CN117426395A - Application of pseudoalteromonas agaropectin strain in crop planting and/or soil improvement - Google Patents
Application of pseudoalteromonas agaropectin strain in crop planting and/or soil improvement Download PDFInfo
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- CN117426395A CN117426395A CN202310671933.2A CN202310671933A CN117426395A CN 117426395 A CN117426395 A CN 117426395A CN 202310671933 A CN202310671933 A CN 202310671933A CN 117426395 A CN117426395 A CN 117426395A
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2101/00—Agricultural use
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
Abstract
The invention provides an application of a pseudoalteromonas agaropectin (Pseudoalteromonas agarivorans) strain in crop planting and/or soil improvement, belonging to the technical field of agricultural microorganisms. The pseudomonas agaropectin strain HJWL2022 with the preservation number of CGMCCNo.26172 has the functions of reducing the heavy metal content in plants, promoting the growth of the plants, increasing the Vc content in the plants, the soluble protein content, increasing the abundance of soil microorganisms, improving the enzyme activity in the soil and the like, reduces the heavy metal content in the plants by using marine bacteria producing extracellular polysaccharide, improves the enzyme activity of the soil, reduces the transportation of the heavy metal into the plants, ensures the food safety, and is a safe, economic and environment-friendly technical means.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganisms, and particularly relates to application of a pseudoalteromonas agaricus strain in crop planting and/or soil improvement.
Background
Cadmium (Cd) is a persistent environmental pollutant, is difficult to degrade and has high accumulation property, cannot be assimilated and utilized by organisms, is extremely easy to enter a human body along with a food chain, seriously endangers the health of the human body, and the cadmium pollution of farmland soil caused by nonstandard industrial and agricultural production activities has become a primary problem to be solved by heavy metal pollution of farmland soil.
The in-situ fixation technology can reduce the transfer of Cd in soil, and the bioavailability of Cd can be reduced by adsorption, complexation and precipitation through applying the heavy metal fixative in the soil, however, the traditional organic and inorganic fixative can have adverse effects on soil properties and ecological environment and even cause secondary pollution.
In the prior art, reports about the application of microorganism strains for heavy metal degradation and plant growth promotion are numerous, but the application of marine bacteria producing extracellular polysaccharide to soil and the use of a single strain for crop planting and soil improvement have few reports on the combination effect, and the development of multifunctional microorganism strains is also a problem which needs to be solved in the technical field of agricultural microorganisms at present.
Disclosure of Invention
Therefore, the invention aims to provide the application of the pseudoalteromonas agaricus, which not only can reduce the heavy metal content in crops, but also can improve the soil.
The invention provides an application of a pseudoalteromonas agaragar (Pseudoalteromonas agarivorans) strain HJWL2022 or fermentation liquor thereof in crop planting, wherein the preservation number of the strain HJWL2022 is CGMCC No.26272.
Preferably, when the crops are planted, the strain HJWL2022 or the fermentation broth thereof has the effects of improving the quality of the crops and/or promoting the growth of the crops.
Preferably, the improving the crop quality comprises increasing the Vc content and/or the soluble protein content in the crop.
Preferably, the crop includes both commercial and agricultural crops.
Preferably, the commercial crop comprises vegetables and/or fruit trees.
The invention provides an application of a pseudoalteromonas agaragar strain HJWL2022 or fermentation liquor thereof in improving heavy metal contaminated soil.
Preferably, the heavy metal contamination comprises Cd 2+ And (5) pollution.
Preferably, the fermentation liquor of the pseudomonas agavorax strain HJWL2022 is prepared by the following steps:
inoculating the pseudoalteromonas agaragar strain HJWL2022 into a seed culture medium for seed culture, and inoculating the obtained seed liquid into a fermentation culture medium for fermentation culture to obtain a fermentation liquid.
Preferably, the seed medium is an aqueous solution comprising: 5g/L peptone, 1g/L yeast extract, 35g/L sea salt and pH value of 7.6-7.8;
the temperature of the seed culture is preferably 23-27 ℃; the rotation speed of the seed culture is 150-200 rpm; the time for culturing the seeds is 7-9 h.
