CN110777085B - Cadmium-resistant nightshade growth-promoting rhizosphere lysine bacillus and application thereof - Google Patents

Abstract

The strain is preserved in China general microbiological culture Collection center (CGMCC for short), and the preservation number is CGMCC NO.16051, and the preservation date is 7, 3 days in 2018. The strain can be used for remedying cadmium-polluted farmland by combining black nightshade and microorganism, promoting the growth and development of black nightshade, enhancing the absorption and enrichment of cadmium by black nightshade, and improving the extraction efficiency of cadmium in black nightshade.

Description

Cadmium-resistant nightshade growth-promoting rhizosphere lysine bacillus and application thereof

Technical Field

The invention relates to a technology in the field of heavy metal soil bioremediation, in particular to cadmium-resistant solanum nigrum L.rhizosphere growth-promoting lysine bacillus and application thereof.

Background

Because of the wide area and the complex particularity of restoring the environment, the farmland soil heavy metal pollution generally adopts the bioremediation technology. The plant-microorganism combined repair technology is a hotspot of current research, and the key of the technology is to obtain microorganisms capable of promoting the repair efficiency of heavy metal super-enriched plants.

The existing combined repair microorganism based on Solanum nigrum (Solanum nigrum L.) is mainly screened from cadmium-resistant microorganisms and endophytes, the screening mode belongs to a wide-spreading type, no pertinence exists, and the separated effective strains are still few at present. In order to obtain a more efficient repairing strain, a more efficient cadmium-resistant combined repairing strain is directionally screened by combining a microorganism-plant repairing mechanism.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the cadmium-resistant nightshade growth-promoting rhizosphere lysine bacillus and the application thereof.

The invention is realized by the following technical scheme:

the invention relates to a lysine bacillus (Lysinibacillus sp.) NT1, which is currently preserved in China general microbiological culture Collection center (CGMCC for short), the preservation number is CGMCC NO.16051, and the preservation date is 7 months and 3 days in 2018.

The preservation address of the China general microbiological culture Collection center is No. 3 of Xilu No.1 of Beijing Kogyo district, morning and evening, and the postal code is as follows: 100101.

the invention relates to application of the bacillus Lysinibacillus sp NT1 in microbial-black nightshade combined soil cadmium pollution remediation, which specifically comprises the following steps: after expanding culture of the lysine bacillus NT1, inoculating the Bacillus lysimachiae NT1 to the rhizosphere of the solanum nigrum for bioremediation of soil in cadmium-polluted farmland, adsorbing heavy metal Cd in the soil by the interaction of microorganism-plant growth promotion and microorganism-heavy metal in the growth process of the thallus ²⁺ Promoting plant growth and absorption and transport of cadmium, and enhancing heavy metal Cd of plant ²⁺ The ability to enrich.

The enlarged culture is that: culturing in an aseptic fermentation medium containing carbon source, nitrogen source, inorganic salt and water at an initial pH of 6.0-7.5 and a culture temperature of 28-37 ℃ for 12-24 hours.

The inoculation is as follows: black nightshade at 1X 10 ⁹ Soaking roots in CFU/mL bacterial solution, transplanting or applying 1 × 10 to nightshade rhizosphere ⁹ CFU/mL of bacterial liquid.

The cadmium-polluted farmland is characterized in that: the total cadmium concentration is 0.3-10 mg/L.

Technical effects

The invention has the technical effects that:

1) The screened lysine bacillus (lysine bacillus sp.) NT1 can stably grow at the rhizosphere of the black nightshade, promotes the growth and development of the black nightshade, enhances the absorption and enrichment of cadmium by the black nightshade, improves the extraction efficiency of cadmium by the black nightshade, has no pathogenicity, can be widely applied to agricultural production, and has good application prospect.

2) Under the water culture condition, after the NT1 strain is applied, the Cd content of the roots of the solanum nigrum can be increased by 45.74%, the Cd content of stems and leaves can be respectively reduced by 10.43% and 30.01%, the plant height and plant height can be respectively increased by 20.64%, the dry weight of the overground part can be increased by 62.65%, and the cadmium enrichment amount of the overground part and the underground part of the single solanum nigrum can be respectively increased by 28.52% and 54.61%.

3) The strain provided by the invention can have excellent Cd ²⁺ Heavy Metal tolerance, cd content at 10mg/L ²⁺ The growth of the Cd in the culture medium is not affected, and the Cd can be adsorbed by the organism ²⁺ Can be absorbed in cells.

