CN115418326B

CN115418326B - Composite microbial agent and application thereof

Info

Publication number: CN115418326B
Application number: CN202210309221.1A
Authority: CN
Inventors: 李鑫; 陶禹; 陈杰; 陈芳; 周池
Original assignee: Hunan Longke Wisdom Agricultural Technology Co ltd; Hunan vegetable research institute
Current assignee: Hunan Longke Wisdom Agricultural Technology Co ltd; Hunan vegetable research institute
Priority date: 2022-03-28
Filing date: 2022-03-28
Publication date: 2024-08-20
Anticipated expiration: 2042-03-28
Also published as: CN115418326A

Abstract

The invention belongs to the technical field of agricultural microorganism application, and relates to a composite microbial agent and application thereof. The invention separates the achromobacter (Achromobacter insuavis) SL8, the enterobacter (Enterobacter cancerogenus) SL12, the alcaligenes (ALCALIGENES FAECALIS) SL7 and the Serratia (SERRATIA MARCESCENS) SL11 from the cadmium polluted site, has the capability of efficiently removing cadmium and can also prevent and treat plant epidemic diseases and gray mold.

Description

Composite microbial agent and application thereof

Technical Field

The invention belongs to the technical field of agricultural microorganism application, and particularly relates to a composite microbial agent and application thereof.

Background

The main method for reducing and repairing the heavy metal pollution of the soil at present comprises the following steps: physical methods (e.g., earth-alien, earth-changing, deep ploughing), chemical methods (leaching, application of modifiers) and biological methods (phytoremediation and microbial remediation). The physical method has large engineering quantity, the chemical method is easy to cause secondary pollution, and the biological method is used for solving the problem without losing an effective method. In addition, the biological restoration method can ensure the long-term effectiveness of soil restoration and maintain the long-term stability of a soil ecological system.

CN112538449A (alcaligenes faecalis DY-8 and application thereof in removing heavy metal cadmium), wherein the strain is used for leaching cadmium-polluted soil, the removal rate of weak acid cadmium, iron-manganese combined cadmium and total cadmium is 65-75%, the removal rate of organic combined cadmium is 50-60%, and the pH value of the soil is obviously improved by 1-2.5 units.

CN112625981A (a Serratia marcescens strain and application thereof) discloses that when the initial concentration of Cd ²⁺ is 10, 20, 40 and 80 mg.L ^-1, the removal efficiency of Cd ²⁺ is 40.08%, 40.74%, 38.71% and 49.81% respectively.

The maximum removal rate of the strain ZXH21 belonging to the genus Achromobacter to cadmium is 36.5% respectively in the screening of 2 cadmium-resistant microorganisms and the adsorption passivation difference mechanism of cadmium on the strain (Zhang Xuhui, 2019, nanjing university of agriculture).

CN104673715A (enterobacter which has fixing effect on cadmium and can promote plant growth) and application thereof, and discloses that the removal rate of the enterobacter to cadmium can reach 70-75%.

The pathogenic bacteria of the pepper phytophthora capsici (Phytophthora capsici leon.) in the fungus of the subgenera of the flagelliforme are of a wider host range, and besides the pepper, other hosts such as tomatoes, melon crops and the like are also included. Zhou Qiming the study reported that the host of phytophthora capsici contained 9 families of 21 cultivars and weeds. The pepper epidemic disease can be developed at each stage of pepper growth and development, and each tissue fluid of the pepper can be developed.

The gray mold of the capsicum damages the whole growing period of the capsicum, and mainly damages the leaves, stems, flowers and fruits. Onset of seedling stage, fading and yellowing of top of primary cotyledon, and water immersion soft rot when humidity is high; and then the seedlings are expanded to young stems, the young stems are thinned and become tan, and the young stems are broken or wilted to death. The plant is ill in the adult stage, the outer edge of the initial leaf is faded to form a water-immersed light brown disease spot, the initial leaf is expanded to form a round or oval shape, the initial leaf is brown and is provided with a large disease spot with light brown wheel lines, a gray mold layer is densely distributed on the disease spot when the humidity is high, and the whole leaf can be rotten and dead in the final stage of the disease; the stalks and the petioles are affected by diseases, the primary water is used for immersing irregular disease spots, the disease parts generate grey white mold-shaped objects, and the disease branches spread downwards to the crotch; the flower organ is infected with diseases, petals are brown, soaked in water and covered with a dense gray mold layer; the fruit is affected, water-immersed brown disease spots are generated locally around the pedicel of the young fruit, the skin becomes dark brown after the skin is gradually enlarged, the skin is recessed and rotten, and irregular rotagray mold is generated on the surface of the skin. The pathogen of the gray mold of the capsicum is botrytis cinerea, which belongs to fungi of the half-known bacteria.