Preferably, the fermentation medium is an aqueous solution comprising: glucose 45g/L, (NH) 4 ) 2 SO 4 2.5g/L and sea salt 35g/L, and the pH value is 8-9;
the temperature of the fermentation culture is preferably 23-27 ℃; the rotation speed of the fermentation culture is 150-200 rpm; the fermentation culture time is 34-37 hours. .
The invention provides an application of a pseudoalteromonas agaragar (Pseudoalteromonas agarivorans) strain HJWL2022 or fermentation liquor thereof in crop planting and/or improving heavy metal contaminated soil, and the preservation number is CGMCC No.26272. Experiments prove that the pseudoalteromonas agaragar strain HJWL2022 provided by the invention has the function of reducing heavy metal Cd in plants 2+ The content, promote plant growth, increase Vc content, soluble protein content, increase soil microorganism abundance, increase enzyme activity in soil and the like, and the marine bacteria producing extracellular polysaccharide are utilized to reduce heavy metal content in plants, improve soil enzyme activity, reduce heavy metal transportation into plants, ensure food safety, and are a safe, economic and environment-friendly technical means.
Drawings
FIG. 1 shows different concentrations of Cd after inoculation of HJWL2022 in the soil filtrate 2+ For bacterial content (OD) 600 Representation) of the effect results;
FIG. 2 shows different concentrations of Cd 2+ The influence result of the polysaccharide content in the soil filtrate under treatment;
FIG. 3 shows different concentrations of Cd 2+ The pH value of the soil filtrate is changed under treatment;
FIG. 4 shows the results of cadmium concentration changes after inoculation of strain HJWL2022 in soil filtrate of different initial cadmium concentrations;
FIG. 5 is a graph showing the effect of strain HJWL2022 on lettuce root cadmium content;
FIG. 6 is a graph showing the effect of strain HJWL2022 on cadmium content in lettuce edible tissues;
FIG. 7 is a graph showing the effect of strain HJWL2022 on leachable cadmium content of lettuce rhizosphere soil DTPA;
FIG. 8 is a graph showing the effect of strain HJWL2022 on the soluble protein content of lettuce edible tissues;
FIG. 9 is a graph showing the effect of strain HJWL2022 on the VC content of lettuce edible tissues;
FIG. 10 is the effect of strain HJWL2022 on alkaline phosphatase content in rhizosphere soil;
FIG. 11 is a graph showing the effect of strain HJWL2022 on urease in rhizosphere soil;
FIG. 12 is a graph showing the effect of strain HJWL2022 on dehydrogenase in rhizosphere soil;
FIG. 13 shows the proliferation effect of strain HJWL2022 on Acidovorax (A), bacteroides (B) and Acidovorax (C);
FIG. 14 is a front 20 heat map of lettuce rhizosphere soil contaminated with different concentrations of cadmium.
Biological material preservation information
Pseudomonas agaricus (Pseudoalteromonas agarivorans) HJWL2022 is preserved in China general microbiological culture Collection center (China General Microbiological Culture Collection Center, CGMCC) with the preservation address of China general microbiological culture collection center: the collection date is 2022, 12 and 26 days, and the strain collection number is CGMCC No. 26172.
Detailed Description
The invention provides an application of a pseudoalteromonas agaricus (Pseudoalteromonas agarivorans) strain HJWL2022 or a fermentation liquor thereof in crop planting, wherein the preservation number of the pseudoalteromonas agaricus strain HJWL2022 is CGMCCNo. 26172.
In the invention, the pseudoalteromonas agaragar strain HJWL2022 is grown on the culture plates of da Ji haliotis discus in the Shandong province, and the morphological characteristics are as follows: the colony is smaller, moist, the surface is more transparent, and the edge is smooth and neat. The species relationship is determined by the comparison of 16S rRNA identification and homology, and belongs to pseudoalteromonas (Pseudoalteromonas agarivorans).
