CN110616169A

CN110616169A - Stratospheric bacillus with growth promoting effect and application thereof

Info

Publication number: CN110616169A
Application number: CN201910876943.3A
Authority: CN
Inventors: 杜秉海; 丁延芹; 汪城墙; 姚良同; 刘凯; 王文团
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2019-09-17
Filing date: 2019-09-17
Publication date: 2019-12-27
Anticipated expiration: 2039-09-17
Also published as: CN110616169B

Abstract

The disclosure belongs to the technical field of plant rhizosphere growth-promoting bacteria, and particularly relates to a stratospheric bacillus with growth-promoting effect and application thereof. The salinization of soil has serious influence on the continuous development of farmland and the yield of crops, and the covering of the salinization soil by the farmland is a commonly used treatment means at present. Wherein, the growth-promoting rhizobacteria can effectively improve the growth condition and saline-alkali tolerance viability of crops. Aiming at the defects that the growth promoting effect of growth promoting bacteria in the prior art is single and the like, the Bacillus stratosphericus 35W2 is obtained by screening a large number of strains, has the effects of producing protease and siderophores and decomposing organic phosphorus in the environment, also has good saline-alkali resistance viability, can improve the survival rate and the agronomic characters of crops in saline-alkali soil, and has important significance when applied to the treatment of farmland salinization.

Description

Stratospheric bacillus with growth promoting effect and application thereof

Technical Field

The disclosure belongs to the technical field of plant rhizosphere growth-promoting bacteria, and particularly relates to a stratospheric bacillus capable of producing protease and siderophores, dissolving phosphorus and improving saline-alkali tolerance of plants 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.

Salinization of soil means that the salt content in the soil is too high (more than 0.3%), so that crops are low in yield or cannot grow. Because groundwater mineralization degree is higher, along with the change of weather, groundwater comes the shallow surface layer through the capillary action, along with the evaporation of moisture, salt constantly to the soil surface layer gathering form salt soil. With the dramatic increase of population and unreasonable reclamation, the original structure of the soil epidermis is destroyed, resulting in the increase of land desertification and salinization. The total area of the Chinese saline soil is 14.87 hundred million acres, wherein the modern saline soil is 5.54 hundred million acres, and the potential saline soil is 2.60 hundred million acres; the cultivated land damaged by salinization in China reaches 1.4 hundred million mu, and is mainly distributed in Xinjiang, the corridor in the river and western, the chai da mu basin, the river jacket plain, the Yinchuan plain, the Huang-Huai-Hai plain, the west part of the northeast plain and the coastal area. Wherein the salinized farmland area of the inland region in the northwest accounts for 15 percent of the total farmland area.

Aiming at the problem of salinization of a farmland, the existing research shows that plant growth-promoting rhizobacteria (PGPR) can build a resistance or tolerance mechanism by inducing plants, so that the survival capability of the plants under the condition of salt stress is enhanced, the salinization degree of the soil is reduced by covering the farmland, and the yield of the salinization farmland is improved. The rhizosphere growth-promoting bacteria (PGPR) refer to a group of beneficial microorganisms which are parasitic on plant roots or rhizosphere soil, directly or indirectly participate in plant growth, improve crop quality or resist adversity stress, at present, 27 species of PGPR separated in China exist, wherein, Pseudomonas (Pseudomonas), Bacillus (Bacillus), Enterobacter (Enterobacter) and Burkholderia (Burkholderia) are common rhizosphere growth-promoting bacteria populations, and the strains of the genera have unique physiological and ecological functions and strong environmental adaptability, can be planted in plant rhizosphere, promote nutrient utilization and improve the capability of plants to resist biological and abiotic stress. However, whether the PGPR can enhance the saline-alkali resistance of the plant is influenced by various factors such as strain specificity, soil environment, host plants and the like, the PGPR exists in various types of soil, but the type and community structure of the PGPR are related to various factors such as soil physicochemical property, strain specificity, saline-alkali resistance of the plant and the like.

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 alleviation of plant growth inhibition by modulating ACC deaminase expression, and the production of plant hormones and iron carriers, 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. Bacillus stratosphericus (Bacillus stratosphericus) is a common growth-promoting bacterium, and Bacillus stratosphericus T-246 with the effect of eliminating zearalenone is reported from Chan; the ligustrum lucidum ait reports a stratospheric bacillus PR1 isolated from the rhizosphere soil of tobacco, and the stratospheric bacillus has the effects of producing protease and dissolving phosphorus. Aiming at the current research situation, the inventor finds that the effect of the rhizosphere growth-promoting bacteria disclosed at present is single, and multiple growth-promoting bacteria are required to be used in a combined manner in order to improve the yield of crops.

