CN106978347A - The screening and its application in improvement booth vegetable soil hardening of one plant of dissolving phosphor and dissolving potassium bacillus - Google Patents

Abstract

The invention relates to the screening of a phosphate-solubilizing and potassium-solubilizing Bacillus JJMSH1 and its application in improving soil compaction of vegetables in greenhouses. The strain was screened from the rhizosphere soil of vegetable greenhouse plants, enriched with organic phosphorus medium, screened with inorganic phosphorus and silicate medium in turn, and detected available phosphorus and available potassium with molybdenum antimony anti-colorimetry and flame photometry content, finally selected JJMSH1 as the ideal strain. It was identified as Gram-positive Bacillus by Gram staining and spore staining. Use this inventive bacterial strain to treat the compacted soil in greenhouses, the content of available phosphorus and available potassium in the soil is relatively significantly improved, and the OD600 reaches 1.5. After the bacterial agent is diluted 100 times and acts on the soil, the content of available phosphorus increases by 35.7%, and the content of available potassium increases by 100 times. 26.1%.

Description

Screening of a Bacillus phosphate-solubilizing potassium-solubilizing bacteria and its compaction in improved greenhouse vegetable soil application in

technical field

The invention is designed in the fields of agriculture and microorganisms, especially the strain has the effect of dissolving phosphorus and potassium, and can improve the phenomenon of soil compaction in greenhouses to a certain extent.

Background technique

Both phosphorus and potassium are essential nutrients for plant growth. Phosphorus is widely involved in plant photosynthesis and biochemical reactions in the body, and potassium is closely related to plant metabolism and growth. Phosphorus in the soil mainly exists in the form of organic phosphorus and inorganic phosphorus. Most of the organic phosphorus exists in the form of phytic acid, nucleic acid, and phospholipids, which cannot be absorbed directly. It must be transformed into the form of inorganic phosphorus under the action of microorganisms to be absorbed. More than 95% of the phosphorus in the soil cannot be directly absorbed and utilized by plants. The soluble potassium content in the soil is relatively low, but the soil contains a large amount of potassium-containing minerals, but they mainly exist in the form of stable aluminosilicates, and more than 90% of the potassium cannot be absorbed and utilized by crops. Potassium-containing silicate minerals in the soil can only gradually release available potassium through differentiation and decomposition under the action of physical and chemical factors and microorganisms for crop growth and utilization. The content of soluble potassium in the soil is still declining at a high rate, resulting in the lack of available potassium in the soil, resulting in a decline in soil fertility. In agriculture, we usually supplement phosphorus and potassium by applying chemical fertilizers. However, the application of chemical fertilizers is likely to cause damage to the soil structure and cause unnecessary pollution. Moreover, the application of chemical fertilizers phosphorus and potassium cannot be fully utilized, especially the utilization rate of phosphorus fertilizer is lower than 30%, and most of the phosphorus is solidified in the soil and cannot be used.

Greenhouse vegetables are an agricultural production method that can create high-quality and efficient economic crops, and it is a very important way for farmers to become rich and well-off. However, with the accumulation of cultivation time and improper fertilization techniques, soil compaction is easy to occur, which seriously affects the sustainable development of greenhouse vegetables. One of the reasons for the large soil compaction is that too much nitrogen fertilizer is applied, and available phosphorus, available potassium and other trace elements are insufficient. Although the content of phosphorus and potassium in the soil is rich, they cannot be absorbed directly. Microorganisms play a great role in improving the soil rhizosphere environment, promoting the release of available phosphorus and potassium in the soil, and improving soil fertility. Therefore, phosphorus and potassium-dissolving strains can effectively It can effectively alleviate the imbalance of soil elements and make full use of soil elements.

Contents of the invention

The first object of the present invention is to obtain a bacterial strain with good phosphorus and potassium dissolving functions.