In the invention, the pseudoalteromonas agaragar strain can also be used as a microorganismThe microbial inoculum is provided in the form of a bacterial agent. The microbial agent comprises the pseudoalteromonas agaragar strain HJWL2022 and auxiliary materials. The concentration of the pseudoalteromonas agaropectin strain HJWL2022 is preferably 1 multiplied by 10 8 ～1×10 9 CFU/mL, more preferably 2X 10 8 CFU/mL. The invention is not particularly limited in the types of the auxiliary materials, and auxiliary materials of microbial agents well known in the art can be adopted. The preparation method of the microbial inoculum is not particularly limited, and the microbial inoculum can be prepared by adopting preparation schemes well known in the art.
In the invention, the culture method of the fermentation broth of the pseudoalteromonas agaropectin strain HJWL2022 comprises the following steps:
inoculating the pseudoalteromonas agaragar strain HJWL2022 into a seed culture medium for seed culture, and inoculating the obtained seed liquid into a fermentation culture medium for fermentation culture to obtain a fermentation liquid.
In the present invention, the seed medium is preferably an aqueous solution comprising: 5g/L peptone, 1g/L yeast extract, 35g/L sea salt and pH value of 7.6-7.8. The method of preparing the seed medium is not particularly limited, and the preparation scheme of the medium known in the art can be adopted. The temperature of the seed culture is preferably 23 to 27 ℃, more preferably 25 ℃. The rotation speed of the seed culture is 150 to 200rpm, more preferably 180rpm. The time for the seed culture is preferably 7 to 9 hours, more preferably 8 hours. During the seed culture, the pseudoalteromonas agaragar strain HJWL2022 is preferably selected for single colony inoculation.
In the present invention, the fermentation medium is preferably an aqueous solution comprising: glucose 45g/L, (NH) 4 ) 2 SO 4 2.5g/L and sea salt 35g/L, and the pH value is 8-9. The method of preparing the fermentation medium is not particularly limited, and the preparation scheme of the medium known in the art can be adopted. The temperature of the fermentation culture is preferably 23 to 27 ℃, more preferably 25 ℃. The rotation speed of the fermentation culture is preferably 150 to 200rpm, more preferably 180rpm. The fermentation time is preferably 34 to 37 hours, more preferably 35 hours. In the fermentation culture, the seed liquid is preferably inoculated in an amount of7 to 10%, more preferably 8%.
According to the invention, after fermentation culture, the pseudoalteromonas agaropectin strain HJWL2022 produces extracellular polysaccharide, and the extracellular polysaccharide can effectively reduce the heavy metal content in plants, improve the enzyme activity in soil, reduce the heavy metal content in soil, further reduce the transportation to plants, ensure the food safety, improve the soil and increase the microorganism abundance of the soil.
In the present invention, the crops preferably include commercial crops and agricultural crops. The cash crop preferably includes vegetables and/or fruit trees.
Experiments prove that the pseudoalteromonas agaricus strain HJWL2022 can promote the growth of crops, increase the Vc content and the soluble protein content in the crops, and improve the planting quality of the crops. Meanwhile, the pseudoalteromonas agaropectin strain HJWL2022 can also reduce the cadmium content in crops.
The invention provides an application of the pseudoalteromonas agaragar strain HJWL2022 or fermentation liquor thereof in improving heavy metal contaminated soil.
In the present invention, the heavy metal contamination preferably includes Cd 2+ And (5) pollution. Cd (cadmium sulfide) 2+ The concentration of contamination is preferably 3mg/L or more, more preferably 6 to 30mg/L, and still more preferably 6 to 20mg/L. In the embodiment of the invention, the pseudoalteromonas agaragar strain HJWL2022 or the fermentation liquor pair Cd obtained by the method 2+ The contaminated soil filtrate was treated, and the result showed that Cd 2+ The strain HJWL2022 is inhibited from growing to a certain extent by pollution, but extracellular polysaccharide is obviously increased compared with a control group, and soluble Cd is also increased 2+ The content is obviously reduced (24 to 37 percent), and the pH value of the soil is improved, which is beneficial to reducing Cd in the soil 2+ The content is as follows. The pseudomonas agaricus bisporus strain HJWL2022 is capable of repairing soil by utilizing the characteristic of extracellular polysaccharide production, and is a soil repairing technology with application prospect.