In the prior art, the existence of a composite microbial inoculum which can realize the effect of reducing cadmium in soil and can prevent and treat plant epidemic diseases and gray mold does not appear.

Disclosure of Invention

In order to overcome the technical problems, the invention provides the following technical scheme:

a composite microbial inoculant comprising enterobacter (Enterobacter cancerogenus) SL12 and serratia (SERRATIA MARCESCENS) SL11; accession number of the enterobacteria (Enterobacter cancerogenus) SL 12: CCTCC NO is M2022288, and is preserved in China center for type culture Collection (China, university of Wuhan, wuhan) at a preservation address of 18 days of 2022; deposit number of Serratia (SERRATIA MARCESCENS) SL 11: CCTCC NO: M2022287, 3.18.2022, with China center for type culture Collection, with the preservation address being university of Wuhan, china.

Preferably, the complex microbial inoculant further comprises alcaligenes (ALCALIGENES FAECALIS) SL7; deposit number of alcaligenes (ALCALIGENES FAECALIS) SL 7: CCTCC NO: M2022285.

Preferably, the complex bacterial agent further comprises Achromobacter (Achromobacter insuavis) SL8 deposit number: CCTCC NO: M2022286.

Preferably, the bacterial liquid ratio of the enterobacteria (Enterobacter cancerogenus) SL12, serratia (SERRATIA MARCESCENS) SL11, alcaligenes (ALCALIGENES FAECALIS) SL7 and achromobacteria (Achromobacter insuavis) SL8 is: 1:1:1:1.

An application of a composite microbial inoculum in preparing a microbial inoculum for passivating cadmium.

An application of a composite microbial inoculum in preparing a medicament for preventing and treating plant diseases.

Preferably, the plant disease is epidemic disease or gray mold.

Preferably, the plant is capsicum.

The invention belongs to the technical field of agricultural microorganism application, and relates to a composite microbial agent and application thereof. The invention separates the Achromobacter (Achromobacter insuavis) SL8 and the enterobacter (Enterobacter cancerogenus) SL12 alcaligenes (ALCALIGENES FAECALIS) SL7 and the Serratia (SERRATIA MARCESCENS) SL11 from the cadmium polluted site, has the capability of efficiently removing cadmium and can also prevent and treat plant epidemic diseases and gray mold.

Drawings

FIG. 1 is a phylogenetic tree of Achromobacter (Achromobacter insuavis) SL 8.

FIG. 2 is a phylogenetic tree of E.coli (Enterobacter cancerogenus) SL 12.

FIG. 3 is a phylogenetic tree diagram of Alcaligenes (ALCALIGENES FAECALIS) SL 7.

FIG. 4 is a phylogenetic tree of Serratia (SERRATIA MARCESCENS) SL 11.

FIG. 5 is a graph of the growth of Achromobacter (Achromobacter insuavis) SL8 in a cadmium removal experiment.

FIG. 6 is a graph of growth of E.coli (Enterobacter cancerogenus) SL12 in a cadmium removal experiment.

FIG. 7 is a graph of the growth of Alcaligenes (ALCALIGENES FAECALIS) SL7 in a cadmium removal experiment.

FIG. 8 is a graph of growth of Serratia (SERRATIA MARCESCENS) SL11 in a cadmium removal experiment.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The invention discloses a method for preparing the enterobacteria (Enterobacter cancerogenus) SL 12: CCTCC NO: M2022288; deposit number of Serratia (SERRATIA MARCESCENS) SL 11: CCTCC NO: M2022287; deposit number of alcaligenes (ALCALIGENES FAECALIS) SL 7: CCTCC NO: M2022285; achromobacter (Achromobacter insuavis) accession number of SL 8: CCTCC NO: M2022286. The 4 strains of microorganisms are preserved in China Center for Type Culture Collection (CCTCC) with the preservation address of university of Wuhan, china, no. 3 and 18.

Example 1 isolation and identification of Alcaligenes SL7 and Serratia SL 11.

