CN110564644B

CN110564644B - Serratia atropurpurea with growth promoting effect and application thereof

Info

Publication number: CN110564644B
Application number: CN201910877843.2A
Authority: CN
Inventors: 杜秉海; 丁延芹; 汪城墙; 王文团; 刘凯; 姚良同
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2019-09-17
Filing date: 2019-09-17
Publication date: 2021-04-02
Anticipated expiration: 2039-09-17
Also published as: CN110564644A

Abstract

The disclosure belongs to the technical field of microbial growth promotion, and particularly relates to serratia furiosus with a growth promoting effect and application thereof. The problem of soil salinization seriously affects the crop yield and the sustainable development of agricultural economy. Aiming at the development problem of saline-alkali soil, some rhizosphere growth-promoting bacteria can improve the salt resistance and alkali resistance of crops, promote the growth condition of the crops, improve the stress resistance of the crops and gradually realize the improvement effect of the soil. The invention provides Serratia rubra (Serratia rubidaea)3004J-3, which is verified to keep good growth in a saline-alkali environment, has the capability of promoting the growth of plants in the saline-alkali environment, further explores the mechanism of the Serratia rubra rubidaea, has the functions of producing protease and siderophores and decomposing organic phosphorus in the environment, and has important significance in improving saline-alkali soil and improving the stress resistance of plants.

Description

Serratia atropurpurea with growth promoting effect and application thereof

Technical Field

The disclosure belongs to the technical field of microbial growth promotion, and particularly relates to Serratia rubra (Serratia rubidaea) with growth promotion effects of producing protease, siderophores, solubilizing phosphorus and the like and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Saline-alkali soil is soil with high salt content and concentration and is not beneficial to plant growth, is distributed in the global range, and along with the sharp increase of population and unreasonable reclamation, the original structure of the soil epidermis is destroyed, so that land desertification and salinization are aggravated. In Huang-Huai-Hai region, the salinization of land seriously affects the local agricultural production. The crop yield is low and unstable, the crop quality is poor, mainly because the local soil salinization is serious, the second national soil general investigation in 1983 finds that the saline-alkali soil area in the Huang-Huai-Hai plain is as high as 63048.8hm²And accounts for 48.58% of the land area available in the county. The salinized soil seriously influences the crop yield and limits the sustainable development of rural economic industry in China.

With the increase of population, the usable cultivated land area is reduced, and the development and utilization of saline-alkali soil are highly emphasized in the state at present. Researches show that the method for treating the saline-alkali soil by combining rhizosphere growth-promoting bacteria (PGPR) with saline-alkali tolerant plants has the advantages of quick response, sustainability, low cost, no pollution and great potential. By using the microbial agent, the adverse effects of the pH value, the salt content and the like of the soil on the plants can be relieved, the root development is promoted, and the plants can thrive. Some researches show that the rhizosphere growth-promoting bacteria can effectively improve the salt resistance and alkali resistance of plants and promote the growth of the plants, and under the stress of salt, alkali and other adverse environments, PGPR can also show certain characteristics of reducing salt and alkali, and the promotion of the growth of the plants under the stress of salt and alkali is mainly shown in that seeds are grownThe germination rate and survival rate are improved, the biomass is increased, the root system is developed, and the stress resistance is enhanced to ensure that Na in soil⁺And Cl^-The concentration decreases and the concentration of other nutrient ions increases.

The protease is a general term for a class of enzymes for hydrolyzing protein peptide chains, can catalyze the hydrolysis of proteins and polypeptides, and is widely present in animal viscera, plant stems and leaves, fruits and microorganisms. The mechanism by which plant growth-promoting rhizobacteria PGPR stimulates plant growth involves the availability of nutrients derived from genetic processes such as biological nitrogen fixation and phosphate degradation or the alleviation of plant growth inhibition by modulating ACC deaminase expression, as well as the production of plant hormones and siderophores, among others. The growth promoting mechanism is generally realized by increasing the dissolution of biological nitrogen fixation and phosphorus, dissolving potassium, synthesizing plant hormone, generating siderophores, biologically preventing and treating and the like.

Disclosure of Invention

Based on the research background, the inventor considers that the expansion of the application of the microorganisms in the aspect of improving the saline-alkali tolerant environment survival of crops has important significance for the sustainable development of agriculture. The inventor obtains a Serratia rubra (Serratia rubidaea) strain through screening, and the Serratia rubra strain can improve the growth condition of wheat in a saline-alkali environment and enhance the adaptability of the wheat to the saline-alkali environment.

Through research, the strain has the function of producing protease, the protease activity of the fermentation liquor can reach 80.728 mu g/mL, the produced protease can degrade proteins in soil to increase the content of amino acids in the soil, and the amino acid fertilizer has the function of promoting the growth of plants. In addition to producing protease, the growth promoting mechanisms of the strains of the present disclosure include the decomposition of organic phosphorus, the production of auxins, siderophores, and the like.