In the invention, the pseudoalteromonas agaragar strain HJWL2022 has multiple functions of improving the enzyme activity in soil and the like. Experiments prove that the strain HJWL2022 can improve the activities of alkaline phosphatase, urease and dehydrogenase in lettuce rhizosphere soil. Meanwhile, the strain HJWL2022 can also increase the abundance of soil microorganisms, particularly has a certain proliferation effect on the bacterial phylum (Acidobacter) of Acidovorax (Gemmatimonetes), and experiments prove that the application of the strain HJWL2022 has an improvement effect on the relative abundance of Streptomyces (Streptomyces), sphinomonas (Sphingomonas), nocardioides (Nocardioides), bacillus (Bacillus), gaiella and rhodobacter (Solirubrobacter).
The application of the pseudoalteromonas agaragar strain provided by the invention in preparing crop planting and/or soil improvement is described in detail below with reference to examples, but they should not be construed as limiting the scope of the invention.
Example 1
Separation and screening method of pseudoalteromonas agaropectin strain
The sample was collected from Shandong adult sea area, and the sampling time was when abalone larvae were peeled off from the attachment base (5 middle ten days). The sample was scraped 10cm on a plate 2 The attachments on the area are stored in a 2ml sterilizing and freezing tube, stored in an ice box in a closed manner and returned to the laboratory within 24 hours. Isolation of heterotrophic bacteria and extraction of DNA were performed. Diluting the collected sample with sterile seawater to 10 -3 、10 -4 、10 -5 、10 -6 Four concentrations were spread evenly on 2216E solid medium plates with a sterilized spreader and incubated at 25℃for 48h to pick colonies with significant morphological differences. Purification was performed by streaking. Detecting whether the strain produces polysaccharide or not by a phenol-sulfuric acid method, and primarily screening polysaccharide with the yield of more than 0.5g/L to obtain the strain which produces polysaccharide, namely HJWL2022.
Example 2
Identification method of pseudoalteromonas agaropectin strain
PCR amplification is carried out by taking genomic DNA of the strain HJWL2022 as a template and adopting universal primers 27F and 1492R, and the length of the amplified sequence is 1413bp, and the sequence is shown as SEQ ID NO. 1 (CATGCAGTCGAGCGGTAACAGAAAGTGTGCTTGCTACTTTGCTGACG AGCGGCGGACGGGTGAGTAATGCTTGGGAACATGCCTTGAGGTGGGGGACAACAGTTGGAAACGACTGCTAATACCGCATAATGTCTACGGACCAAAGGGGGCTTCGGCTCTCGCCTTTAGATTGGCCCAAGTGGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCAACGATCCCTAGCTGGTTTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTCAGGAGGAAAGGTTAGTAGTTAATACCTGCTAGCTGTGACGTTACTGACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTACGCAGGCGGTTTGTTAAGCGAGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATTTCGAACTGGCAAACTAGAGTGTGATAGAGGGTGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAATACCGATGGCGAAGGCAGCCACCTGGGTCAACACTGACGCTCATGTACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTAGAAGCTCGGAGCCTCGGCTCTGTTTTTCAAAGCTAACGCATTAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACACTTGACATACAGAGAACTTACCAGAGATGGTTTGGTGCCTTCGGGAACTCTGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTTAGTTGCTAGCAGGTAATGCTGAGAACTCTAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGTGTAGGGCTACACACGTGCTACAATGGCGCATACAGAGTGCTGCGAACTCGCGAGAGTAAGCGAATCACTTAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCATATCAGAATGATGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGATAGTCTAACCCTCGGGAGGACGTTACCACGGAG). The strain HJWL2022 was systematically classified by BLAST tool alignment in NCBI.