4) The strain provided by the invention has excellent growth promoting characteristics, has the capacity of producing auxin, siderophores and decomposing organic phosphorus and inorganic phosphorus, can be used in combination with other enrichment plants, and can be widely applied to different plant-microorganism combination heavy metal Cd ²⁺ And (3) repairing the system.

Drawings

FIG. 1 is a photograph showing the colony morphology of the lysine bacillus NT1 of the present invention;

FIG. 2 is a photograph (100X) of a spore stain of Bacillus lysinate NT1 of the present invention;

FIG. 3 is a photograph (100X) of a gram stain of Bacillus lysinate NT 1;

FIG. 4 is a phylogenetic tree of the present invention, bacillus lysinate NT1.

Detailed Description

Example 1

Isolation and purification of Bacillus Lysinibacillus sp NT1

The embodiment comprises the following steps:

step 1, taking nightshade rhizosphere soil, collecting plants with roots, shearing off overground parts, removing large soil blocks around the roots, brushing rhizosphere soil within 1cm of root systems into a sterile fresh-keeping bag by using a brush, taking the sterile fresh-keeping bag back to a laboratory, storing the sterile fresh-keeping bag in a refrigerator at 4 ℃, and screening the plant in 2 days. Weighing 5g of soil sample, adding into a conical flask containing glass beads and 50mL of sterile water, performing shake culture on a shaking table at 37 ℃ and 200rpm for 30min, and keeping the culture solution in a water bath kettle at 80 ℃ for 10min; transferring the above 10mL suspension into an Erlenmeyer flask containing 100mL LB-Cd (Cd content 10 mg/L) liquid culture medium, and performing enrichment culture at 37 deg.C and 200rpm for 24h. Repeating the operation for 2 times, and carrying out enrichment culture.

Step 2, taking the culture solution to dilute in a super-clean workbench, and taking 10 ^-4 、10 ^-5 、10 ^-6 Respectively coating 200 mu L of the single colonies on LB-Cd (Cd content is 10 mg/L) solid plates, inversely placing the plates in a constant temperature box at 37 ℃ for culture, when more single colonies appear on the plates, selecting the single colonies with different properties such as colony morphology, color and the like on an ultra-clean bench, streaking and purifying the single colonies on the corresponding solid plates to prepare glycerol bacteria, and preserving the glycerol bacteria at-80 ℃ to finally obtain the strain lysine bacillus (Lysinibacillus sp.) NT1.

As shown in FIGS. 2 to 4, the lysine bacillus NT1 is cultured for 24 hours in LB solid medium at 35 ℃, and the bacterial colony is round, light opaque, light yellow and flat in surface; culturing at 35 deg.c for 2-3 days to form spore, spherical spore or elliptic spore. The culture temperature is positive in gram staining, the puncture test shows that the strain has motility, does not produce indole, does not produce hydrogen sulfide, and cannot reduce nitrate, beta-galactosidase, urease and arginine double hydrolase experiments are negative, catalase experiments are positive, glucose acid production experiments are positive, and lactose, sucrose, maltose and D-fructose acid production experiments are negative.

The lysine bacillus NT1 (Lysinibacillus sp.) is detected by a 16S rDNA sequence, the sequence is shown as SEQ ID No.1, the sequence is compared with a gene sequence in GenBank for homology, the homology reaches 99 percent, and a phylogenetic tree (shown as figure 4) is constructed by utilizing MEGA 5.0 (Molecular evolution Genetics Analysis) software and adopting an adjacency method (Neighbor-Joining) clustering Analysis.

Example 2

Isolation and growth promotion identification of lysine bacillus (Lysinibacillus sp.) NT1

The embodiment comprises the following steps:

1) And (3) identification of IAA production capacity: the strains obtained by separation are respectively inoculated into 20mL of TSB liquid culture medium containing tryptophan (100 mg/L) by adopting a Salkowski's reagent microplate colorimetric method and referring to the method of Loper, and centrifuged at 8000r/min for 10min after shaking culture is carried out for 48h at the temperature of 28 ℃ and at the speed of 200 r/min. After 2mL of the supernatant obtained by the above centrifugation was added 50. Mu.L of 83% volume orthophosphoric acid and 4mL of Salkowski reagent, and the solution became pink, indicating the generation of IAA. Taking the above color development solution, developing at 25 deg.C in dark for 30min, and measuring light absorption value at wavelength of 530 nm. And (3) replacing the culture solution with distilled water for the same reaction to be used as a reference for zero adjustment, preparing 10, 20, 30, 40 and 50mg/L standard IAA gradient solutions, measuring the light absorption value at 530nm by the same method, making a standard curve, and calculating the IAA concentration of the bacteria liquid.

