fig. 7 represents that different saltness are to the influence of ZZ18 bacterial strain product IAA;
Biomaterial preservation information
Subtilis ZZ18, classification called after subtilis Bacillus subtilis, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date is on March 15th, 2013, the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preserving number CGMCC No.7324.
Embodiment
Below in conjunction with accompanying drawing the present invention is done further explanation.
At first prepare following three kinds of substratum.
The LB substratum: peptone 10g, yeast extract 5g, sodium-chlor 10g, agar 20g, distilled water 1000ml, pH7.0 ?7.2,121 ℃ of sterilizations, 20min.
The LB liquid nutrient medium: do not add agar, other condition is the same.
Inorganic phosphorus bacteria substratum (PKO substratum): tricalcium phosphate 5g, glucose 10g, ammonium sulfate 0.5g, sodium-chlor 0.3g, bitter salt 0.3g, Repone K 0.3g, manganous sulfate 0.03g, ferrous sulfate 0.03g, pH7.0 agar 20g, distilled water 1000ml, pH7.0～7.2.121 ℃ of sterilizations, 20min.
Minimal medium: ammonium sulfate 2.0g; SODIUM PHOSPHATE, MONOBASIC 0.5g; Dipotassium hydrogen phosphate 0.5g; Magnesium sulfate heptahydrate 0.2g; Calcium dichloride dihydrate 0.1g, distilled water 1000mL, pH7.0,121 ℃ of sterilizations, 20min.
To choose from the plant rhizosphere soil that take at the Zijin foot of the hill, Nanjing and take by weighing the triangular flask that l0g places the 250ml that fills the 100ml aqua sterilisa after root sieves, in shaking table, 30 ℃, 150rmin
?1Vibration 20min leaves standstill 10min, obtains soil suspension.Contain some kinds of short livings bacterium in this soil suspension, be applied to the LB substratum after adopting the dilution method dilution, flat board is inverted, in 30 ℃, in the thermostat container behind the cultivation 24h, the single bacterium colony of the dissimilar typical cases of picking, behind dull and stereotyped purifying, 4 ℃ to be kept at the LB inclined-plane stand-by.
Filter out the bacterium that can secrete indolylacetic acid by qualitative test and quantitative assay more below.
Qualitative test: with the microbionation after the separation and purification in employing contain L ?the LB liquid nutrient medium of tryptophane (200mg/L), 30 ℃, 180rmin
?1Shaking table is cultivated 1d.Get 50 μ L bacteria suspensions and drip on the whiteware plate, add 50 μ L Salkowski color solution (50mL35%HClO simultaneously
4+ 1mL0.5M FeCl
3).To add the color solution of 50 μ L50mg/L indolylacetic acids as positive control.The whiteware plate is observed after the room temperature lucifuge is placed 30min, and the color person of reddening represents to secrete indolylacetic acid.
Quantitative assay: the bacterium of the secretion IAA that primary dcreening operation is obtained carries out quantitative assay, and culture condition is the same.At first use the OD of spectrophotometry bacteria suspension
600The value, then with bacteria suspension with 10000rmin
?1Centrifugal 10min gets supernatant liquor and adds equal-volume Salkowski color solution, and lucifuge leaves standstill 30min, measures its OD
530Value.Calculate bacteria concentration OD
600Value is 1 o'clock, the content of indolylacetic acid in the unit volume fermented liquid.Analytically pure indolylacetic acid gradient dilution preparation is adopted in the drafting of typical curve.
The product IAA bacterium that obtains is carried out the screening assay of molten phosphorus situation, strains tested is inoculated in the 150mL triangular flask that fills 30mL inorganic phosphorus bacteria liquid nutrient medium, 30 ℃, 180rmin
?1After cultivating 4d, with the nutrient solution 4 ℃ of following 10000rmin of centrifuge tube that pack into
?1Centrifugal, 15min.Get supernatant liquor and measure available phosphorus content with molybdenum blue colorimetric method.