(1) Sample collection: the cadmium-polluted soil sample containing microorganisms is collected from 5-20cm surface soil of a cadmium-polluted farmland in Liuyang city of Hunan province.

(2) Enrichment of microorganisms: 10g of soil sample is weighed, placed in a triangular flask containing sterilized 90mL of liquid LB medium under aseptic operation, and placed in a shaking table at 200rpm/min for 12 hours at 28 ℃.

(3) Separation of cadmium resistant bacteria: sucking 1mL of the soil sample in the step (2) into 9mL of sterile normal saline, sequentially diluting to 10 ^-3,10^-4,10^-5 according to a stepwise dilution method, respectively taking 0.1mL of the above diluted concentration soil sample to be coated on a screening culture medium containing 50mg/L CdCl ₂, coating 3 repeated plates on each diluted concentration, and culturing at a constant temperature of 28 ℃ until a required candidate colony with cadmium resistance grows. The liquid LB culture medium comprises the following preparation components: yeast extract 5g/L, tryptone 10g/L, sodium chloride 10g/L, naOH to adjust pH to 7.0. Sterilizing in a 121 ℃ high pressure steam sterilizing pot for 30 minutes. The solid LB culture medium is prepared as the same as the liquid LB culture medium, and 2% of agar is needed to be added into the solid LB culture medium.

(4) And (3) scribing, separating and purifying: and (3) carrying out streaking separation on different colonies obtained in the step (3) according to a streaking plate method to obtain a purified single colony.

(5) Screening of cadmium resistant bacteria: and (3) inoculating the single colony obtained in the step (4) to LB solid medium with cadmium concentration of 50mg/L for preliminary screening, setting 3 repeats for each group, and placing the flat plate in a 28 ℃ incubator for inverted culture for 3 days. The colonies growing on the plates are sequentially streaked and inoculated on LB solid medium plate culture media with the concentration of cadmium (Cd ²⁺) of 50mg/L, 100mg/L, 200mg/L, 300mg/L, 400mg/L and 500mg/L for re-screening, and the colonies are reversely cultured for 3 days in a 30 ℃ incubator, and the operations are repeated for 3 times, so that the growth condition of the colonies is observed. Solid plates with colony growth were stored in a refrigerator at 4 ℃.

(6) Screening of cadmium removal bacteria: and (3) monoclonal inoculating the strain obtained in the step (5) into a liquid LB culture medium containing 100mg/L cadmium (Cd ²⁺), and measuring the content of Cd ²⁺ remained in the solution of single strain and different combination strains. A strain or combination of strains with high efficiency cadmium removal is determined.

Classifying and identifying cadmium removing bacteria: 1. identification of 16S ribosomal RNA: the sequence of the hypervariable region of the 16S rRNA gene is selected for bacterial community sequencing, the sequencing primer is 27F (5-AGAGTTTGATCCTGGGCTCAG-3) -1492R (5-TACCTTGTTACGACTT-3), and the sequencing region is a V3-V4 region. PCR amplification was then performed, primer adaptors were pooled and the PCR products were purified, quantified and homogenized using agarose electrophoresis and sequencing libraries were synthesized. After the 16S ribosomal RNA of the alkali bacillus SL7 and the Serratia SL11 are amplified, the obtained sequence amplified product is sequenced, and the gene sequence of the 16S rRNA of the leucobacter (Achromobacter insuavis) SL8 is shown as SEQ ID NO. 1; the gene sequence of 16S rRNA of enterobacter (Enterobacter cancerogenus) SL12 is shown as SEQ ID NO. 2, the gene sequence of 16S rRNA of Alcaligenes (ALCALIGENES FAECALIS) SL7 is shown as SEQ ID NO. 3, the gene sequence of 16S rRNA of Serratia (SERRATIA MARCESCENS) SL11 is shown as SEQ ID NO. 4, and the sequencing result is BLAST analysis in GenBank nucleic acid sequence library. Phylogenetic tree construction was performed on Achromobacter (Achromobacter insuavis) SL8, enterobacter (Enterobacter cancerogenus) SL12 Alcaligenes (ALCALIGENES FAECALIS) SL7 and Serratia (SERRATIA MARCESCENS) SL11 strains with Mega.7, and classification was determined. The phylogenetic tree (FIG. 2) was constructed, and as a result, it was found that Achromobacter (Achromobacter insuavis) SL8 and Enterobacter (Enterobacter cancerogenus) SL12 were clustered together with the strains of Achromobacter and Enterobacter, respectively, and that the nucleotide sequence homology was 99.79% and 98.97%, respectively; the alcaligenes (ALCALIGENES FAECALIS) SL7 and Serratia (SERRATIA MARCESCENS) SL11 strains can be clustered together with the alcaligenes and Serratia strains respectively, and the nucleotide sequence homology is 99.72% and 99.73% respectively. Thus, the two strains were identified as Alcaligenes ALCALIGENES FAECALIS SL, serratia SERRATIA MARCESCENS SL, respectively. 2. The cadmium-removed strain was morphologically observed using an electron scanning microscope.