The results showed that the strain HJWL2022 was most similar to Pseudoalteromonas agarivorans, reaching 99.67%, and it was determined that the strain HJWL2022 belongs to Pseudomonas (Pseudoalteromonas agarivorans).
Example 3
Fermentation culture method of pseudoalteromonas agaropectin strain
The strain HJWL2022 is inoculated in a solid culture medium (seed culture medium containing 17g/L of agar) for culture, single colonies are selected and inoculated in the seed culture medium, shaking culture is carried out for 8 hours at 25 ℃ at 180rpm, then the selected colonies are inoculated in a fermentation culture medium according to the inoculation amount of 8%, shaking culture is carried out for 36 hours at 25 ℃ at 180rpm, and the strain HJWL2022 fermentation liquor is obtained.
Wherein, the seed culture medium is: 5g/L peptone, 1g/L yeast extract, 35g/L sea salt and the balance of water, and the pH value is 7.6. The fermentation medium is as follows: glucose 45g/L, (NH) 4 ) 2 SO 4 2.5g/L, 35g/L sea salt and the balance of water, and the pH value is 8.5. Sterilizing at 121deg.C for 20 min.
Example 4
Soil filtrate experiment
1. Preparation of soil filtrate
Uniformly mixing a soil sample with deionized water according to a ratio of 1:4 (m: v, unit g: mL), placing the mixture in a shaking table (180 rpm) at 25 ℃ for shaking culture for 48 hours, centrifuging the mixture for 15 minutes (5000 rpm) by a centrifuge, collecting supernatant, filtering and sterilizing the supernatant by a Millipore filter with a pore diameter of 0.45 mu m to obtain a soil filtrate, uniformly mixing the soil filtrate with a prepared sterile fermentation medium according to a volume ratio of 4:1 (200 mL total), simulating a soil environment, and respectively filling the soil filtrate into 500mL sterile conical flasks for later use.
2. Treatment of Strain HJWL2022
The activated strain HJWL2022 is picked up to be inoculated into a sterilized seed culture medium, shake flask culture (25 ℃,180 rpm) is carried out for 8 hours, sterile physiological saline is used for resuspension, and the bacterial suspension after resuspension is inoculated into a fermentation broth containing 200mL of sterile soil filtrate and fermentation medium. Configuring CdCl with different concentrations 2 Solutions are respectively added into fermentation liquor, and Cd in the fermentation liquor is set 2+ The concentration gradients were 0, 3, 6, 10 and 20mg/L, four replicates were set per group, the non-inoculated strain was used as control, the shaker was set at 25℃and 180rpm, and the culture was performed on the shaker for 96h. According to the characteristics of the bacterial growth curve, five sampling points are set, and the sampling points are respectively: 0h, 24h, 48h, 72h and 96h.
Samples were taken at 5 time points, respectively. Monitoring bacterial growth (OD) by spectrophotometry (600 nm) of the optical density of the bacterial suspension 600 Representation). The pH value of the fermentation broth was measured using a pH meter.
The method for detecting the polysaccharide content comprises the following steps: the polysaccharide content of the soil filtrate was determined (The antioxidant activities of carboxymethylated garlic polysaccharide and its derivatives) by reference to the method of Hao Cheng et al (2019).
The method for detecting the Cd content comprises the following steps: cadmium concentration was determined by inductively coupled plasma mass spectrometry (ICP-MS), reference Liu Xiaojun (2019) (ICP-MS method to determine six heavy metal elements in soil).
The experimental results are shown in fig. 1 to 4.
Determination of OD in soil filtrate by five-point sampling 600 pH, exopolysaccharide (EPSs) content and soluble Cd 2+ The content is as follows. The results show that in Cd 2+ Under stress, although the strain growth is inhibited, the EPS content is increased compared with the control group, and the soluble Cd is soluble 2+ The content is obviously reduced (24 to 37 percent), and the pH value of the soil filtrate is also increased, thereby being beneficial to reducing the Cd in the soil filtrate 2+ . The in vitro experimental result of the soil filtrate simulation shows that the use of the exopolysaccharide-producing strain HJWL2022 for restoring heavy metals in soil can be a restoration technical scheme with wide application prospect.