Namely filter out a plant height by above mensuration and produce indolylacetic acid and the strong bacterial strain of molten phosphorus ability, as shown in Figure 1, the bacterium colony that this bacterial strain forms is white, protuberance, neat in edge, and smooth surface is moistening, and is opaque, produces gemma, plant type called after ZZ18.This bacterial strain shakes under bottle condition in the laboratory, and described subtilis reaches 148.09mgL to the inversion quantity of tricalcium phosphate
?1With respect to the result of blank, described subtilis exceeds 96.08mgL to the inversion quantity of tricalcium phosphate than blank
?1(Fig. 2).
The bacterial strain that the aforesaid method screening and separating is gone out, the handsome biotechnology company limited order-checking through Shanghai, sequencing result according to 16SrDNA, analyze in http://www.ncbi.nlm.nih.gov online query, utilize Blast software in GenBank, to carry out the homology comparison with other 16S rDNA sequence, select the 16SrDNA systematic evolution tree of the sequence usefulness MEGA version3 software building ZZ18 of close sequence and ZZ18.According to the physiological and biochemical property of this bacterial strain, be accredited as subtilis (Bacillus subtilis), homology is 99%.With this bacterial strain on March 15th, 2013 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number CGMCC No.7324.
The aerobic test
The LB substratum of the bacterium of going out is poured in 3 sterilized test tubes, greatly about 2/3 place, on the aseptic technique platform, with the ZZ18(CGMCC No.7324 of inoculating needle picking slant culture), percutaneous puncture-inoculation (must be punctured to the pipe end) in above-mentioned substratum.30 ℃ of cultivations are respectively 3 days to 7 days observationss.Giving birth to the elder on the agar column surface is aerobic bacteria, is anerobe or facultative anaerobe as giving birth to the elder along the puncture line.
Catalatic mensuration
Drip 1 3%H at clean slide
2O
2, the LB slant culture 1 of getting 18～24h encircles, at H
2O
2In smear, if there have bubble to produce to be then positive, otherwise negative.
Methyl red test (M.R test)
A. substratum and reagent: peptone 5g, glucose 5g, sodium-chlor 5g, distilled water 1000mL regulates pH7.0～7.2, the packing test tube, every pipe is adorned 4～5mL, 121 ℃ of sterilization 20min.Reagent: methyl red 0.1g, 95% alcohol 300mL, distilled water 200mL.
B. spawn culture and result observe inoculating strain in above-mentioned nutrient solution, cultivate l～2 day for 30 ℃.In nutrient solution, add several methyl red reagent, present redness as nutrient solution, be the methyl red positive, yellow negative (methyl red color change interval 4.4 redness～6.0 yellow).
Second phthalein carbinol methine test (VP test)
A. the same methyl red test of culture medium culturing base.
B. spawn culture and result observe inoculation and cultivate same methyl red test.When doing the VP test, get nutrient solution (about 2mL) and mix mutually with the 40%NaOH of equivalent, add a small amount of creatine, behind the 2～5min that fully vibrates, redness occurs as nutrient solution, be the VP positive.
The starch hydrolysis experiment
A. substratum and reagent add 0.2% Zulkovsky starch in meat soup peptone agar, the packing triangular flask, and 121 ℃ of sterilization 20min are standby.Road Ge Shi iodine liquid: crystalline flake of iodine 1g, potassiumiodide 2g, earlier with a small amount of (3～5mL) dissolved in distilled water potassiumiodides now add the crystalline flake of iodine, treat that iodine dissolves fully after, thin up is to 300mL.
B. spawn culture and result observe and to get the bacterial classification point and be connected on the flat board, cultivate 2～4 days for 30 ℃, form bacterium colony after, drip road Ge Shi iodine liquid at flat board, to be paved with periphery of bacterial colonies degree of being, it is blue that flat board is, and periphery of bacterial colonies is irised out now if any water white transparency, illustrates that starch is hydrolyzed.The size of the big or small general remark hydrolyzed starch ability of transparent circle.
The gelatin hydrolysis test
A. substratum and reagent peptone 5g, gelatin 120g, distilled water 1000mL.Regulate pH7.2～7.4, the packing test tube, the substratum height is about 4～5cm, 121 ℃ of sterilization 20min.