In order to achieve the technical effects, the present disclosure provides the following technical solutions:

in a first aspect of the disclosure, there is provided a Serratia furiosus (Serratia rubidaea)3004J-3, which has been deposited in the china general microbiological culture collection center on 8/6 th 2019, abbreviated as CGMCC, and the address is: the biological preservation number of the Xilu No.1 Hospital No. 3 of the Chaojing Chaoyang district is: CGMCC No. 18361.

The serratia furiosus of the first aspect has the 16S rDNA sequence shown in SEQ ID NO. 1.

The strain sequence provided by the disclosure has 99-100% similarity with other known homologous Serratia rubra 16S rDNA sequences, is identified as Serratia rubra according to physiological and biochemical characteristics, and is named as Serratia rubra (Serratia rubidaea) 3004J-3. The morphological characteristics of the strain are as follows:

the characteristics of the thallus are as follows: the shape of the thallus is rod-shaped, gram negative and has no spore;

colony characteristics: after 24h of culture on LB plate medium (yeast powder, 0.5%, peptone, 1%, sodium chloride, 1%, distilled water, agar, 1.6%, distilled water, sterilized at 121 ℃ for 20min, pH7.0), the colonies were round, smooth in surface, moist, flat, pale yellow, and regular in edge.

The physiological and biochemical characteristics of the strain are as follows: positive catalase, negative catalase in V-P experiment, positive D-glucose, positive L-arabinose, positive D-mannitol, positive amylase and positive citrate utilization.

Preferably, the strain is cultured by adopting an LB culture medium; and the LB culture medium is sterilized at the temperature of 115-116 ℃ for 15-25 min, and the pH value is 6.8-7.2.

Preferably, the culture temperature of the strain is 35-39 ℃.

The rhizosphere growth-promoting bacteria which are disclosed at present comprise more than 20 species such as pseudomonas, bacillus, agrobacterium, flavobacterium, serratia and the like, wherein the serratia is an important biocontrol bacterium and has good development significance as a biocontrol microbial inoculum. At present, related researches on serratia rubra as a biocontrol microbial inoculum are few, moreover, the serratia rubra provided by the disclosure has a multi-aspect growth promoting effect, in the prior art, the serratia rubra which has the functions of producing protease, siderophores, decomposing organic phosphorus, promoting the growth of plants in saline-alkali environment and the like is rarely reported, and the serratia rubra has a relatively comprehensive growth promoting effect and has important significance for improving the stress-resistant growth of crops and improving saline-alkali soil.

In a second aspect of the present disclosure, there is provided a microbial inoculum of the Serratia furiosa (Serratia rubidaea)3004J-3 of the first aspect and/or a culture of the strain of the first aspect.

Preferably, the culture of the bacterium is prepared as follows: inoculating the activated strain into a culture medium for culturing.

Further preferably, the activation is performed as follows: inoculating the strain into a liquid culture medium, and culturing at constant temperature for a period of time.

In some specific embodiments, the volume of the liquid medium is 4-6 mL.

In some specific embodiments, the constant temperature is 35-39 ℃, or the culture time is 12-14 h.

Preferably, the activated strain is inoculated in a culture medium and cultured for 22-26 h at 35-39 ℃.

In a third aspect of the disclosure, the application of the strain of the first aspect and the microbial inoculum of the second aspect in crop growth promotion is provided.

Preferably, the application comprises the application in producing protease, the application in producing siderophore, the application in dissolving phosphorus, the application in promoting the survival of crops in saline-alkali soil, or the effect in improving the agronomic characters of crops.

Preferably, the crop is wheat.

Compared with the prior art, the beneficial effect of this disclosure is:

1. the Serratia rubra (Serratia rubidaea)3004J-3 provided by the disclosure has multiple growth promoting functions, can be subjected to root irrigation treatment after wheat seedling setting, can obviously improve the growth condition of wheat seedlings, and has a good effect of improving the yield of crops.

2. The strain has high growth speed, exceeds fungi, can tolerate high-concentration saline-alkali environment, can still keep the growth condition in the environment of pH 12-10% NaCl, is used for improving saline-alkali soil and relieving the saline-alkali soil of farmlands, and has good application prospect.

3. Serratia is a common biocontrol bacterium, but few studies are currently conducted on Serratia rubra as a biocontrol bacterium. In particular, Serratia rubra (Serratia rubidaea)3004J-3 provided by the disclosure has various growth promoting effects of producing protease, siderophores, decomposing phosphorus, improving stress resistance growth of crops and the like, and research results of the disclosure enrich strain resources of plant growth promoting functions.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.

