CN107099477B

CN107099477B - Endophytic curvularia lunata with salt tolerance and application thereof

Info

Publication number: CN107099477B
Application number: CN201710416367.5A
Authority: CN
Inventors: 秦盛; 王田田; 冯维维; 邢珂; 王玉; 丁鹏; 蒋继宏
Original assignee: Jiangsu Normal University
Current assignee: Jiangsu Normal University
Priority date: 2017-06-05
Filing date: 2017-06-05
Publication date: 2021-02-02
Anticipated expiration: 2037-06-05
Also published as: CN107099477A

Abstract

The invention discloses a plant endogenetic curvatus Bacillus (Bacillus flexus) PE4941 with salt tolerance, which is preserved in Guangdong collection of microorganisms in 2017 for 5 months and 10 days, wherein the preservation number is GDMCC 60176; and its use for promoting plant growth under salt stress. The separated and screened endophytic bacteria Bacillus flexus PE4941 of the limonium has various growth promoting characteristics of nitrogen fixation, siderophore generation and the like, can tolerate 8 percent of NaCl, is inoculated back to the limonium seedlings by root irrigation, has the effects of remarkably slowing down the salt damage symptom and increasing the biomass under the salt stress so as to promote the growth of the limonium seedlings, and is beneficial to improving the salt tolerance of plants and reducing the use of chemical fertilizers.

Description

Endophytic curvularia lunata with salt tolerance and application thereof

Technical Field

The invention belongs to the field of microbial fertilizer and crop stress protection research, and relates to a plant endophytic curvularia bacillus and application thereof.

Background

The soil salinization is one of the main abiotic stress factors threatening the global agricultural production and crop yield, nearly 50% of the irrigated land in the world is threatened by salt stress of different degrees at present, and China has about 9.9 multiplied by 10⁷hm²The land has the problem of salt damage and the threatened area is positiveIncreasing year by year. How to improve and utilize large-area salinized soil and protect and improve the salt stress resistance of important economic crops is an important scientific and technological problem to be solved urgently in the international and life science fields. At present, the main biological improvement method measures are to cultivate and plant saline-alkali tolerant crops, such as beet, corn, soybean, salt-tolerant plant soda ash, tamarix chinensis and the like, and research on salt-tolerant transgenic plants, but the methods are usually long in period and high in cost, and the plant transgenic method is not widely accepted by the society. The endophytic bacteria refer to a new microbial resource which exists in healthy plant tissues and does not cause plant diseases. The microorganisms are colonized in plants and are not easily influenced by external environment, researches find that a plurality of endophytic bacteria have the functions of fixing nitrogen, dissolving phosphorus, generating plant growth hormone and siderophores, generating antibiotics and inducing plant resistance, and the action potential of the endophytic bacteria in the aspects of promoting plant growth and protecting adverse environmental stress is concerned by more and more researchers.

The halophyte limonium is a medicinal herb plant in the blue and snow family, belongs to a pioneer plant which is known as saline-alkali land improvement, simultaneously contains a plurality of effective components such as flavone and polysaccharide, has a plurality of efficacies of clearing heat and detoxicating, resisting aging, resisting cancer and the like, is one of the traditional Chinese medicines in China, and has great clinical medicinal value. However, the number of wild Limonium sinense oliver is reduced rapidly at present, and needs to be protected urgently. At present, the beneficial bacteria have been reported to promote the growth of wild Limonium sinense and improve the stress resistance under the high salt stress. The microorganism has the advantages of small volume, quick growth, easy control and the like, so that beneficial microorganism strain resources are discovered from the blood-supplementing grass growing in saline-alkali soil, and the method has important significance for promoting the growth and stress protection of wild medicinal herbaceous plant blood-supplementing grass.

Disclosure of Invention

The invention aims to solve the technical problems and provide a plant endogenous campylobacter with salt tolerance.

Another object of the present invention is to provide a plant growth promoting agent produced by the endophytic bacterium.

Still another object of the present invention is to provide the use of the endophytic bacterium for promoting plant growth under salt stress.

The purpose of the invention can be realized by the following technical scheme:

the invention provides a strain of endophytic Bacillus flexus (Bacillus flexus) PE4941 with salt tolerance, which is preserved in Guangdong collection of microorganisms in 2017 in 5 months and 10 days, and the preservation number is GDMCC 60176.

The salt-tolerant endophytic Bacillus flexus PE4941 is characterized in that the 16S rRNA gene sequence of the strain is shown in SEQ ID NO. 1.

A plant salt-resistant growth promoter containing the PE4941 strain with the accession number of GDMCC 60176.