B. spawn culture and result observe and are seeded in test tube central authorities with puncture method.In 30 ℃ of incubators, cultivated one month, observe gelatin and whether liquefy.
Nitrate reduction test
A. substratum and reagent peptone 10g, KNO
31g, distilled water 1000mL, pH7.0～7.4.Ge Lisishi (Gries) reagent: A liquid: Sulphanilic Acid 0.5g, dilute acetic acid (about 10%) 150mL; B liquid: Cai's amine 0.1g, distilled water 20mL, dilute acetic acid (about 10%) 150mL.Pentanoic reagent: pentanoic 0.5g is dissolved in the l00mL vitriol oil, uses the 20mL distilled water diluting.
B. spawn culture and result observe test organisms are inoculated in the nitrate liquid nutrient medium, cultivate 1,3,5 day for 30 ℃.In white porcelain dish aperture, pour a little nutrient solution into, drip 1 reagent A and B liquid then therein respectively, when nutrient solution become pink, rose, when orange or brown etc., expression has nitrite to exist, and is the nitrate reduction positive, otherwise negative.
The utilization of Citrate trianion
A. substratum and reagent Trisodium Citrate 2g, NaCl5g, MgSO
47H
2O0.2g, (NH
4)
2 HPO
41g, 1% Australia thymol blue aqueous solution 10mL, agar 20g, distilled water 1000mL, pH6.8 ?7.0,121 ℃ the sterilization 20min.
B. spawn culture and result observe and to get children's bacterial classification inoculation in age on the inclined-plane, 30 ℃ cultivate 3 ?7 days, substratum is the positive reaction of alkalescence (blueness) person, constant person is then negative.
The physiological and biochemical property of table 1 subtilis (Bacillus subtilis) ZZ18
+: positive reaction positivereaction; ?: negative reaction negativereaction
The subtilis ZZ18(CGMCC No.7324 that obtains for further checking embodiment 1) ability and the optimum condition of producing indolylacetic acid explored the influence to indolylacetic acid output at different pH, liquid amount, different carbon source, different nitrogen sources below.
To contain L ?tryptophane (200mg/L) LB liquid nutrient medium press 15ml, 30ml, 45ml, 60ml, 75ml, 90ml are loaded in the triangular flask of 150mL, by 1%(v/v) after the inoculum size inoculation is in the ZZ18 of logarithmic phase, place 30 ℃, 180rmin
?1Shaking table is cultivated 24h, measures the amount of producing IAA by the method for quantitative assay.The result as shown in Figure 3 because bacterial strain ZZ18 is facultative good oxygen metabolism, air flow influence the efficient that bacterial strain produces IAA, bacterial strain produces IAA and measures and diminish gradually after increase along with liquid amount become greater to maximum value earlier again, during the 45mL liquid amount, IAA generation maximum.
To contain L ?the LB substratum of tryptophane (200mg/L) be adjusted to different pH(4,5,6,7,8,9,10 respectively), get in the triangular flask that 30mL is loaded on 150mL, by 1%(v/v) after inoculum size inoculation is in the ZZ18 of logarithmic phase, place 30 ℃, 180rmin
?1Shaking table is cultivated 24h, method by quantitative assay is measured the amount of producing IAA, the result as shown in Figure 4, show that pH is 4 and did not produce IAA at 5 o'clock, in strong acid environment, thalline can't carry out growth metabolism, is to have in 6～10 o'clock to produce the IAA ability more by force at pH, be also can stablize IAA at 10 o'clock at pH, stronger alkaline-resisting ability is arranged.The optimal pH of this bacterial classification high yield IAA is 6～10.
Contain L ?add 1%(w/v respectively in tryptophane (200mg/L) minimal medium) carbon source, carbon source has glucose, wood sugar, sucrose, fructose, N.F,USP MANNITOL, lactose, maltose etc., get in the triangular flask that 30ml is loaded on 150ml, by 1%(v/v) after inoculum size inoculation is in the ZZ18 of logarithmic phase, place 30 ℃, 180rmin
?1Shaking table is cultivated 24h, measures the amount of producing IAA by the method for quantitative assay.The result as shown in Figure 5, this bacterial strain is when supplying with N.F,USP MANNITOL, the ability of producing IAA is the strongest, the utilization ratio of lactose is minimum, produces IAA hardly.