The second purpose of the present invention is that the screened bacterial strain can improve the hardened soil in greenhouses, enhance the growth of crops, and provide technical support for the development of related bacterial agents. Correspondingly reduce relevant economic losses and increase income, which is conducive to the healthy development of ecology and the sustainable development of agriculture.

Embodiment one, strain screening

Bacillus JJMSH1 was isolated from the soil of vegetable greenhouses. In order to realize this invention, organic phosphorus medium was used for primary screening to enrich, and then inorganic phosphorus medium was used for rescreening, and then the strains with phosphorus-solubilizing function were spot-connected to silicate bacterial medium . Therefore, strains that may have the effect of dissolving phosphorus and potassium can be obtained at the same time.

Firstly, use organic phosphorus medium for enrichment, take 1g greenhouse vegetable rhizosphere soil sample in 100ml enrichment medium, enrich at 28°C and 200r/min for 2 days. After that, dilute the gradient with sterile normal saline to 10-8, then spread the three gradients of -6, -7, and -8 on the inorganic phosphorus medium, culture at 30°C, observe the growth every day, observe the hydrolysis circle of the inorganic phosphorus medium, and Record the diameter ratio of its phosphorus-dissolving circle to the diameter of the colony (the JJMSH1 diameter ratio is 4.1), connect the colony with the hydrolysis circle to the silicate medium and pay attention to the colony shape, and select the vigorous colony on the medium. Save the obtained strains on the LB slope, and finally select 5 strains with larger hydrolysis circles and grow well on the potassium ore medium

Media for screening, culturing, and storage

1. Organic phosphorus medium, Montina basal medium plus egg yolk liquid (1000ml): glucose 10g, NH ₄ SO ₄ 0.5g, NaCl 0.3g, KCl 0.3g, 7H _{2 O. MgSO 40.3g, 7H 2 O.} FeSO ₄ 0.03g, 4H ₂ O.MnSO 0.03g, CaCO ₃ 5g, (NH ₄ ) ₃ PO ₄ 0.5g, yeast extract 0.4g, adjust pH to 7, 121°C for 20min, then add 10ml egg yolk liquid (sterile normal saline and Egg yolk 1:1) (solid plus 20g agar)

2. Inorganic phosphorus medium, Montina basal medium plus Ca ₃ (PO ₃ ) ₂ : Glucose 10g, NH ₄ SO ₄ 0.5g, NaCl 0.3g, KCl 0.3g, 7H ₂ O.MgSO4 0.3g, 7H ₂ O.FeSO ₄ 0.03g, 4H ₂ O.MnSO 0.03g, CaCO ₃ 5g, Ca ₃ (PO ₃ ) ₂ 2g, (solid plus 20g agar) adjust pH to 7, sterilize at 121°C for 20min

3. Silicate bacteria medium: sucrose 5g, MgS04 7H20 0.5g, FeCl30.005g, Na2HPO4 2g, potassium feldspar powder (800 mesh, containing K2O: 9.8%) 2g, water 1000mL, (agar 20g), adjust pH7 , sterilized at 121°C for 20min

4. LB: tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar (solid) to adjust the pH to 7, sterilize at 121°C for 20min, then place it at room temperature with an inclination of about 15° to cool and solidify naturally, and place it at 4°C Refrigerator for spare.

The isolated strains were activated with LB medium, and then inserted into three kinds of liquid medium, namely inorganic phosphorus, organic phosphorus, and silicate bacterial medium, with an inoculum size of 2%, 200r/min, and cultured for 5 days. its phosphorus and potassium content.

Embodiment two, the determination of available phosphorus and available potassium

The present invention uses the molybdenum antimony anti-colorimetric method to measure the available phosphorus content, and the flame photometry to measure the available potassium content

Determination of available phosphorus by molybdenum antimony anti-colorimetric method

The principle of molybdenum antimony anticolorimetric method: 2,4-dinitrophenol is used as indicator, and soluble phosphate can react with molybdenum antimony antichromogenic agent to generate phosphomolybdenum blue. The colorimetric determination is carried out at a wavelength of 660nm, according to the standard The curve calculates the content of available phosphorus, and the concentration of available phosphorus indicates the level of microbial phosphorus-dissolving ability.