The application of the plant endophytic curvularia lunata with salt tolerance in promoting plant growth under salt stress is disclosed, wherein the plant is Limonium sinense.

The plant salt-tolerant growth promoter contains the PE4941 strain with the accession number of GDMCC 60176. Is produced by the following method:

1) inoculating Bacillus curvatus PE4941 with a collection number of GDMCC 60176 in 250ml of LB liquid medium (10g of tryptone, 5g of yeast powder, 30g of NaCl, pH 7.2, 1000ml of water);

2) shaking the flask at 37 deg.C and 150rpm for 48h, centrifuging at 8000rpm when the thallus grows to logarithmic phase, and collecting thallus;

3) washing the thallus with sterile water twice, adjusting OD value of thallus to 0.5-0.6 with sterile water, and thallus concentration to 10⁷-10⁸cfu/mL is the salt-tolerant growth promoter.

The plant salt-tolerant growth promoter containing the PE4941 strain is applied to promoting plant growth under salt stress.

The plant salt-tolerant growth promoter containing the PE4941 strain is applied to promoting the growth of the limonium sinense kuntze under the salt stress.

The method for promoting the growth of the plants under the condition of salt stress by using the plant salt-tolerant growth promoter containing the PE4941 strain is characterized in that seedling root irrigation treatment is carried out by using bacterial liquid (50ml) containing PE4941 thalli with the OD value of 0.5-0.6, the root irrigation is carried out once per week, and the root irrigation is carried out continuously for four times.

Detailed description of the invention:

the invention obtains a strain with stronger salt-tolerant growth-promoting activity by separating endophytic bacteria of medicinal herbaceous plants, namely the limonium sinense, and screening salt-tolerant growth-promoting characteristics, and the strain is numbered as PE 4941. The taxonomic status of the gene is determined by morphological characteristics, physiological and biochemical characteristics, 16S rRNA gene sequencing and phylogenetic analysis. The potted plant experiment verifies that the endophytic bacterium Bacillus flexus PE4941 provided by the invention is a method capable of promoting the growth of Limonium sinense and obviously improving the adaptability under the salt stress, and can be used for bacterial manure and improving the salt tolerance of plants through fermentation.

The isolated strain is Bacillus flexus with code number PE4941, and is deposited in Guangdong province microorganism culture Collection with the deposit number GDMCC 60176 in 2017, 5 and 10 months.

Meanwhile, the invention provides a 16S rRNA gene sequence (1400bp) of the Bacillus curvatus, which is shown as SEQ ID NO. 1.

In addition, the invention provides a preparation method of a bacterial suspension of Bacillus curvatus PE4941, which comprises the following specific steps: selecting a single colony of Bacillus flexus PE4941 which grows on an LB solid plate for 24-48h, inoculating the single colony into 250ml of LB liquid culture medium (10g of tryptone, 5g of yeast powder, 30g of NaCl, pH 7.2 and 1000ml of water), carrying out shake-flask culture at 37 ℃ and 150rpm for 48h, centrifuging at 8000rpm when the thallus grows to a logarithmic growth phase, collecting the thallus, washing the thallus with sterile water twice, adjusting the OD value of the thallus to be 0.5-0.6 by using the sterile water, and adjusting the thallus concentration to be 10⁷-10⁸cfu/mL。

Meanwhile, the method for fertilizing the bacterial suspension is provided, and comprises the following steps: mixing nutrient soil and vermiculite according to a volume ratio of 4:1 to obtain a potting matrix, sterilizing at 121 ℃ for one hour, cooling, and subpackaging into potting flowerpots (9cm in length, 9cm in width and 9cm in height) with the weight of about 200g per pot. Selecting full and consistent-size blood-enriching grass seeds, sterilizing the seeds on the surface (75% ethanol for 15 seconds and 0.1% HgCl for 6-7 minutes), putting the seeds into sterilized soil for seedling culture, selecting consistently-growing seedlings for pot experiment after the seeds germinate and grow two true leaves, performing one seedling in each pot, growing the seedlings in a greenhouse (25 ℃, 12h illumination/12 h dark treatment), performing seedling root irrigation treatment by using bacterial liquid (50ml) with an OD value of 0.5-0.6, performing root irrigation once a week, performing salt stress treatment by using 200mM NaCl solution after continuously irrigating the roots for 4 times. Through pot experiments, the bacillus flexus PE4941 under salt stress has obvious promotion effect on the growth of the seedling of the limonium sinense, and the plant height and the fresh weight are respectively improved by 1.4 and 1.93 times.