FIG. 1 is a microscopic photograph of Serratia furiosus 3004J-3 in example 1;

FIG. 2 is a photograph showing colonies of Serratia furiosus 3004J-3 in example 1;

FIG. 3 is a photograph showing the proteolytic ability of Serratia furiosperma 3004J-3 in example 1;

FIG. 4 is a photograph showing the organophosphorus decomposing ability of Serratia furiosaensis 3004J-3 in example 1;

FIG. 5 is a photograph showing the siderophore productivity of Serratia furiosperma 3004J-3 in example 1;

FIG. 6 is a comparative view of root scanning of the experiment of potting Serratia furiosaefolia 3004J-3 in example 1;

wherein, FIG. 6A is a control group of potted wheat; FIG. 6B shows experimental group potted wheat.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As introduced in the background art, soil salinization seriously affects the yield of crops, and the existing research shows that rhizosphere growth-promoting bacteria can relieve the adverse effect of the saline-alkali soil on the plants and promote the development of root systems. The disclosure provides Serratia furiosus 3004J-3, which is saline-alkali resistant, has multiple growth promoting functions and has good promoting effect when applied to wheat growth.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.

Example 1

As shown in FIGS. 1 and 2, after the strain is cultured on LB plate medium (yeast powder, 0.5%, peptone, 1%, sodium chloride, 1%, distilled water, agar, 1.6%, distilled water, sterilized at 121 ℃ for 20min, pH7.0) for 24h, the bacterial colony is round, smooth in surface, moist, flat, light yellow and regular in edge; the shape of the thallus is rod-shaped, gram negative and has no spore.

In addition, the physiological and biochemical characteristics of the strain are as follows through the detection of physiological and biochemical experiments: positive catalase, negative catalase in V-P experiment, positive D-glucose, positive L-arabinose, positive D-mannitol, positive amylase and positive citrate utilization.

In this embodiment, a fermentation solution of a bacterium is provided, wherein Serratia furiosus (Serratia rubidaea)3004J-3 stored on a slant is transferred to a test tube containing 5ml of a liquid medium, and cultured in a constant temperature shaking table for 12-14h at a temperature: 37 ℃, rotation speed: 180 ℃ and 200 rpm; inoculating the activated bacterial liquid into 100-200mL LB culture medium according to the inoculation amount of 1%, and placing the culture medium into a shaking table for 24h at the temperature: 37 ℃, rotation speed: 180 ℃ and 200rpm to prepare the fermentation liquid.

LB culture medium: yeast extract, 0.5%, peptone, 1%, sodium chloride, 1%, distilled water, agar, 1.6%, distilled water, sterilizing at 121 deg.C for 20min, and pH 7.0.

In the embodiment, the growth promotion effect of the strain on wheat is verified by pot experiments by taking wheat planted in saline-alkali soil as a test sample. The saline-alkali soil is saline-alkali soil from saline-alkali soil in junkerer county in coast. The pH value of the soil is more than 8.6, and the soil is alkaline. The control group (CK) is wheat after seedling setting in saline-alkali soil, no fertilizer is applied, and 1ml of LB liquid culture medium is diluted to 20ml with water for root irrigation treatment. The experimental group adopts the fermentation liquor to dilute 1ml of fermentation liquor to 20ml after wheat seedling setting for root irrigation treatment, and the dosage during root irrigation is as follows: in potting experiments 1ml of fermentation broth (20ml of dilution) was used per strain.

The results of 20d and 40d after root irrigation treatment by detecting the plant height of potted wheat, the biomass of the overground part, the biomass of the root part, the root development and the chlorophyll content are shown in tables 1-3.

TABLE 1 growth promoting effect of 20d bacteria application

TABLE 2 growth promoting effect of 40d bacteria application

TABLE 3 detection results of chlorophyll content in wheat leaves

The results show that after the strain fermentation liquor is applied, the agronomic characters of wheat are obviously improved compared with those of a control group, and the strain is proved to have a practical growth promoting effect. As shown in fig. 6, the wheat roots of the experimental group were more vigorous than those of the control group.

In order to further clarify the growth promoting mechanism of the strain, the protease producing capability, the phosphate solubilizing capability and the siderophore producing capability of the strain are examined, the results are shown in tables 4-6, and the protease detection results in table 4

TABLE 5 results of examination of phosphorus-solubilizing ability

TABLE 6 detection results of siderophore productivity of strains

Respectively inoculating the bacterial colonies into a plate culture medium, dipping a small amount of bacterial sludge with a sterilized toothpick, inoculating the bacterial sludge onto a skimmed milk powder plate culture medium, and culturing in a 37 ℃ constant-temperature incubator for 72 h. The results are shown in fig. 3-5, the Serratia furiosus (Serratia rubidaea)3004J-3 has the effect of producing protease, the protease activity can reach 80.728 mu g/mL through detection, and the strain also has the functions of producing siderophore and decomposing organophosphorus in the environment.