Contain L ?add 0.1%(w/v respectively in tryptophane (200mg/L) minimal medium (not comprising ammonium sulfate)) nitrogenous source, nitrogenous source comprises ammonium nitrate, ammonium sulfate, saltpetre, peptone, yeast powder, L-glutamic acid etc., get in the triangular flask that 30ml is loaded on 150ml by 1%(v/v) after the inoculum size inoculation is in the ZZ18 of logarithmic phase, place 30 ℃, 180rmin
?1Shaking table is cultivated 24h, measures the amount of producing IAA by the method for quantitative assay.The result as shown in Figure 6, when illustrating that getting yeast powder is nitrogenous source, the amount of producing IAA is maximum.
To contain L ?tryptophane (200mg/L) LB liquid nutrient medium (not containing NaCl) be adjusted to different saltness (1%, 2%, 3%, 4%, 5%, 7%, 9%) respectively, get in the triangular flask that 30mL is loaded on 150mL, by 1%(v/v) inoculum size inoculation is in the ZZ18(CGMCC No.7324 of logarithmic phase) after, place 30 ℃, 180rmin
?1Shaking table is cultivated 24h, method by quantitative assay is measured the amount of producing IAA, the result as shown in Figure 7, show ZZ18(CGMCC No.7324) upgrowth situation variation along with the increase of saltness all, be almost can not grow in 9% o'clock to saltness, its interval of producing IAA content is 1%～3%, and change of production is little.This bacterial strain has stronger salt resistance ability.
In order further to verify subtilis ZZ18(CGMCC No.7324) to the adaptation of highly basic condition, under stronger alkaline condition, cultivate bacterial strain, explore the anti-highly basic ability of this bacterium by its upgrowth situation and product IAA ability.To contain L ?tryptophane (200mg/L) LB liquid nutrient medium be adjusted to different pH(10,10.5,11,11.5,12 respectively), get in the triangular flask that 30mL is loaded on 150mL, by 1%(v/v) inoculum size inoculation is in the ZZ18(CGMCC No.7324 of logarithmic phase) after, place 30 ℃, 180rmin
?1Shaking table is cultivated 24h, measures the amount of producing IAA by the method for quantitative assay, the results are shown in Table 2.As shown in the table, ZZ18(CGMCC No.7324) being 10 o'clock well-growns and can stablizing product IAA at pH, is almost can not grow in 10.5 o'clock to produce IAA to pH, can not grow especially afterwards and can not produce IAA.
Table 2
Claims (2)
1. a strain salt tolerant alkali produces subtilis (Bacillus subtilis) ZZ18 of indolylacetic acid, on March 15th, 2013 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number CGMCC No.7324.
2. the application of the described subtilis of claim 1 (Bacillus subtilis) ZZ18 in the preparation bio-feritlizer.
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| CN111363694A (en) * | 2020-01-21 | 2020-07-03 | 中国科学院城市环境研究所 | Stagnant corynebacterium, screening method and application thereof |
| CN111808776A (en) * | 2020-07-28 | 2020-10-23 | 德州八虎生物科技有限公司 | Saline-alkali-tolerant air bacillus and preparation method and application of viable bacteria preparation thereof |
| CN112831437A (en) * | 2021-01-20 | 2021-05-25 | 河北冀微生物技术有限公司 | Bacillus subtilis and fermentation method for high yield of indoleacetic acid and high spore formation rate |
| CN112831437B (en) * | 2021-01-20 | 2021-11-23 | 河北冀微生物技术有限公司 | Bacillus subtilis and fermentation method for high yield of indoleacetic acid and high spore formation rate |
| CN117431195A (en) * | 2023-12-21 | 2024-01-23 | 北京绿氮生物科技有限公司 | Saline-alkali resistant growth-promoting bacterium, growth-promoting bacterium agent and application thereof |
| CN117431195B (en) * | 2023-12-21 | 2024-05-17 | 北京绿氮生物科技有限公司 | Saline-alkali resistant growth-promoting bacterium, growth-promoting bacterium agent and application thereof |
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