Example 4
Potting experiment
To dispose the prepared CdCl 2 The solution was thoroughly mixed with the collected soil sample, setting 3 concentration gradients: 0. 0.5 and 1mg/kg, 6 replicates per gradient were set. Placing into flowerpots with diameter of 28cm and height of 35cm, each pot containing 4.8kg of tested soil, adding deionized water to maintain the water content of the soil at about 70% of the field water holding capacity, balancing for 45 days, placing into a greenhouse (25+ -3deg.C), humidity of 70%, and photoperiod of 10 or 12h/d. Selecting purchased lettuce seeds with uniform size by a floatation method, soaking and sterilizing the lettuce seeds with full particles by using 5% sodium hypochlorite solution for 10min, and cleaning the lettuce seeds with sterile water for three times. And selecting complete unbroken seeds to sow 30 seeds in each pot. After germination of the seeds, the seeds were diluted, 15 seedlings of similar growth and maturity were selected per pot, and the cultivation was continued until the third season of lettuce, and the P agarivarorans HJWL2022 strain was inoculated in a seed medium for 10 hours (25 ℃) and then resuspended in sterile water to a concentration of 1X 10 8 cells mL. 90mL of the bacterial suspension was injected into the root zone with a sterile syringe for root irrigation, and 3 replicates were set for 45d with sterile water as a control.
After the cultivation is finished, collecting the edible tissues and roots of each lettuce basin for subsequent analysis; lettuce roots and edible tissues were washed with 0.01M ethylenediamine tetraacetic acid (EDTA) and distilled water and divided equally into two parts. Wherein one part is deactivated at 105 ℃ for 30min, dried to constant weight at 65 ℃, and the weight after drying is recorded; the dried edible tissue and roots were separately ground and digested to determine Cd content. The vitamin C (Vc) and soluble protein content of fresh edible tissue were determined using standard methods.
The method for measuring the Cd content comprises the following steps: referring to the method disclosed in the prior art (C.VOICA1, A.DEHELEAN1, A.IORDACHE2, I.GEANA.METHOD VALIDATION FOR DETERMINATION OF METALS IN SOILS BY ICP-MS), 0.1g of plant sample is accurately weighed for microwave digestion, and 5% HNO is used for digestion 3 And (3) determining the volume, and determining the Cd content in the digestion solution by adopting ICP-MS.
Method for determining vitamin C: the vitamin C content of the edible vegetable tissue is determined by the methods disclosed in the prior art (Zhao Xiaomei, jiang Ying, wu Yupeng, liu Kuan and Zhang Zhijiang. Study of the determination of VC content in fruits and vegetables [ J ]. Food science, 2006,27 (3): 197-199.).
Method for determining the content of soluble proteins: the soluble protein content of the edible tissues of vegetables was determined by the method disclosed in reference to the prior art (Zhao Yingyong, dycloud, cui Xiuming, zhang Wen and Ma Ni. Coomassie Brilliant blue G-250 staining method for determining the soluble protein content of kusnezoff monkshood [ J ]. University of Yunnan national university, nature science edition, 2006,15 (3): 235-237.).
Collecting soil tightly combined with the root system of the potting experiment (used as rhizosphere soil) for subsequent analysis; the urease activity, dehydrogenase activity and alkaline phosphatase activity of the collected soil were determined by reference to the methods of the host soldier et al, the wheat rhizosphere soil enzyme activity and the response of microbial biomass to the coverage mode, and the cotton and several crops rhizosphere soil enzyme activity and bacterial diversity analysis by the methods of Liu Haiyang et al.