Example 2 cadmium removal curve of strain.

(1) Purified Achromobacter (Achromobacter insuavis) SL8, enterobacter (Enterobacter cancerogenus) SL12, alcaligenes (ALCALIGENES FAECALIS) SL7 and Serratia (SERRATIA MARCESCENS) SL11 strains were inoculated into 100mL of LB liquid medium and cultured with shaking in a shaker at 200rpm/min at 30 ℃. After shaking culture until the OD ₆₀₀ of the bacterial liquid was 0.8, 300uL (bacterial liquid ratio of each strain was 3 times as designed by referring to 16 groups of experiments in Table 1) was taken and cultured in 30mL of 100mg/L liquid LB medium containing Cd ²⁺ at 200rpm/min in a shaking table at 30 ℃. Samples were taken on days 0,1,3, and 7 of the experiment, 1mL each time in a centrifuge tube, centrifuged at 12000rpm for 10min, and 1mL of supernatant was taken. The filtrate was filtered through a 45um filter and the concentration of Cd ²⁺ in the filtrate was determined by atomic spectroscopy (AAS).

(2) Description of experimental results: when the initial concentration of cadmium added was 100mg/L, the cadmium concentration in the experimental system solutions containing SL7, SL8, SL11 and SL12 after 7 days was reduced to 32.23mg/L, 30.45mg/L, 37.98mg/L and 29.19 mg/L, respectively, in the systems to which SL7, SL8, SL11 and SL12 were added, respectively, and the cadmium removal rates were 67.77%, 69.55%, 62.02% and 70.81%, respectively. The cadmium removal rate reaches 67.77 percent. Meanwhile, SL11 and SL12 are added into a cadmium-containing experimental system of 100mg/L according to a bacterial liquid ratio of 1:1, and the cadmium removal rate reaches 78.03% after seven days. When SL11, SL12 and SL7 are added into a cadmium-containing experimental system of 100mg/L according to a bacterial liquid ratio of 1:1:1, the cadmium removal rate reaches 81.03% after seven days. When SL11, SL12, SL7 and SL8 are added into a cadmium-containing experimental system of 100mg/L according to the bacterial liquid ratio of 1:1:1, the cadmium removal rate reaches 82.44 percent after seven days. Thus, the co-use of the composite strain has a synergistic effect on the removal of cadmium compared to the use of a single strain.

Table 1 compounding Table and results thereof

Example 3 growth curve of strain in cadmium solution.

(1) Purified Achromobacter (Achromobacter insuavis) SL8, enterobacter (Enterobacter cancerogenus) SL12, alcaligenes (ALCALIGENES FAECALIS) SL7 and Serratia (SERRATIA MARCESCENS) SL11 strains were inoculated into 100mL of LB liquid medium and cultured with shaking in a shaker at 200rpm/min at 30 ℃. After shaking culture until the OD ₆₀₀ value of the bacterial liquid is 0.8, 300uL is taken in 30mL of 0mg/L, 50mg/L, 100mg/L or Cd ²⁺ -containing liquid LB culture medium, and shaking culture is carried out in a shaking table at 200rpm/min and at 30 ℃. Samples were taken at 0,6, 24, 30, 54, and 70 hours of the experiment, observed with an optical microscope, counted with a hemocytometer, and the change in bacterial concentration during the experiment was recorded, and growth curves of Achromobacter alcaligenes (Achromobacter insuavis) SL8, enterobacter (Enterobacter cancerogenus) SL12, alcaligenes (ALCALIGENES FAECALIS) SL7, and Serratia (SERRATIA MARCESCENS) SL11 in cadmium solution were plotted.