2) And (3) identifying the phosphate solubilizing capability: inoculating the strain to an LB culture medium, culturing for 24h at 30 ℃ and 200r/min, absorbing 1mL of the strain, inoculating the strain to an inorganic phosphorus and organic phosphorus culture medium, culturing for 7d at 28 ℃ and 200r/min, adopting a molybdenum-antimony colorimetric resistance method, taking 10mL of bacterial liquid, centrifuging for 10min at 8000r/min, taking 2.5-5mL of supernatant, transferring the supernatant to a 50mL volumetric flask, diluting to about 3/5 of the total volume with water, adding 1-2 drops of dinitrophenol indicator, adjusting the solution to be just yellowish by using 100g/L of sodium carbonate solution or 50mL/L of sulfuric acid solution, accurately adding 5mL of antimony color-resisting agent, shaking up, adding water to fix the volume, keeping the room temperature to more than 15 ℃, and standing for 30min (within 8 h). And (3) carrying out colorimetric determination on the chromogenic sample solution on a microplate reader for the absorbance value of 700nm, and taking a blank (a blank culture medium without inoculated thalli) as a reference to adjust the zero point. Preparing standard solutions containing phosphorus (K2 HPO 4) of 0.0, 0.2, 0.4, 0.8 and 1.0mg/L, measuring absorbance value at 700nm, making a standard curve, and calculating the corresponding phosphorus content.

3) And (3) identifying the siderophore production capacity: inoculating the strain picked by a platinum wire inoculating loop into an MKB liquid culture medium, carrying out shake culture at 28 ℃ and 200r/min for 48h, taking the bacterial liquid, centrifuging for 15min at 1500r/min, adding 3mL of CAS detection liquid and centrifugal supernatant into a 20PA bottle, fully and uniformly mixing, measuring the light absorption value (A) at the wavelength of 630nm after 1h, and taking double distilled water as a control for zero adjustment. And another 3mL CAS detection solution is fully and uniformly mixed with 3mL MKB liquid culture medium, and the light absorption value is determined by the same method to be the reference value (Ar).

Example 3

Bacillus Lysinibacillus sp NT1 enhanced cadmium extraction test for black nightshade

The embodiment comprises the following steps: taking full solanum nigrum seeds, disinfecting and soaking, pre-culturing for 4 weeks, selecting solanum nigrum seedlings with strong and consistent growth vigor in a water culture tank, selecting Hoagland nutrient solution as water culture solution, and adding CdCl from an external source ₂ Treating with concentration of 4mg/L, culturing growth promoting bacteria in LB liquid culture medium for 24 hr, adding into hydroponic liquid (the concentration of bacteria liquid in hydroponic liquid is about 1 × 10) ⁷ CFU/mL), replacing the hydroponic solution and the bacterial solution every 5d, and continuously culturing for 8 weeks in a glass greenhouse. Setting a blank Control (CK) without adding cadmium and bacteria, and a control (CK-Cd) without adding cadmium and bacteria.

The growth index of the black nightshade is measured: gently taking out the whole plant of Solanum nigrum, measuring the plant height and root length of Solanum nigrum with a ruler, sucking off water from root, stem and leaf of Solanum nigrum with stainless steel scissors, weighing, putting into kraft paper envelopes, deactivating enzyme at 105 deg.C for 10min, drying at 70 deg.C to constant weight, and measuring the dry matter weight of the plant sample.

Treatment of	Plant height (cm)	Root length (cm)	Dry weight of aerial parts (g)	Underground dry weight (g)
					CK	6.75	5.36	2.12	0.49
CK-Cd	5.62	5.43	1.58	0.41
					NT1	6.78	16.70	2.57	0.52

And (3) determining the Cd content of the black nightshade: dividing the plant sample into root, stem and leaf each 5g, 20mmol/L Na ₂ And (3) performing hybridization in the EDTA solution for 15min, and then washing with deionized water for several times to remove heavy metals adsorbed on the surface. Removing surface water, deactivating enzyme at 105 deg.C for 10min, drying at 70 deg.C to constant weight, measuring dry matter weight of plant sample, and pulverizing the dried sample. HNO for plant samples ₃ -H ₂ O ₂ Digesting by a (3:1) method, diluting the digested sample by deionized water, and determining the content of Cd in the sample by an ICP-AES method.

The foregoing embodiments may be modified in many different ways by one skilled in the art without departing from the spirit and scope of the invention, which is defined by the appended claims and not by the preceding embodiments, and all embodiments within their scope are intended to be limited by the scope of the invention.