Has the advantages that:

compared with the prior art, the separated and screened endophytic bacteria Bacillus flexus PE4941 of the limonium has various growth promoting characteristics of nitrogen fixation, siderophore generation and the like, can tolerate 8 percent of NaCl, has the effects of remarkably alleviating the salt damage symptom and increasing the biomass under the salt stress by the root irrigation and the reinjection of the limonium seedlings, thereby promoting the growth of the limonium seedlings, and is beneficial to improving the salt tolerance of plants and reducing the use of chemical fertilizers.

Drawings

FIG. 1 photograph of colonies of strain PE4941 cultured on LB plate for 48 hours

FIG. 2 phylogenetic tree of strain PE4941 constructed based on 16S rRNA gene

FIG. 3 comparison of growth status of inoculated and non-inoculated Limonium sinense seedlings under high salt stress conditions

FIG. 4 comparison of fresh weights of inoculated and non-inoculated Limonium sinense seedlings under high-salt and salt-free stress conditions

FIG. 5 comparison of the height of inoculated and non-inoculated Limonium sinense seedlings under high salt and salt-free stress conditions

Biological material preservation information

The salt-tolerant endophytic campylobacter flexus PE4941 is classified and named as campylobacter flexus (Bacillus flexus), is preserved in Guangdong microbial strain preservation center (GDMCC), is located at Guangdong microbial strain preservation center No. 59 of Zhonglu No. 100 Mc. of Guangzhou city, Guangdong province, has the preservation number of GDMCC 60176, and has the preservation date of 2017, 5 months and 10 days.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Example 1:

1. endophyte isolation

The plant sample is collected from saline-alkali soil of Jiangsu coastal beach, and the strain separation is completed within 48 hours after the plant collection is brought back to a laboratory. Washing the sample with tap water, removing surface mud and attachments, cutting the plant sample into 2cm × 2cm, soaking the sample in 5% sodium hypochlorite in sterile ultraclean bench for 3-5min, adding sterile 2.5% sodium thiosulfate for 10min, washing the surface-sterilized plant with sterile water for 5 times, adding 75% alcohol, soaking for 3min, washing the sample with sterile water for 3-5 times, grinding with sterile mortar, adding a certain amount of sterile water, and diluting to 10 deg.C^-1-10^-4Gradient of concentration, take 10^-2-10^-4The sample diluent with the concentration is coated on an LB separation culture medium (10g of tryptone, 5g of yeast powder, 30g of NaCl, pH 7.2, 18g of agar and 1000ml of water), cultured for 2-7d at 37 ℃, and grown colonies are purified in time and preserved at 4 ℃ by using an LB solid inclined plane.

2. Identification of strains

Morphology and physiological-biochemical characteristics of strain PE 4941: the bacterial strain grows well on various culture media such as tryptone-yeast extract culture medium (LB), Nutrient Agar (NA), yeast extract-malt extract (ISP 2) and the like, and bacterial colonies are round, wet, about 2-3mm in diameter, near white and opaque after the bacterial colonies grow on an LB solid plate for 48 hours (figure 1). Can tolerate NaCl with the highest concentration of 8 percent, and can reduce nitrate into nitrite to generate arginine hydrolase, urease and protease.

Using NA liquid culture medium to perform shaking culture at 37 ℃ and 150rpm for 48h, then centrifuging at 8000rpm to collect thalli, extracting genome DNA of the thalli by a microwave method, and performing microwave amplification by using bacterial universal primers 27f 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492 r: 5'-AAGGAGGTGATCCAGCCGCA-3' PCR amplification of the 16S rRNA gene was performed. The PCR reaction system is as follows: template DNA 2. mu.L, 10 XBuffer 5. mu.L, MgCl₂mu.L (25mmol), 1. mu.L dNTP (10mmol/L), 1. mu.L 27f (10. mu. mol/L), 1. mu.L 1492r (10. mu. mol/L), 0.5. mu.L Taq enzyme (5 u/. mu.L), and the PCR product was detected by 0.8% agarose gel electrophoresis and sent to Producer (Shanghai) Co., Ltd for sequencing. Sequencing results the sequencing results were aligned on NCBI and EzBio Cloud websites, followed by sequence alignment and analysis with Mega 6.0 software, and finally by the neighbor joining method (Neigh)bor-Joining, NJ) and carrying out phylogenetic analysis, finding that the strain has the highest similarity with the effective seed sequence of the Bacillus and has the highest similarity with Bacillus flexus NBRC15715^TThe similarity of (c) was 100% (fig. 2). Thus, strain PE4941 should belong to a member of the genus Bacillus, designated Bacillus flexus PE4941(Bacillus flexus PE 4941).

