CN108102958B

CN108102958B - Plant rhizosphere growth promoting Bacillus aryabhattai for heavy saline-alkali soil and application thereof

Info

Publication number: CN108102958B
Application number: CN201711396251.6A
Authority: CN
Inventors: 井大炜; 马海林; 刘方春; 刘幸红; 马丙尧; 杜振宇; 杨庆山; 周健; 彭琳
Original assignee: Shandong Academy of Forestry
Current assignee: Shandong Academy of Forestry
Priority date: 2017-12-21
Filing date: 2017-12-21
Publication date: 2020-05-22
Anticipated expiration: 2037-12-21
Also published as: CN108102958A

Abstract

The invention discloses a bacillus aryabhattai capable of promoting rhizosphere growth in heavy saline-alkali soil and application thereof. The strain can improve the saline-alkali tolerance of the tamarix chinensis in a heavy salt-alkali environment. The bacillus aryabhattai for promoting rhizosphere growth of the heavy saline-alkali soil is prepared into a biological microbial inoculum, the biological microbial inoculum is inoculated to the periphery of the root system of the tamarix chinensis, and after salt stress for a certain time, compared with different inoculated microbial inoculants, the biological microbial inoculum can promote the growth of the tamarix chinensis and improve the root system of the tamarix chinensis and the water content of plants. Therefore, the invention can be used as an inoculant to be applied to a heavy salt and alkali habitat, and provides excellent strain resources for improving the salt and alkali resistance of the tamarix chinensis.

Description

Plant rhizosphere growth promoting Bacillus aryabhattai for heavy saline-alkali soil and application thereof

Technical Field

The invention belongs to the technical field of agriculture and forestry microorganisms, and particularly relates to a plant rhizosphere growth promoting Bacillus aryabhattai strain for heavy saline-alkali soil and application thereof.

Background

The salinization of the land is one of important factors for restricting the development of agriculture and forestry. Salinization severely limits plant growth, thereby having a great impact on crop yield. Statistically, crop losses in irrigated areas due to salination worldwide are as much as $ 110 billion, and are increasing. At present, salinized soilThe soil occupies about 30 percent of the total land area, and the soil is about 10 hundred million and more hm all over the world²The land is in a salinized state. According to the statistical report of the 2 nd national census data of the ministry of agriculture organization, the total area of the salinized soil in China accounts for 4.88 percent of the available land area in China, wherein the salinized area in cultivated land reaches 920.9 ten thousand hm²And accounts for 6.62 percent of the cultivated land area in China. NaHCO 2₃With Na₂CO₃Is a major component of inland soda saline-alkali soil, and plants growing on such soil are susceptible to Na⁺Multiple stresses of high pH and low water potential cause the reduction of water permeability and air permeability of soil, the increase of osmotic pressure of soil solution, the reduction of effectiveness of soil nutrients, and the serious limitation of normal growth of plants, resulting in the continuous reduction of diversity of earth surface vegetation communities.

The microorganism plays an important role in improving the saline-alkali tolerance of plants. Yanqingshan et al applied for a Enterobacter cloacae TIA062(CN201610330288.8), which can effectively improve the salt tolerance of white wax under certain salt condition; douglas et al, by using a method combining temperature screening and surface-directed culture, isolated and screened bacillus halodurans culturable in soil in the northeast region, considered that bacillus curvatus and bacillus subtilis have good salt tolerance; the research of Wanhuijie et al shows that the tomato inoculated with the pseudomonas aurantiaca can resist acid and alkali and has higher antibacterial activity on germs.

Plant growth-promoting rhizobacteria (PGPR) are a group of beneficial bacteria that live in the rhizosphere and root surface of plants and promote Plant growth or antagonize pathogenic bacteria. A large number of experiments show that a plurality of PGPR strains have obvious effects of promoting plant growth, preventing and treating soil-borne diseases, improving the stress resistance of plants to abiotic stress, promoting soil bioremediation and the like. PGPR may promote plant growth directly or indirectly through one or several mechanisms of action, including the direct mechanisms of PGPR: produce phytohormone, dissolve scales, fix nitrogen, produce siderophins, secrete ACC deaminase and the like. However, most plants and plant rhizosphere growth-promoting bacteria do not have the capability of resisting high salt and high saline alkali, and particularly in the case of severe saline alkali, beneficial microorganisms in the rhizosphere environment of the plants are reduced, so that the bioactivity of the rhizosphere of the plants is reduced, and the salt tolerance is reduced. The method screens the plant rhizosphere growth-promoting bacteria suitable for severe saline-alkali environment, improves the saline-alkali resistance of the plants, plays a role in microorganism-plant combined restoration, and has great application potential in ecological restoration of severe saline-alkali soil.