Bacterial genomic DNA from the rhizosphere soil samples were extracted using a rapid DNA extraction kit (MP Biomedicals, santaAna, CA) and stored at-20 ℃ before further analysis; the amount and quality of the extracted DNA were determined by spectrophotometry (NanoDrop 1000, thermo Scientific, USA) and gel electrophoresis, respectively. The extracted DNA was amplified by 338F (5'-ACTCCTAGGGGGCAGCA-3', SEQ ID NO: 2) and 806R (5 '-GGACTACHVGGGTWTCTAAT-3', SEQ ID NO: 3) primers, which target the V4 region of the bacterial 16s rRNA. High throughput sequencing was done in Illumina Hiseq 2000 (Illumina inc., usa, san diego).
The results are shown in FIGS. 5 to 14.
FIGS. 5 and 6 are different concentrations Cd 2+ The edible tissue of lettuce and the cadmium content of root are treated. Cadmium content in lettuce roots and edible tissues increases significantly with increasing cadmium concentration in the soil. The cadmium content of the root is obviously higher than that of the aerial part. Biological enrichment factor (BCF) is often used to reflect the ability of plants to absorb heavy metals. After inoculation of the strain HJWL2022 in cadmium contaminated soil, the leaf enrichment index for cadmium decreased with increasing cadmium concentration, while the root enrichment index for cadmium tended to increase, decreasing and increasing by 5% and 10%, respectively. After the strain HJWL2022 is applied in the soil, the cadmium content in the leaves treated with different cadmium concentrations is obviously reduced by 40% and 12% respectively compared with the control group, and the cadmium content in the roots is obviously reduced (23% -26%).
FIG. 7 is a graph showing the effect of HJWL2022 on leachable cadmium content of lettuce rhizosphere soil DTPA. After HJWL2022 is added, the content of cadmium in the effective state in the soil is obviously reduced, and Cd is 0.5mg/kg 2+ And 1mg/kg Cd 2+ The effective cadmium content in the two treatment groups is reduced by 21% and 25%, respectively, and the Cd concentration is high 2+ The decrease of the cadmium content in the effective state in the treatment group is larger and is reduced to 0.58mg/kg.
FIG. 8 is the effect of HJWL2022 on the soluble protein content of lettuce edible tissues. Soluble protein content in edible lettuce tissue grown in cadmium contaminated soil with Cd 2+ The concentration is increased and is obviously reduced, compared with a blank group, when Cd in soil 2+ When the content was 1mg/L and the strain HJWL2022 was not applied, the soluble protein content was reduced by 14%, and by applying the strain HJWL2022 at different concentrations, the soluble protein content was increased by 6%, 12% and 7% respectively as compared with the blank control group.
FIG. 9 is HJWL2022 effect on VC content of lettuce edible tissues. In the potting experiments, the Vc content in the lettuce edible tissues in three potting plants per treatment group was calculated. High concentration Cd 2+ (1 mg/kg) can significantly inhibit Vc content in lettuce edible tissues, and compared with two groups of controls, the Vc content is reduced by 43% and 39%, and Cd concentration is low 2+ (0.5 mg/L) also reduced Vc content in lettuce, but by applying strain HJWL2022, vc content in lettuce edible tissues could be increased to be recovered to be free of Cd 2+ And the level of poisoning. Next, cd was added at 0mg/L and 1mg/L 2+ Under the stress condition, the strain HJWL2022 is applied to promote the synthesis of Vc content in lettuce edible tissues and relieve the influence of cadmium stress on Vc content.
FIG. 10 is a graph showing the effect of HJWL2022 on alkaline phosphatase content in rhizosphere soil. Under the condition of no inoculation HJWL2022, low concentration Cd is found 2+ The alkaline phosphatase activity of the treated group was not significantly different from that of the control group without cadmium pollution, but the Cd concentration was high 2+ Under the condition that the alkaline phosphatase activity is obviously reduced by 6%, the heavy metal cadmium pollution can inhibit the alkaline phosphatase activity. In the soil-treated group containing strain HJWL2022, it was found that alkaline phosphatase activity was significantly improved by 18%, 25% and 35%, respectively, as compared to the group without HJWL2022.