Sequence listing

<110> Shanghai university of transportation

<120> cadmium-resistant black nightshade root growth promoting lysine bacillus and application thereof

<130> f-b314e

<141> 2018-07-25

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1457

<212> DNA

<213> Bacillus Lysinibacillus sp

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cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgg catgctgatc cgcgattact 120

agcgattccg gcttcatgta ggcgagttgc agcctacaat ccgaactgag aacgacttta 180

tcggattagc tccctctcgc gagttggcaa ccgtttgtat cgtccattgt agcacgtgtg 240

tagcccaggt cataaggggc atgatgattt gacgtcatcc ccaccttcct ccggtttgtc 300

accggcagtc accttagagt gcccaactaa atgatggcaa ctaagatcaa gggttgcgct 360

cgttgcggga cttaacccaa catctcacga cacgagctga cgacaaccat gcaccacctg 420

tcaccgttgc ccccgaaggg gaaactatat ctctacagtg gtcaacggga tgtcaagacc 480

tggtaaggtt cttcgcgttg cttcgaatta aaccacatgc tccaccgctt gtgcgggccc 540

ccgtcaattc ctttgagttt cagtcttgcg accgtactcc ccaggcggag tgcttaatgc 600

gttagctgca gcactaaggg gcggaaaccc cctaacactt agcactcatc gtttacggcg 660

tggactacca gggtatctaa tcctgtttgc tccccacgct ttcgcgcctc agcgtcagtt 720

acagaccaga aagtcgcctt cgccactggt gttcctccaa atctctacgc atttcaccgc 780

tacacttgga attccacttt cctcttctgc actcaagtcc cccagtttcc aatgaccctc 840

cacggttgag ccgtgggctt tcacatcaga cttaaaggac cgcctgcgcg cgctttacgc 900

ccaataattc cggacaacgc ttgccaccta cgtattaccg cggctgctgg cacgtagtta 960

gccgtggctt tctaataagg taccgtcaag gtacagccag ttactactgt acttgttctt 1020

cccttacaac agagttttac gatccgaaaa ccttcttcac tcacgcggcg ttgctccatc 1080

aggctttcgc ccattgtgga agattcccta ctgctgcctc ccgtaggagt ctgggccgtg 1140

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gccgttacct caccaactag ctaatgcgcc gcgggcccat cctatagcga cagccgaaac 1260

cgtctttcag tctttcacca tgaagtaaaa gagattattc ggtattagcc ccggtttccc 1320

ggagttatcc caaactatag ggtaggttgc ccacgtgtta ctcacccgtc cgccgctaac 1380

gtcaaaggag caagctcctt ttctgttcgc tcgacttgca tgtattaggc acgccgccag 1440

cgttcgtcct gagccag 1457

Claims (6)

1. A lysine bacillus, characterized in that, the lysine bacillus (B), (B) and (C)Lysinibacillus sp.) NT1 is preserved in China general microbiological culture Collection center (CGMCC for short), the preservation number is CGMCC NO.16051, and the preservation date is 7 months and 3 days in 2018.

2. The use of lysinibacillus NT1 according to claim 1, in a solanum nigrum-microorganism combination for remediating cadmium contaminated farmland soil.

3. Use according to claim 2, characterized in that lysine is reacted with ammoniaBacillus acidificans (Lysinibacillus sp.) NT1 is inoculated to the rhizosphere of the solanum nigrum after being subjected to expanded culture, the growth and development of the solanum nigrum are promoted by the growth promoting effect of the bacterial strain on the solanum nigrum, and heavy metal Cd is utilized ²⁺ The biological adsorption effect of the black nightshade can change the absorption, enrichment and transfer characteristics of the black nightshade to cadmium, thereby achieving the purpose of removing heavy metals.

4. The use according to claim 3, wherein said scale-up culture is: bacillus lysinate: (Lysinibacillus sp.) The NT1 is inoculated in an aseptic fermentation medium containing a carbon source, a nitrogen source, inorganic salt and water, and is cultured for 12 to 24 hours under the environment that the initial pH is 6.0 to 7.5 and the culture temperature is 28 to 37 ℃.

5. The use of claim 2, prepared by mixing said black nightshade at 1 x 10 ⁹ Soaking roots in CFU/mL bacterial solution, transplanting or applying 1 × 10 to nightshade rhizosphere ⁹ CFU/mL of bacterial liquid.

6. The use of claim 2, wherein the cadmium pollution of the farmland soil is as follows: the total cadmium concentration is 0.3-10 mg/L.

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