Disclosure of Invention

Aiming at the problems in the prior art, the inventor screens plant rhizosphere soil of the heavy saline-alkali soil to obtain a heavy saline-alkali soil rhizosphere growth-promoting bacillus aryabhattai from the tamarisk rhizosphere soil, and can improve the saline-alkali tolerance of the tamarisk in a heavy saline-alkali environment.

Specifically, the invention relates to the following technical scheme:

one of the purposes of the invention is to provide a heavy saline-alkali soil rhizosphere growth-promoting bacillus aryabhattai (Bacillus aryabhattai) GPR018 which is preserved in China general microbiological culture Collection center (CGMCC) at 1 month and 7 days of 2016, and the preservation number is CGMCC No. 11973.

The invention also aims to provide a method for culturing the growth-promoting Bacillus aryabhattai (Bacillusaryabhattai) GPR018 in the heavy saline-alkali soil, in particular to a process for fermenting on a skimmed milk powder culture medium.

The invention further aims to provide a fermentation culture of the GPR018 of the heavy saline-alkali soil rhizosphere growth-promoting Bacillus aryabhattai (Bacillus aryabhattai), wherein the fermentation culture comprises GPR018 thalli of the Bacillus aryabhattai.

In a preferred embodiment, the number of B.aryabhattai GPR018 colonies in the fermentation culture is 10³-10⁸cfu/mL。

It is a further object of the invention to provide a use product comprising the GPR018 strain or a fermentation culture thereof, including in the form of a microbial inoculant or composition.

In a preferred embodiment, the product comprises a carrier, which may be a solid carrier or a liquid carrier; for example, the solid carrier may be selected from clay, talc, kaolin, montmorillonite, zeolite, silica, corn flour. Bean powder, polyvinyl alcohol or polyethylene glycol, trehalose, etc.; the liquid carrier can be vegetable oil, mineral oil, water, etc. In preferred embodiments, the product may be in powder form, solution form, granular form, and the like. For example, the powder form of the product may be a lyophilized powder of the strain or a fermentation product thereof.

The fourth purpose of the invention is to provide the bacterial strain or the fermentation culture thereof and the application of the product in promoting the growth of plants, increasing the water content of plant leaves or reducing the peroxidation damage of plant membranous substances in heavy salt alkali environment. Preferably, the plant is tamarix chinensis.

In a preferred embodiment, the above-mentioned strains or fermentation cultures thereof, as well as the products, are applied around the root system of the plant in the application.

The saline-alkali environment is mainly NaHCO₃With Na₂CO₃The main component is soda saline-alkali soil, and plants growing on the soil are easily subjected to Na⁺Multiple stresses of high pH and low water potential cause the reduction of water permeability and air permeability of soil, increase of osmotic pressure of soil solution, reduction of effectiveness of soil nutrients, and serious limitation of normal growth of plants.

The invention achieves the following beneficial effects:

the rhizosphere growth-promoting bacillus aryabhattai in the heavy saline-alkali soil and the application thereof can improve the saline-alkali resistance of the tamarix chinensis in the heavy saline-alkali environment. The bacillus aryabhattai for promoting rhizosphere growth of the heavy saline-alkali soil is prepared into a biological microbial inoculum, the biological microbial inoculum is inoculated to the periphery of the root system of the tamarix chinensis, and after salt stress for a certain time, compared with different inoculated microbial inoculants, the biological microbial inoculum can promote the growth of the tamarix chinensis and improve the water content of the root system, leaves and plants of the tamarix chinensis. Therefore, the invention can be used as an inoculant to be applied to a heavy salt and alkali habitat, and provides excellent strain resources for improving the salt and alkali resistance of the tamarix chinensis.