Disclosure of Invention

In view of the research background, the inventor believes that further research on rhizosphere growth-promoting bacteria and screening of growth-promoting bacteria with multiple growth-promoting functions can effectively reduce the planting cost, improve the yield of crops, improve the salinization degree of soil and maintain the sustainable development of agricultural economy. The invention obtains a Bacillus stratosphericus (Bacillus stratosphericus)35W2 with growth promoting effect through continuous screening and research, the strain has the effects of producing protease and siderophores and decomposing organic phosphorus in soil environment, and the strain also has good saline-alkali resistance viability and improves the survival rate and the agronomic characters of crops in saline-alkali soil.

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

in a first aspect of the disclosure, a strain of Bacillus stratosphericus (Bacillus stratosphericus)35W2 is provided, which has been deposited in the common microorganism center of the china committee for culture collection and management of microorganisms, abbreviated as CGMCC, at 2019 on 8/6 th day, and has the address: north west road No.1 institute of north jing, chaoyang district, with biological preservation number: CGMCC No. 18362.

The Bacillus stratosphericus (Bacillus stratosphericus)35W2 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 the 16S rDNA sequence of other known homoeothermic Bacillus, is identified as serratia rubra according to physiological and biochemical characteristics, and is named as homoeothermic Bacillus (Bacillus stratosphericus)35W 2. 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-positive and has spores;

colony characteristics: after 24h of culture on an LB plate culture medium (yeast powder, 0.5 percent, peptone, 1 percent, sodium chloride, 1 percent, distilled water, agar, 1.6 percent, distilled water, sterilization at 121 ℃ for 20min, pH7.0), the colony is round, smooth in surface, free of bulges, moist, glossy, milky white and jagged in edges.

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

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 ℃.

In a second aspect of the present disclosure, there is provided a bacterial agent which is a culture of the Bacillus stratosphericus (Bacillus stratosphericus)35W2 of the first aspect and/or 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 the aspect of producing protease, the application in the aspect of producing siderophores, the application in the aspect of dissolving phosphorus, the application in the aspect of promoting the crops to survive in saline-alkali soil, or the effect in the aspect of improving the agronomic traits of the crops.

Preferably, the crop is wheat.

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

1. aiming at the problem of salinization of farmland soil in the prior art, the invention provides a growth-promoting strain with good salinization resistance, the strain can still survive in a high-concentration salinization environment (pH 12-10% NaCl), the planting rate of crops in the salinization soil can be obviously improved, the strain is suitable for the treatment of moderate salinization soil, and the strain has important significance for improving the salinization of the farmland.

2. The Bacillus stratosphericus provided in the prior art generally only has single growth promoting effects of producing protease or dissolving phosphorus, potassium and the like. The bacillus stratosphericus provided by the disclosure has the effect of promoting saline-alkali tolerant growth of plants, and also shows the characteristics of producing protease and siderophores and decomposing organophosphorus components in the environment in a plate test. The strain has multiple growth promoting effects, can realize the promotion effect on the growth of crops and the improvement of the saline-alkali tolerant survival capability of plants by single use, and has important significance for improving the yield of the crops in the saline-alkali soil and reducing the planting cost.

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 without limiting the disclosure in any way.

FIG. 1 is a microscopic photograph of Bacillus stratosphericus 35W2 in example 1;

FIG. 2 is a photograph showing a colony of Bacillus stratosphericus 35W2 in example 1;

FIG. 3 is a photograph showing the protein-solubilizing ability of Bacillus stratosphericus strain 35W2 in example 1;

FIG. 4 is a photograph showing the organophosphorus decomposing ability of Bacillus stratosphericus 35W2 in example 1;

FIG. 5 is a comparative example of root scanning of a pot culture experiment using Bacillus stratosphericus 35W2 in example 1;

wherein, FIG. 5A shows control group potted wheat; FIG. 5B 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/or "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the features, steps, operations, devices, components, and/or combinations thereof.

As introduced in the background art, the rhizosphere growth-promoting bacteria disclosed in the prior art have single functions, and the present disclosure provides a strain of Bacillus stratosphericus (35W 2) which has excellent saline-alkali tolerance and crop growth-promoting effects.

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 broth of a strain is further provided, the Bacillus stratosphericus (Bacillus stratosphericus)35W2 preserved on the inclined plane is transferred into a test tube filled with 5ml of liquid culture medium, and the test tube is cultured in a constant temperature shaking table for 12 to 14 hours at the 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 culturing for 24h in a shaking bed 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 taken from saline-alkali soil of juneberry saline-alkali soil in coastal areas. 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 the fermentation liquor to 20ml after wheat seedling setting, and then root irrigation treatment is carried out, wherein the dosage during root irrigation is as follows: in potting experiments 1ml of fermentation broth (20ml of diluted solution) 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 performance of wheat is obviously improved compared with that of a control group, and the strain is proved to have a practical growth promoting effect. As shown in fig. 5, the wheat roots of the experimental group were more vigorous than the control group.