3. Growth promoting performance of strain

And (3) nitrogen fixation potential detection: a small amount of the cells were picked up and inoculated onto a nitrogen-free medium (sucrose 10.0g, CaCO)₃ 1.0g，K₂HPO₄ 0.5g，MgSO₄·7H₂O 0.2g，NaCl 0.2g，agar 15g，H₂O1000 mL, pH 7.2), placing in a constant temperature incubator at 28 ℃ for about 7 days, observing whether the strain grows, and if the strain can grow after being transferred for 5 times, indicating that the strain has nitrogen fixation potential activity. The strain PE4941 can still grow well on a nitrogen-free culture medium after 5 passages, which indicates that the PE4941 has the potential of fixing nitrogen elements in the air.

Detecting ammonia production activity: selecting a small amount of strains to be detected, inoculating the strains to 5mL of peptone liquid (10g/L) culture medium, repeating the steps for three times, adding 0.5mL of Nessler's reagent into each test tube after shaking culture for 3 days at 28 ℃ and 120r/min, wherein if yellow brown precipitates appear, the strains have ammonia production activity, and if the yellow brown precipitates do not appear, the strains do not produce ammonia. The coloration reaction is positive after the strain PE4941 is cultured, which shows that PE4941 has the capability of producing ammonia.

Detecting the activity of the siderophore: selecting a small amount of thallus, inoculating the thallus onto a CAS detection culture medium by a spot planting method, culturing in a constant temperature incubator at 28 ℃ for about 7 days, and observing whether yellow halos appear around colonies or not, wherein if yellow halos appear, the strains have the activity of an iron-producing carrier. Strain PE4941 developed a yellow halo after growth on CAS plates, indicating its ability to produce siderophores.

4. Strain application test

The preparation method of the bacterial suspension comprises the following steps: selecting a single colony of the campylobacter bacillus PE4941 which grows on an LB solid plate for 24-48h, inoculating the single colony into 250ml of LB liquid culture medium, carrying out shake-flask culture at 37 ℃ and 150rpm for 48h, centrifugally collecting the thallus at 8000rpm when the thallus grows to a logarithmic phase, washing the thallus twice by using sterile water, and adjusting the OD value of the thallus to be 0.5-0.6 to serve as a biological agent.

Potting fertilization experiment: mixing nutrient soil and vermiculite according to a volume ratio of 4:1 to obtain a potting matrix, sterilizing at 121 ℃ for one hour, cooling, and subpackaging into potting flowerpots (9cm in length, 9cm in width and 9cm in height) with the weight of about 200g per pot. Selecting full and consistent-size blood-enriching grass seeds, sterilizing the surfaces of the seeds (75% ethanol for 15 seconds, 0.1% HgCl for 6-7 minutes), putting the seeds into sterilized soil for seedling culture, selecting seedlings with consistent growth for pot experiment after the seeds germinate and grow two true leaves, carrying out pot culture test on the seedlings, wherein each pot contains one seedling, the seedlings grow in a greenhouse (25 ℃, 12h illumination/12 h dark treatment), carrying out root irrigation treatment by using bacterial liquid (50ml) with OD value of 0.5-0.6, using isometric sterile water as a control, carrying out treatment once a week, carrying out salt stress treatment by using 200mM NaCl solution after one month, continuing root irrigation on the control, and randomly selecting plants for photographing, measuring and detecting 30 days after the salt stress. As shown in FIG. 3, the seedlings of Limonium sinense inoculated with Bacillus flexus PE4941 under salt stress were better grown from the first row to the right, the third row from left to right was Limonium sinense under no salt stress, the second row was the plant treated with the strain PE4941 under 200mM NaCl stress, and the third row was the seedlings of the plant treated with the strain PE4941 under 200mM NaCl stress. As shown in Table 1 and FIG. 4, the fresh weight of the seedlings of Limonium sinense with the addition of Bacillus curvatus PE4941 was increased by 1.93 times compared to the control without inoculation of endophytes after the high salt stress treatment. As shown in FIG. 5, the height of the Limonium wrightii seedling inoculated with the Bacillus flexus PE4941 after the high-salt stress treatment is increased by 1.4 times compared with the control, and simultaneously, the root length and the leaf number are both increased remarkably.