Commonly used reagents for molybdenum antimony anti-colorimetric method

1. Molybdenum antimony anti-storage solution: Take 153ml of 98% concentrated sulfuric acid and slowly pour it into about 400ml of distilled water, stir and cool. Then weigh 10.0g of ammonium molybdate, dissolve it in 300ml of water at about 60°C, and cool it. Then slowly add the above sulfuric acid solution into the ammonium molybdate solution, then add 100ml of 5g/L antimony potassium tartrate solution, and finally dilute to 1L with water, put it in a brown bottle, and store it in a dark place

2. Molybdenum-antimony antichromogenic agent: 1.5g of ascorbic acid, dissolved in 100ml molybdenum-antimony anti-coloring agent, ready for immediate use

3. Phosphorus standard solution (10mg/L): Potassium dihydrogen phosphate was dried in an oven at 100°C for 2 hours, cooled, 0.439g was taken, dissolved in 100ml of distilled water, added 5ml of 98% concentrated sulfuric acid, dilute to 1L, and diluted 10 times. 10mg/L phosphorus standard solution

Phosphorus standard curve drawing: draw 0, 2, 4, 6, 8, 10, 12ml of 5mg/L phosphorus standard solution respectively, add water to dilute to 26.3m1 respectively, add 5ml of molybdenum antimony anti-chromogenic agent, shake well and make up to volume, that is Get 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mg/L phosphorus solutions, place them at 20°C for 4 hours, use 0 mg/L phosphorus standard solution as the control solution, and compare the color of the remaining phosphorus standard solutions with the test solution at the same time. And record the absorbance at 660nm. Take the measured absorbance as the ordinate and the phosphorus concentration (mg/L) as the abscissa to draw a standard phosphorus curve

Take 50ml of fermentation broth, centrifuge at 4000r/min for 30min, take the supernatant, dilute it with distilled water to 50 times, take 25ml, add dropwise 2-3 drops of 2,4-dinitrophenol indicator solution, and use 1mol/L hydrogen Sodium oxide solution or sulfuric acid solution to adjust the pH until the solution just appears slightly yellow, add molybdenum antimony anti-color agent 5m1 shake well, dilute to the mark. The blank test fermentation broth was treated in the same way. Colorimetric at 660nm, measure the absorbance, set the zero point with the blank test fermentation broth as the control solution, read the absorbance value, find out the concentration of the phosphorus standard solution on the working curve, and calculate it.

Through testing, the available phosphorus content of the inorganic phosphorus medium of the strain JJMSH1 is 59.81 mg/L medium, and the other four strains JJMSH2, 3, 4, and 5 are 23.10, 41.32, 39.28, and 65.45 mg/L, respectively.

The available phosphorus content of strain JJMSH1 strain organic phosphorus medium is 41.23 mg/L medium, and the other four strains JJMSH2, 3, 4, and 5 are 29.88, 40.51, 39.11, and 17.82 mg/L respectively

Determination of available potassium by flame photometry

1.1mol/L neutral ammonium acetate solution: Weigh 77.09g of pure ammonium acetate, add water to dissolve, set the volume to nearly 1L, check the pH, adjust to pH7 with acetic acid and ammonia water

2. Potassium standard solution preparation: KCl was dried at 100°C for 2 hours, weighed 0.1907g and dissolved in 1mol/L ammonium acetate solution, and set the volume to 1L, which was 100ug/mL K standard solution 0, 2.5, 5, 7.5 , 10, 15, 20mL into a 50mL volumetric flask, with 1mol/L ammonium acetate solution to volume, that is, 0, 5, 10, 15, 20, 30, 40ug/mL K standard series solutions

Pour all 25ml of the bacterial solution into an evaporating dish, concentrate on a water bath to about 5ml, add 2ml of hydrogen peroxide to continue evaporating, keep stirring, and add several times until the mucus substance is completely digested. Centrifuge at 3500r/min for 10min, collect the supernatant in a 50ml volumetric flask, and distill it to volume. The control medium was treated in the same way. The filtrate and the standard solution of potassium are measured on a flame photometer

The available potassium content of the inorganic phosphorus medium of the strain JJMSH1 was detected to be 28.56 mg/L medium, and the other four strains JJMSH2, 3, 4, and 5 were 23.11, 7.32, 19.18, and 22.37 mg/L, respectively.