In order to further clarify the growth promoting mechanism of the strain, the present disclosure examines the protease producing ability, phosphate solubilizing ability and siderophore producing ability of the strain, and the results are shown in tables 4-6,

TABLE 4 protease detection results

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 FIGS. 3-4, the Bacillus stratosphericus (Bacillus stratosphericus)35W2 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

TABLE 9 saline-alkali tolerance of the strains

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> a stratospheric bacillus with growth promoting effect and application thereof

<130> 2010

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1430

<212> DNA

<213> Bacillus stratosphericus (35W 216S rDNA)

<400> 1

ctataatgca agtcgagcgg acagaaggga gcttgctccc ggatgttagc ggcggacggg 60

tgagtaacac gtgggtaacc tgcctgtaag actgggataa ctccgggaaa ccggagctaa 120

taccggatag ttccttgaac cgcatggttc aaggatgaaa gacggtttcg gctgtcactt 180

acagatggac ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca aggcgacgat 240

gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct 300

acgggaggca gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc 360

gtgagtgatg aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtgcaaga 420

gtaactgctt gcaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca 480

gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agggctcgca 540

ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt cattggaaac 600

tgggaaactt gagtgcagaa gaggagagtg gaattccacg tgtagcggtg aaatgcgtag 660

agatgtggag gaacaccagt ggcgaaggcg actctctggt ctgtaactga cgctgaggag 720

cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt aaacgatgag 780

tgctaagtgt tagggggttt ccgcccctta gtgctgcagc taacgcatta agcactccgc 840

ctggggagta cggtcgcaag actgaaactc aaaggaattg acgggggccc gcacaagcgg 900

tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatcctct 960

gacaacccta gagatagggc tttcccttcg gggacagagt gacaggtggt gcatggttgt 1020

cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt 1080

agttgccagc attcagttgg gcactctaag gtgactgccg gtgacaaacc ggaggaaggt 1140

ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga 1200

cagaacaaag ggctgcgaga ccgcaaggtt tagccaatcc cacaaatctg ttctcagttc 1260

ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg ctagtaatcg cggatcagca 1320

tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg 1380

caacacccga agtcggtgag gtaaccttta tggagccagc cgccgaaggt 1430

Claims

1. A strain of Bacillus stratosphericus (Bacillus stratosphericus)35W2 is characterized in that the strain is preserved in China general microbiological culture Collection center (CGMCC) in 8-6.2019, and the address is as follows: the biological preservation number of the Xilu No.1 Hospital No. 3 of the Chaojing Chaoyang district is: CGMCC M18361.

2. The Bacillus stratosphericus (35W 2) according to claim 1, wherein the bacterial characteristics are: the shape of the thallus is rod-shaped, gram negative and has no spore;

colony characteristics: after 24 hours of culture on an LB plate culture medium, the colony is round, smooth in surface, moist, flat, light yellow and regular in edge.

3. The Bacillus stratosphericus (Bacillus stratosphericus)35W2 of claim 1, wherein the strain has the physio-biochemical characteristics of: positive catalase, negative catalase in V-P experiment, positive D-glucose, positive L-arabinose, positive D-mannitol, positive amylase and positive citrate utilization.

4. The Bacillus stratosphericus (35W 2) according to claim 1, wherein the strain is cultured in LB medium; sterilizing the LB culture medium at 115-116 ℃ for 15-25 min, wherein the pH is 6.8-7.2; or the culture temperature of the strain is 35-39 ℃.

5. A bacterial agent, characterized in that it is Bacillus stratosphericus (Bacillus stratosphericus)35W2 and/or a culture of said strain as claimed in any of claims 1 to 4.

6. The microbial inoculum of claim 5, which is prepared by a method comprising: inoculating the activated strain into a culture medium for culturing.

7. The microbial inoculum of claim 6, wherein the activation is performed by: inoculating the strain into a liquid culture medium for constant-temperature culture for a period of time; preferably, the constant temperature is 35-39 ℃, or the culture time is 12-14 h.

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

9. Use of the Bacillus stratosphericus (Bacillus stratosphericus)35W2 of any one of claims 1 to 4 and the microbial inoculum of any one of claims 5 to 8 for growth promotion of crops.

10. The use according to claim 9 for promoting growth of crops, wherein the use comprises the use for producing protease, or for producing siderophores, or for solubilizing phosphorus, or for promoting survival of crops in saline and alkaline soils, or for enhancing agronomic performance of crops.