Drawings

FIG. 1 the morphology of GPR018 colonies of the strain of the invention;

FIG. 2 is a diagram of the field of view of the strain GPR018 according to the invention under the optical microscope;

FIG. 3 phylogenetic Tree of the inventive Strain GPR018

FIG. 4 example 3 measured OD600 values of different strains at different salt concentrations

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1 screening, separation and preservation of growth-promoting rhizobacteria in heavy saline-alkali soil

6 samples of the rhizosphere soil of Tamarix chinensis of yellow river Delta are collected. Bacterial isolates were isolated from rhizosphere soil samples by gradient dilution. Purifying the separated bacteria by a three-region scribing method, judging whether the strains are purified by microscopic examination, numbering the purified bacteria, picking out single colonies, and inoculating the single colonies onto a skimmed milk powder culture medium plate by using a sterilized toothpick. After culturing at 37 ℃ for 2 days, selecting bacterial colonies with transparent circles, measuring the diameter D of the bacterial colonies and the diameter D of the transparent circles, calculating D/D, selecting strains with large ratio, purifying, repeatedly verifying the effect of protease production, and selecting strains with stable functions for further re-screening.

The secondary screening adopts Folin phenol method. Adding 2mL of casein solution with mass fraction of 0.5% into 1mL of original fermentation broth, heating in water bath at pH 7.0 and 37 deg.C for 15min, inactivating with 10% trichloroacetic acid, collecting 1mL of centrifugal supernatant of the above reaction solution, adding 1mL of Folin phenol into 5mL of 0.55mol/L Na₂CO₃Under the alkaline condition of (2), measuring the absorbance value at 660nm in a water bath at 37 ℃ for 15 min. The reaction system with the inactivated enzyme solution is used as a blank. And (3) performing seedling raising and pot culture experiments on the strains obtained by re-screening to determine a bacterium with the best effect of promoting the growth of the tamarix chinensis, wherein the number of the bacterium is GPR 018.

Example 2 physiological and biochemical and molecular identification of multiple saline-alkali soil rhizosphere growth-promoting bacteria GPR018

Bacterial colony characteristics and thallus morphology: the colony is round and regular, large, has a convex surface, is wet, yellowish, opaque, and has a small thallus, a rod shape, spores, a colony morphology as shown in FIG. 1, and an optical microscope as shown in FIG. 2.

Physiological and biochemical characteristics: positive catalase test, positive citrate utilization test, negative starch hydrolysis test, negative V-P determination test, negative D-glucose test, negative D-mannitol test, negative trehalose test, negative D-sorbitol test, negative sucrose test and negative glucose gassing test.

Functional qualitative and quantitative determination: adding 2mL of casein solution with mass fraction of 0.5% into 1mL of original fermentation broth, performing water bath at pH 7.0 and 37 deg.C for 15min, inactivating with 10% trichloroacetic acid, collecting 1mL of centrifugal supernatant of the above reaction solution, adding 1mL of Folin phenol into 5mL of 0.55mol/L Na₂CO₃Under the alkaline condition of (2), measuring the absorbance value at 660nm in a water bath at 37 ℃ for 15 min. The reaction system with the inactivated enzyme solution is used as a blank. Under these reaction conditions: the amount of enzyme required to produce 1ug of tyrosine every 1min was defined as one enzyme activity (IU). The protease activity in the fermentation liquor is 47.22 mug/ml by determination.

GPR018 16S rDNA sequence

ACATTGGGGGCATGCCTATAATGCAGTCGAGCGAACTGATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACAAGAGTAACTGCTTGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTCAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGAGTAACCGTAAGGAGCTAGCCGCCTAAGGTGGACAGGATAATGG(SEQ ID NO:1)

Identification and analysis: phylogenetic tree as shown in fig. 3, it can be seen from the phylogenetic tree that the similarity between GPPR018 and Bacillus aryabhattai (Bacillus aryabhattai) on the same branch is 98.57%, and therefore GPR018 was preliminarily identified as Bacillus aryabhattai.