The embodiment further verifies the salt and alkali resistance of the strain, and the results are shown in tables 7-9:

TABLE 7 detection results of salt tolerance of strains

Note: "+ + + +" indicates vigorous growth; "+ +" indicates better growth; "+" indicates growth; "+" indicates micro-growth;

"-" indicates no growth

TABLE 8 detection results of alkali resistance of strains

"-" indicates no growth

TABLE 9 saline-alkali tolerance of the strains

"-" indicates no growth

As can be seen from the results in tables 7-9, the strain can still grow in the soil under the conditions of 7-10% NaCl and pH 9-12.

The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.

SEQUENCE LISTING

<110> Shandong university of agriculture

<120> serratia rubra with growth promoting effect and application thereof

<130> 2010

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1422

<212> DNA

<213> Serratia rubra (Serratia rubidaea) 3004J-316S rDNA

<400> 1

taccatgcaa gtcgagcggc agcgggagga agcttgcttc ctcgccggcg agcggcggac 60

gggtgagtaa tgtctgggga tctgcccgat ggagggggat aaccactgga aacggtggct 120

aataccgcat aacgtcgcaa gaccaaagtg ggggaccttc gggcctcaca ccatcggatg 180

aacccagatg ggattagcta gtaggtgggg taatggctca cctaggcgac gatccctagc 240

tggtctgaga ggatgaccag ccacactgga actgagacac ggtccagact cctacgggag 300

gcagcagtgg ggaatattgc acaatgggcg caagcctgat gcagccatgc cgcgtgtgtg 360

aagaaggcct tcgggttgta aagcactttc agcgaggagg aaggtggtga gcttaatacg 420

ctcatcaatt gacgttactc gcagaagaag caccggctaa ctccgtgcca gcagccgcgg 480

taatacggag ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcac gcaggcggtt 540

tgttaagtca gatgtgaaat ccccgagctt aacttgggaa ctgcatttga aactggcaag 600

ctagagtctc gtagaggggg gtagaattcc aggtgtagcg gtgaaatgcg tagagatctg 660

gaggaatacc ggtggcgaag gcggccccct ggacgaagac tgacgctcag gtgcgaaagc 720

gtggggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat gtcgatttgg 780

aggttgtgcc cttgaggcgt ggcttccgga gctaacgcgt taaatcgacc gcctggggag 840

tacggccgca aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc ggtggagcat 900

gtggtttaat tcgatgcaac gcgaagaacc ttacctactc ttgacatcca gagaactttc 960

cagagatgga ttggtgcctt cgggaactct gagacaggtg ctgcatggct gtcgtcagct 1020

cgtgttgtga aatgttgggt taagtcccgc aacgagcgca acccttatcc tttgttgcca 1080

gcggttcggc cgggaactca aaggagactg ccagtgataa actggaggaa ggtggggatg 1140

acgtcaagtc atcatggccc ttacgagtag ggctacacac gtgctacaat ggcgtataca 1200

aagagaagcg aacctgcgag gcaagcggac ctcataaagt acgtcgtagt ccggattgga 1260

gtctgcaact cgactccatg aagtcggaat cgctagtaat cgtagatcag aatgctacgg 1320

tgaatacgtt cccgggcctt gtacacaccg cccgtcacac catgggagtg ggttgcaaaa 1380

gaagtaggta gcttaacctt cgggagggcg ctaccacttg ga 1422

Claims

1. Serratia profunda (A)Serratia rubidaea)3004J-3, wherein said strain has been deposited in China general microbiological culture Collection center (CGMCC) at 8.6.2019, with the addresses: the biological preservation number of the Xilu No.1 Hospital No. 3 of the Chaojing Chaoyang district is: CGMCC M18361.

2. A bacterial agent characterized in that it is the serratia furiosus (R) as claimed in claim 1Serratia rubidaea)3004J-3 and/or a culture of said strain.

3. The microbial inoculum of claim 2, wherein the culture of the strain is prepared by: inoculating the activated strain into a culture medium for culturing.

4. The microbial inoculum of claim 3, wherein the activation is performed by: inoculating the strain into a liquid culture medium for constant-temperature culture for a period of time; or the culture time is 12-14 h.

5. The microbial inoculum according to claim 4, wherein the constant temperature is 35-39 ℃.

6. The microbial inoculum according to claim 3, wherein the activated strain is inoculated in a culture medium and cultured for 22-26 h at 35-39 ℃.

7. Serratia atropurpurea (R) of claim 1Serratia rubidaea)3004J-3 and any one of the microbial inoculum of claims 2-6 for promoting wheat growth in saline alkali soil.