TABLE 1 Effect of inoculation of endophyte PE4941 on growth of Limonium sinense seedlings

Example 2, a plant salt-tolerant growth promoter, prepared by the following method:

selecting a single colony of the Bacillus flexus PE4941 which grows on an LB solid plate for 24-48h, inoculating the single colony into 250ml of LB liquid culture medium, carrying out shake-flask culture at 37 ℃ and 150rpm for 48h, centrifugally collecting the thallus at 8000rpm when the thallus grows to a logarithmic growth phase, washing the thallus twice by using sterile water, and adjusting the OD value of the thallus to be 0.5-0.6 by using the sterile water to obtain the plant salt-tolerant growth promoter.

<110> university of Jiangsu profession

<120> endophytic curvulus with salt tolerance and application thereof

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1400

<212> DNA

<213> Bacillus flexus PE4941

<400> 1

cgagcgaact gattagaagc ttgcttctat gacgttagcg gcggacgggt gagtaacacg 60

tgggcaacct gcctgtaaga ctgggataac tccgggaaac cggagctaat accggataac 120

attttctctt gcataagaga aaattgaaag atggtttcgg ctatcactta cagatgggcc 180

cgcggtgcat tagctagttg gtgaggtaac ggctcaccaa ggcaacgatg catagccgac 240

ctgagagggt gatcggccac actgggactg agacacggcc cagactccta cgggaggcag 300

cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg tgagtgatga 360

aggctttcgg gtcgtaaaac tctgttgtta gggaagaaca agtacaagag taactgcttg 420

taccttgacg gtacctaacc agaaagccac ggctaactac gtgccagcag ccgcggtaat 480

acgtaggtgg caagcgttat ccggaattat tgggcgtaaa gcgcgcgcag gcggtttctt 540

aagtctgatg tgaaagccca cggctcaacc gtggagggtc attggaaact ggggaacttg 600

agtgcagaag agaaaagcgg aattccacgt gtagcggtga aatgcgtaga gatgtggagg 660

aacaccagtg gcgaaggcgg ctttttggtc tgtaactgac gctgaggcgc gaaagcgtgg 720

ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgagt gctaagtgtt 780

agagggtttc cgccctttag tgctgcagct aacgcattaa gcactccgcc tggggagtac 840

ggtcgcaaga ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 900

gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcctctg acaactctag 960

agatagagcg ttccccttcg ggggacagag tgacaggtgg tgcatggttg tcgtcagctc 1020

gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct tagttgccag 1080

catttagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg tggggatgac 1140

gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg atggtacaaa 1200

gggctgcaag accgcgaggt caagccaatc ccataaaacc attctcagtt cggattgtag 1260

gctgcaactc gcctacatga agctggaatc gctagtaatc gcggatcagc atgccgcggt 1320

gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt gtaacacccg 1380

aagtcggtgg ggtaaccttt 1400

Claims

1. The salt-tolerant endophytic curvularia Lemans strain is a plant endophytic curvularia Lemans strain with the preservation number of GDMCC 60176, and the 16S rRNA gene sequence of the strain is shown in SEQ ID NO. 1.

2. The salt-tolerant endophytic curvulus strain according to claim 1, wherein the PE4941 strain grows well on an LB solid plate, and after 48 hours of growth, the colony is round, wet, 2-3mm in diameter, nearly white and opaque.

3. A plant salt-tolerant growth promoter comprising the salt-tolerant Bacillus flexus endophytic bacterium according to claim 1.

4. The use of the salt tolerant Campylobacter endophytic strain of claim 1 for promoting plant growth under salt stress.

5. The use of Campylobacter endophytic bacteria with salt tolerance according to claim 4, wherein the plant is Limonium sinense.

6. The plant salt-tolerant growth promoter of Campylobacter endophytic bacteria having salt-tolerant ability according to claim 3, which is produced by the following method:

1) inoculating Campylobacter with the collection number GDMCC 60176 into 250ml of LB liquid medium;

7. The use of the plant salt tolerance growth promoter of the salt tolerance-containing plant endophytic curvulus according to claim 3 for promoting the growth of Limonium sinense (Girard) Kuntze under salt stress.

8. The method for promoting the growth of the limonium sinense kuntze under the salt stress by using the plant salt-tolerant growth promoter of the plant endophytic curvulus according to claim 3, wherein 50ml of a bacterial solution with the OD value of 0.5-0.6 of the thalli of the plant endophytic curvulus with the salt tolerance is used for carrying out the root irrigation treatment on the limonium sinkuntze seedlings, the root irrigation is carried out once per week, and the root irrigation is carried out continuously for four times.

9. The salt tolerant endophytic Bacillus flexus strain of claim 1, wherein the strain is capable of producing siderophores, nitrogen fixation.