Biological preservation information: a heavy saline-alkali soil rhizosphere growth-promoting Bacillus aryabhattai is classified and named as Bacillus aryabhattai GPR018, is preserved in China general microbiological culture Collection center (CGMCC for short, address: Beijing city Shangyang district Beichen Xilu No.1 institute of microbiology 3, China academy of sciences, postal code 100101) in 2016, 1 month and 7 days, and has a preservation number of CGMCC No. 11973.

Example 3 salt tolerance of GPR018 and growth promoting Effect in heavy saline alkaline Environment

1. Salt tolerance

Selecting 4 kinds of Bacillus aryabhattai, namely Bacillus aryabhattai BPR078 (with the preservation number of (CGMCC No.11971), Bacillus aryabhattai BPR088 (with the preservation number of (CGMCC No.11972), Bacillus aryabhattai JIA2 (with the preservation number of (CGMCC No. 11967)) and Bacillus aryabhattai GPR018 (with the preservation number of (CGMCC No. 11973)), inoculating 4 strains of auxin strains into a liquid LB culture medium, culturing at 180rpm and 37 ℃ for 24h, then taking 200 microliter of activated bacterium liquid into the LB liquid culture medium with the sodium chloride concentrations of 1%, 3%, 6% and 9%, respectively, and then placing the liquid culture medium into 180rpm and 37 ℃ for 24h, using a spectrophotometer, taking samples periodically by turbidimetry with the corresponding culture medium without inoculation of bacteria as a blank control, and obtaining OD600 results of parallel 3 times of determination as shown in figure 4, wherein when the sodium chloride concentration is increased to 6%, the OD600 of GPR018 is obviously larger than that of other three Bacillus aryabhattai, which shows that the salt tolerance of the strain is obviously higher than that of other strains.

2. Application effect in heavy salt alkali environment

GPR018 preserved on the inclined plane was activated by three-zone streaking on LB (peptone 10 g; NaCl 10 g; yeast extract 5 g; agar 15-20 g; water 1000 ml; pH 7.0-7.2, split-charged, sterilized at 121 ℃ for 20min) plates. Single colonies were picked into the corresponding 10ml liquid medium and incubated at 37 ℃ for 24h at 180 rpm. Inoculating the seed liquid into corresponding 50ml liquid culture medium according to the inoculation amount of 1 percent for fermentation, and culturing for 2d at 37 ℃ and 180 rpm.

Planting the cultivated container seedlings of the tamarix chinensis in plastic pots, and carrying out stress treatment on the tamarix chinensis after culturing for 2 months. 2 alkaline salt NaHCO₃And Na₂CO₃Mixing according to a molar ratio of 9:1, setting a concentration of 400mmol/L, and carrying out stress treatment on the basis of pouring saline water. Inoculating GPR018 bacterial liquid while stressing, diluting 5ml of bacterial liquid by 10 times, and inoculating the diluted bacterial liquid to the periphery of the root of the tamarix chinensis. 10 strains were treated and managed normally, with no inoculum (same amount of medium poured) as a control. The stress treatment lasts for 40 days, and then the same part of mature leaves is taken for the determination of physiological indexes. Measuring the plant height by using a tape measure respectively, and calculating the growth amount of the plant height (the plant height at the end of alkali stress-the plant height at the beginning of alkali stress); the control plant height determination and calculation methods were consistent with stress treatment. The relative water content of the tamarix chinensis leaves is measured by adopting a drying method, and the Malondialdehyde (MDA) content of the tamarix chinensis leaves is measured by adopting a thiobarbituric acid colorimetric method. The results are shown in table 1, and it can be seen that after tamarix chinensis is stressed for 40 days, GPR018 inoculation can increase the relative water content of leaves by 12.29%, malondialdehyde content is reduced by 22.15%, plant height growth amount is increased by 27.0%, GPR018 under salt stress can increase the salt tolerance of tamarix chinensis and promote tamarix chinensis growth.