(2) Experimental results: experiments show that the addition of 100mg/L Cd ²⁺ has an effect on the growth of Achromobacter (Achromobacter insuavis) SL8, enterobacter (Enterobacter cancerogenus) SL12, alcaligenes (ALCALIGENES FAECALIS) SL7 and Serratia (SERRATIA MARCESCENS) SL11, and at this concentration, the bacterial growth is inhibited, but the concentration can reach more than 10 ¹¹ cells/mL. Thus, the cadmium environment of appropriate concentration has little effect on the survival of four bacteria.

Example 4 control effect of composite microbial inoculum on plant diseases.

Setting up 16 treatment, namely selecting peppers with consistent 6-7 weeks and adopting a compound combination mode of numbers 1-16 in table 1 to treat pepper soil (setting up 3 repeats, each repeat of 30 pepper strains), wherein the application amount is 50 mu L/each pepper strain; treating with phytophthora liquid (3×10 ⁸ cfu/mL) and gray mold pathogenic bacteria liquid (3×10 ⁸ cfu/mL) after 3 days, observing the disease condition of the capsicum after 20 days, and counting as disease when the capsicum trunk, leaf, root or fruit is diseased; otherwise, it is normal. The control effect was calculated according to the following formula: control effect= ((control incidence-treatment incidence)/control incidence) x 100%; the results are shown in Table 2;

Table 2 table of control effects of the composite microbial inoculum on plant diseases

Experimental results: the treatment of group number 1-10 has 93.02-97.67% of control effect on pepper epidemic disease; the control effect on gray mold is 92.77-98.80%; compared with the control group 16, the disease rate of pepper epidemic disease and gray mold is obviously reduced, and the control effect is good.

Sequence listing

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ttcctccaga tctctacgca tttcaccgct acacctggaa ttctaccccc ctctacgaga 840

ctctagcttg ccagtttcaa atgcagttcc caggttgagc ccggggattt cacatctgac 900

ttaacaaacc gcctgcgtgc gctttacgcc cagtaattcc gattaacgct tgcaccctcc 960

gtattaccgc ggctgctggc acggagttag ccggtgcttc ttctgcgagt aacgtcaatt 1020

gatgaacgta ttaagctcac caccttcctc ctcgctgaaa gtgctttaca acccgaaggc 1080

cttcttcaca cacgcggcat ggctgcatca ggcttgcgcc cattgtgcaa tattccccac 1140

tgctgcctcc cgtaggagtc tggaccgtgt ctcagttcca gtgtggctgg tcatcctctc 1200

agaccagcta gggatcgtcg cctaggtgag ccattacccc acctactagc taatcccatc 1260

tgggcacatc tgatggcaag aggcccgaag gtccccctct ttggtcttgc gacgttatgc 1320

ggtattagct accgtttcca gtagttatcc ccctccatca ggcagtttcc cagacattac 1380

tcacccgtcc gccgctcgtc acccagggag caagctcccc tgtgctaccg ctcgacttgc 1440

atgtgttaag cctgccgcca gcgttcatct 1470

Claims

1. A composite microbial agent, characterized in that the composite microbial agent comprises enterobacter (Enterobactercancerogenus) SL12 and serratia (SERRATIA MARCESCENS) SL11; accession number of the enterobacteria (Enterobactercancerogenus) SL 12: CCTCC NO: M2022288; deposit number of Serratia (Serratia marcescens) SL 11: CCTCCNO: M2022287.

2. The composite microbial inoculant of claim 1, further comprising alcaligenes (ALCALIGENES FAECALIS) SL7; deposit number of alcaligenes (ALCALIGENES FAECALIS) SL 7: CCTCCNO: M2022285.

3. The composite microbial inoculant of claim 2, further comprising achromobacter (Achromobacterinsuavis) SL8, wherein the achromobacter (Achromobacterinsuavis) SL8 has a accession number of: CCTCC NO: M2022286.

4. The composite microbial agent according to claim 3, wherein the bacterial liquid ratio of enterobacteria (Enterobactercancerogenus) SL12, serratia (SERRATIA MARCESCENS) SL11, alcaligenes (ALCALIGENESFAECALIS) SL7 and achromobacteria (Achromobacterinsuavis) SL8 is: 1:1:1:1.

5. Use of the composite microbial inoculant of any one of claims 1-4 in the preparation of a cadmium-passivated microbial inoculant.

6. Use of a compound microbial inoculant according to any one of claims 1-4 for the preparation of a medicament for controlling phytophthora capsici and grey mould of capsicum.