The selected strain JJMSH1 is an ideal strain

Embodiment three, bacterial strain JJMSH1 strain identification

The surface of the colony is rough, opaque, and the color is milky white and slightly yellowish, similar to the colony morphology of certain strains of Bacillus

Gram stain and spore stain

Gram staining: fix the smear, stain with crystal violet for 1 minute, rinse with tap water, be careful not to wash off the bacteria, mordant with iodine solution for about 1 minute, wash with water, absorb water with absorbent paper, 1 drop of 95% alcohol, 15~ After 20 seconds, wash with water, absorb the water, counterstain with safranin staining solution for 1 minute, rinse with tap water, and observe with an oil microscope.

The bacterial strain of the present invention is identified as a positive bacterium through Gram staining, and the bacterium is long rod-shaped, and similar uncolored spores are occasionally produced.

Spore staining

Smear, dry, and fix, add 3-5 drops of malachite green dyeing solution dropwise, heat the glass slide on the flame, make the dyeing solution steam but do not boil, do not let the dyeing solution evaporate dry, about 4-5 minutes, wash with water until Until the malachite green no longer fades, safranin counterstained for 1 minute, washed with water, dried, and observed with an oil lens

A few green spores and a large number of red bacteria were observed

The present invention is identified as Bacillus

Embodiment four, bacillus fermentation

The strain JJMSH1 was activated in 100ml LB liquid medium, activated at 35°C and 200r/min, and then inoculated in a 10L fermenter. The medium included tryptone: 5-10g/L, yeast extract: 5-10g/L, sodium chloride: 2-10g/L, speed 300rpm, temperature 25-35°C, culture for 12-24h, then take a suitable gradient for colony counting to 10 billion/mL.

Embodiment four, greenhouse soil application test

The bacterial solution was diluted 100, 200, 400 times respectively.

Sieve through a 4000-mesh sieve for compacted soil in greenhouses

Take 12 1L beakers, labeled 1~12 respectively, sterilize at 121°C for 20 minutes, and fill them with 0.5 kg of the same sieved greenhouse compaction soil, 1~3 is the control group, 4~12 is the experimental group, and each experimental group Spray 50ml of bacterial solution, every 3 is a dilution factor, each spray the same volume of sterile water in the control group

Culture at 25°C for 15 days

Determination of available phosphorus content in soil

Take 2g of soil sample for each sample

Add 50ml of 0.5mol/L NaHCO ₃ solution, shake at 180r/min at 25°C for 30min, take 25ml of the supernatant at 8000r/min, make a set of blanks for zeroing in the same way, see Example 2 for details

The available phosphorus content of the control group was 9.75mg/kg, the available phosphorus content of the experimental group was about 13.23mg/kg, and the effective phosphorus content of the soil sample was about 12.11mg/kg after spraying and diluting the 100-fold bacterial agent in the experimental group. The effective phosphorus content of the soil sample diluted 400 times is about 10.87mg/kg

The effective phosphorus content increased by 35.7%, 24.2%, and 11.9% when diluted 100, 200, and 400 times

Soil Available Potassium Determination

Take 2g of soil sample for each sample

Add 50ml of 1mol/L ammonium acetate solution, shake at 180r/min at 25°C for 30min, take 25ml of the supernatant at 8000r/min, make a set of blanks for zeroing in the same way, see Example 2 for details