TABLE 1 growth promoting Effect of GPR018 in heavy salt alkaline Environment

SEQUENCE LISTING

<110> scientific research institute of forestry in Shandong province

<120> plant rhizosphere growth promoting Bacillus aryabhattai strain for heavy saline-alkali soil and application thereof

<130>

<160>1

<170>PatentIn version 3.5

<210>1

<211>1461

<212>DNA

<213>Bacillus aryabhattai

<400>1

acattggggg catgcctata atgcagtcga gcgaactgat tagaagcttg cttctatgac 60

gttagcggcg gacgggtgag taacacgtgg gcaacctgcc tgtaagactg ggataacttc 120

gggaaaccga agctaatacc ggataggatc ttctccttca tgggagatga ttgaaagatg 180

gtttcggcta tcacttacag atgggcccgc ggtgcattag ctagttggtg aggtaacggc 240

tcaccaaggc cacgatgcat agccgacctg agagggtgat cggccacact gggactgaga 300

cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg acgaaagtct 360

gacggagcaa cgccgcgtga gtgatgaagg ctttcgggtc gtaaaactct gttgttaggg 420

aagaacaagt acaagagtaa ctgcttgtac cttgacggta cctaaccaga aagccacggc 480

taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttatccg gaattattgg 540

gcgtaaagcg cgcgcaggcg gtttcttaag tctgatgtga aagcccacgg ctcaaccgtg 600

gagggtcatt ggaaactggg gaacttgagt gcagaagaga aaagcggaat tccacgtgta 660

gcggtgaaat gcgtagagat gtggaggaac accagtggcg aaggcggctt tttggtctgt 720

aactgacgct gaggcgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca 780

cgccgtaaac gatgagtgct aagtgttaga gggtttccgc cctttagtgc tgcagctaac 840

gcattaagca ctccgcctgg ggagtacggt cgcaagactg aaactcaaag gaattgacgg 900

gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960

ggtcttgaca tcctctgaca actctagaga tagagcgttc cccttcgggg gacagagtga 1020

caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080

agcgcaaccc ttgatcttag ttgccagcat ttagttgggc actctaaggt gactgccggt 1140

gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta 1200

cacacgtgct acaatggatg gtacaaaggg ctgcaagacc gcgaggtcaa gccaatccca 1260

taaaaccatt ctcagttcgg attgtaggct gcaactcgcc tacatgaagc tggaatcgct 1320

agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg 1380

tcacaccacg agagtttgta acacccgaag tcggtggagt aaccgtaagg agctagccgc 1440

ctaaggtgga caggataatg g 1461

Claims

1. A heavy saline-alkali soil rhizosphere growth-promoting Bacillus aryabhattai GPR018 has been preserved in the China general microbiological culture Collection center in 2016, 1 month and 7 days, and the preservation number is CGMCC No. 11973.

2. The method for culturing the heavy saline-alkali soil rhizosphere growth-promoting Bacillus aryabhattai (Bacillus aryabhattai) GPR018 comprises a fermentation process on a skimmed milk powder culture medium.

3. The heavy saline-alkali soil rhizosphere growth-promoting Bacillus aryabhattai (Bacillus aryabhattai) GPR018 fermentation culture as claimed in claim 1, wherein the fermentation culture comprises Bacillus aryabhattai GPR018 cells.

4. The fermentation culture of claim 3, wherein the number of GPR018 colonies of Bacillus aryabhattai in the fermentation culture is 10³-10⁸cfu/mL。

5. A use product comprising the strain of claim 1 or the fermentation culture of any one of claims 3-4, comprising a microbial agent or composition.

6. The product of claim 5, wherein the product comprises a carrier that is a solid carrier or a liquid carrier.

7. The product according to claim 6, wherein the solid carrier is selected from one or more of clay, talc, kaolin, montmorillonite, zeolite, silica, corn flour, bean flour, polyvinyl alcohol, polyethylene glycol, trehalose;

the liquid carrier is selected from one of vegetable oil, mineral oil and water.

8. The product according to claim 5, characterized in that it is in the form of a powder, a solution or a granulate.

9. The product of claim 8, wherein the product is in the form of a lyophilized powder of the strain or fermentation culture.

10. Use of the strain of claim 1 or the fermentation culture of any one of claims 3 to 4 or the product of any one of claims 5 to 9 for promoting plant growth, increasing plant leaf water content or reducing plant membranous peroxidation damage in heavy salt alkaline environments.

11. Use according to claim 10, characterized in that said plant is tamarix chinensis.

12. Use according to claim 10 or 11, wherein the strain or fermentation culture, product is applied around the root system of the plant.