FIG. 11 is a graph showing the effect of HJWL2022 on urease activity in rhizosphere soil, as shown by Cd 2+ The concentration is increased, the urease activity in the soil is obviously improved, and compared with the condition without cadmium stress, 0.5mg/kg Cd is added 2+ The urease activity was increased by 33% when 1mg/kg Cd was added 2+ After that, the urease activity is greatly increased by one time. After inoculation of HJWL2022, each concentration Cd 2+ Urease activity in the treatment group was also increased to varying degrees, especially at low concentrations of Cd 2+ Under the condition, the urease activity is obviously improved by 35 percent.
FIG. 12 shows the effect of HJWL2022 on dehydrogenase in rhizosphere soil. After the strain HJWL2022 is inoculated in the soil, the dehydrogenase activity in the soil is obviously improved compared with that of a non-inoculated control group, and the presence of the strain HJWL2022 can improve the dehydrogenase activity in the soil. In the low concentration cadmium contaminated soil, the activity of dehydrogenase is slightly reduced compared with the non-contaminated soil.
FIG. 13 shows the effect of HJWL2022 on the abundance of rhizosphere microorganisms, strains with a certain proliferation effect on Acidobacter (Acidobacter), bacteroides (Bacteroides) and Acidovorax (Gemmatimonades).
FIG. 14 is a front 20 heat map of lettuce rhizosphere soil contaminated with different concentrations of cadmium. The administration of cadmium reduces the relative abundance of most dominant bacteria. With inoculation of HJWL2022, the relative abundance of dominant species increased. For example, in rhizosphere soil inoculated with HJWL2022, the relative abundance of Streptomyces (Streptomyces), sphingomonas (sphingamonas), nocardiopsis (Nocardioides), bacillus (Bacillus), gaiella, and rhodobacter (Solirubrobacter) is high.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. Application of a pseudoalteromonas agaragar (Pseudoalteromonas agarivorans) strain HJWL2022 or fermentation liquor thereof in crop planting, wherein the preservation number of the strain HJWL2022 is CGMCC No.26272.
2. Use according to claim 1, wherein the strain HJWL2022 or a fermentation broth thereof has the effect of improving crop quality and/or promoting crop growth when the crop is planted.
3. The use according to claim 2, wherein the improvement in crop quality comprises an increase in Vc content and/or soluble protein content in the crop.
4. The use according to claim 1, wherein the crops include cash crops and agricultural crops.
5. The use according to claim 4, wherein the cash crop comprises vegetables and/or fruit trees.
6. An application of a pseudoalteromonas agaragar strain HJWL2022 or a fermentation liquor thereof in improving heavy metal contaminated soil.
7. The use according to claim 6, wherein the heavy metal contamination comprises Cd 2+ And (5) pollution.
8. The use according to any one of claims 1 to 7, characterized in that the fermentation broth of the pseudoalteromonas agaricus strain HJWL2022 is prepared by the following steps:
inoculating the pseudoalteromonas agaragar strain HJWL2022 into a seed culture medium for seed culture, and inoculating the obtained seed liquid into a fermentation culture medium for fermentation culture to obtain a fermentation liquid.
9. The use according to claim 8, wherein the seed medium is an aqueous solution comprising: 5g/L peptone, 1g/L yeast extract, 35g/L sea salt and pH value of 7.6-7.8;
the temperature of the seed culture is preferably 23-27 ℃; the rotation speed of the seed culture is 150-200 rpm; the time for culturing the seeds is 7-9 h.
10. The use according to claim 8, wherein the fermentation medium is an aqueous solution comprising: glucose 45g/L, (NH) 4 ) 2 SO 4 2.5g/L and sea salt 35g/L, and the pH value is 8-9;
the temperature of the fermentation culture is preferably 23-27 ℃; the rotation speed of the fermentation culture is 150-200 rpm; the fermentation culture time is 34-37 hours.
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