The content of available potassium in the control group was 5.01mg/kg, the available phosphorus content of the experimental group was about 6.32mg/kg when spraying and diluting 100 times the bacteria agent, and the available phosphorus content of the soil sample when spraying and diluting 200 times the bacteria agent was about 5.77mg/kg. The effective phosphorus content of the soil sample diluted 400 times is about 5.27mg/kg

Diluted 100, 200, 400 times the available potassium content increased by 26.1%, 15.2%, 5.2% respectively

<110> Institute of Process Engineering, Chinese Academy of Sciences

<120> Screening of a strain of Bacillus phosphate and potassium solubles and its application in improving soil compaction of vegetables in greenhouses

<130> 2017

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1442

<212>DNA

<213> Bacillus subtilis JJMSH1

<400> 1

ggggcgggtg ctatacatgc agtcgagccc acagatggga gcttgctccc tgatgttagc 60

ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag actgggataa ctccgggaaa 120

ccggggctaa taccggatgg ttgtttgaac cgcatggttc agacataaaa ggtggcttcg 180

gctaccactt acagatggac ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca 240

aggcgacgat gcgtagccga cctgagaggg tgatcggcca cactgggact gagacaccgc 300

ccagactcct acgggagcca gcagtaggga atcttccgca atggacgaaa gtctgacgga 360

gcaacgccgc gtgagtgatg aaggttttcg gatcgtattg ctctgttgtt agggaagaac 420

aagtgccgtt caaatagggc ggcaccttga cggtacctaa ccagaaagcc acggctaact 480

acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggaatt attgggcgta 540

aagggctcgc aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg 600

tcattggaaa ctggggaact tgagtgcaga agaggagagt ggaattccac gtgtagcggt 660

gaaatgcgta gagatgtgga ggaacaccag tggcgttggc gactctctcc tctgtaactg 720

acgctgagga gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg 780

taaacgatga gtgctaagtg ttagggggta accgcccctt agtgctgcag ctaacgcatt 840

aagcactccg cctggggaga acggtcgcaa gactgaaact caaacgaatt gacggggcccc 900

cgcacaagcg gtggagcatg acctttaatt cgaagcaacg cgaagaacct taccaggtct 960

tgacatcctc tgacaatcct agagatagga cgtccccttc gggggcagag tgacaggtgg 1020

tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1080

cccttgatct tagttgccag cattcagttg ggcactctaa ggtgactgcc ggtgacaaac 1140

cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt 1200

gctacaatgg acagaacaaa gggcagcgaa accgcgaggt taagccaatc ccacaaatct 1260

gttctcagtt cggatcgcag tctgcaactc gactgcgtga agctggaatc gctagtaatc 1320

gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc 1380

acgagagttt gtaacacccg aagtcggtga ggtaacctaa aggagccagc cgccgaagaa 1440

GT 1442

Claims (6)

1. the screening of one plant of dissolving phosphor and dissolving potassium bacillus, fermentation and its application in improvement booth vegetable soil hardening.

2. the screening technique of dissolving phosphor and dissolving potassium bacillus, first exists the soil from booth vegetable according to claim 1 Enrichment 2-6 days is carried out in organophosphor culture medium, then Phos culture medium and silicic acid salt culture medium are screened, and are chosen at training Growing way is vigorous on foster base, contain the larger bacterium colony for hydrolyzing circle.

3. bacterial strain is Gram-positive bacillus according to claim 1.

4. the 16SRNA gene orders of bacterial strain such as SEQ ID NO according to claim 1:Shown in 1.

5. the fermentation condition of bacterial strain is according to claim 1：Culture medium：Tryptone:5-10g/L, yeast extract:5- 10g/L, sodium chloride:2-10g/L, rotating speed 200-400rpm, 25-35 DEG C of temperature cultivate 12-24h.

6. the application conditions in improvement booth vegetable soil hardening are according to claim 1：Microbial inoculum is diluted into 100-400 Apply again, drip irrigation to plastic shed soil.