CN111100813A - Salt-tolerant growth-promoting bacterium JP-JH for relieving salt damage of plants and application thereof - Google Patents
Salt-tolerant growth-promoting bacterium JP-JH for relieving salt damage of plants and application thereof Download PDFInfo
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Abstract
The invention discloses a salt-tolerant growth-promoting bacterium JP-JH for relieving plant salt damage and application thereof, wherein the strain belongs to Kushneria indalina, has been deposited in China center for type culture collection No. 12 in 2019 in 12 months, and has a deposition number of CCTCC M20191039. The strain has strong salt-tolerant and alkali-resistant characteristics, the growth-tolerant NaCl concentration is 1-15% (w/v), the tolerant pH value is 7.0-9.0, and IAA can be secreted without L-tryptophan induction. In addition, the strain can remarkably promote the growth (leaf length and overground fresh weight) of wheat in a living body state, an inactivated body state, a salt-free stress condition and a salt stress condition, and obviously relieves the salt damage of wheat in the salt stress condition. Therefore, compared with the reported salt-tolerant strains, the salt-tolerant growth-promoting bacterium JP-JH disclosed by the invention can reasonably reduce the requirements on the conditions of culture, storage, transportation, colonization and the like of the strains, and is beneficial to reducing the production cost and facilitating popularization and application.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a salt-tolerant growth-promoting bacterium for relieving salt damage of plants and application thereof.
Background
The saline-alkali soil is the main soil type of low-yield fields in China, and the resources of the saline-alkali soil are reasonably developed and utilized, so that the method has important significance for guaranteeing national food safety, promoting agricultural sustainable development, improving ecological environment and promoting regional economic and social coordination development. The biological improvement measures for the saline-alkali soil can overcome some limitations of physical, chemical and engineering improvement measures and meet the requirement of green development of the current saline-alkali soil. The relatively mature biological improvement measures are mainly expressed as plant-based measures, including planting salt-tolerant trees (such as narrow-leaved oleaster, populus diversifolia, and the like) and salt-tolerant pasture (such as alfalfa, and the like), improving the soil structure, improving the soil fertility, and inhibiting the salt accumulation on the soil surface; and breeding new salt-resistant plant varieties. Based on the measures of microorganisms, most of bacteria which are separated by researchers and can promote plant growth belong to Halomonas, Bacillus, Pseudomonas and other strains, and novel phylosphaera and endopyhytica of Kushneria. At present, aiming at the systematic classification identification of Kushneria indalina strains limited to gene level and the basic salt tolerance characteristics, no research indicates that the strains belonging to Kushneria indalina have the capability of promoting plant growth.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a salt-tolerant growth-promoting bacterium JP-JH for relieving plant salt damage, wherein the strain has strong salt-tolerant and alkali-tolerant characteristics, the NaCl concentration which can be tolerated by growth of the strain is 1-15% (w/v), the pH value which can be tolerated is 7.0-9.0, and the strain can secrete IAA.
The invention also aims to provide an application of a microbial agent or a biological fertilizer prepared by utilizing the salt-tolerant growth-promoting bacteria JP-JH in plant salt damage alleviation.
The invention also aims to provide an application of a microbial agent or a biological fertilizer prepared by utilizing the salt-tolerant growth-promoting bacteria JP-JH in plant growth promotion.
The invention is realized by the following technical scheme:
the invention provides a salt-tolerant growth-promoting bacterium Kushneria indalina JP-JH separated from suaeda glauca rhizosphere soil in saline-alkali land of Haxing county of Cangzhou city, Hebei province, wherein the strain is preserved and the preservation unit is as follows: china center for type culture Collection, collection address: china, wuhan university, date of preservation: 12 month 12 in 2019, accession number: CCTCC M20191039.
The bacterial colony of the salt-tolerant growth-promoting bacterium JP-JH is orange and protruded, and the edge of the bacterial colony is slightly transparent.
The 16S rDNA sequence of the salt-tolerant growth-promoting bacterium JP-JH is shown in SEQ ID NO.1 and belongs to Kushneria indalina strains.
The salt-tolerant growth-promoting bacterium JP-JH has the growth-tolerant NaCl concentration of 1-15% (w/v) and the tolerant pH value of 7.0-9.0.
The salt-tolerant growth-promoting bacterium JP-JH has the capability of secreting IAA, and can secrete IAA without L-tryptophan induction.
The salt-tolerant growth-promoting bacterium JP-JH obviously relieves the salt damage of wheat under the condition of salt stress, and mainly shows that the growth vigor of the wheat is better, the color of leaves is greener, and the length of the wheat leaves and the fresh weight on the ground are increased.
The salt-tolerant growth-promoting bacterium JP-JH can remarkably promote the growth of wheat in a living state, an inactivated state, a salt-free stress condition and a salt stress condition, and mainly shows that the leaf length of the wheat or the fresh weight on the ground is increased by 10.00-21.48%.
The invention has the advantages and beneficial effects that:
the salt-tolerant growth-promoting bacterium Kushneria indalina JP-JH disclosed by the invention can play a remarkable promoting role in the growth (leaf length and overground fresh weight) of wheat in a living state, an inactivated state and under the conditions of no salt stress and salt stress, and obviously relieves the salt damage of wheat under the condition of salt stress. Therefore, compared with other reported strains, the salt-tolerant growth-promoting bacterium JP-JH disclosed by the invention can reasonably reduce the requirements on the conditions of culture, storage, transportation, colonization and the like of the strains, and is beneficial to reducing the production cost and facilitating popularization and application; on the other hand, the method has wider environmental adaptability, is suitable for the conditions of no salt stress and salt stress, including moderate and severe saline-alkali soil (with the salt content of 0.4-1.0 percent), even severe saline-alkali soil with the salt content of 1-15 percent, and is favorable for realizing multiple purposes of one bacterium.
In addition, the salt-tolerant growth-promoting bacterium Kushneria indalina JP-JH disclosed by the invention has excellent characteristics of plant growth promotion and plant salt damage alleviation, on one hand, resource and technical support are provided for preparing microbial agents and fertilizers for promoting the application of plant growth, the application of replacing or partially replacing chemical fertilizers is realized, and the problems of cost increase and a series of environmental pollution caused by excessive application and blind application of the chemical fertilizers are solved; on the other hand, the method provides resources and technical support for the application of preparing the microbial agent and the fertilizer for relieving the salt damage of the plants, develops the biological measures for improving the saline-alkali soil based on the microorganisms, overcomes the limitations of physical, chemical and engineering measures in the improvement of the saline-alkali soil, and simultaneously reduces the time cost for cultivating the biological measures of new salt-resistant varieties. Furthermore, a safe, environment-friendly and efficient biotechnology support is provided for saline-alkali soil improvement and utilization.
Drawings
FIG. 1 is a colony observation diagram of the salt-tolerant growth-promoting bacterium JP-JH of the present invention;
FIG. 2 is a phylogenetic tree of the 16S rDNA sequence of the salt-tolerant growth-promoting bacterium JP-JH of the present invention;
FIG. 3 is a qualitative detection diagram of the salt tolerant growth-promoting bacterium JP-JH IAA secretion;
FIG. 4 is an observation picture of salt tolerant growth promoting bacteria JP-JH of the present invention for relieving wheat salt damage and promoting wheat growth;
FIG. 5 shows the effect of the salt-tolerant growth-promoting bacterium JP-JH of the present invention on wheat growth (leaf length);
FIG. 6 shows the effect of the salt-tolerant growth-promoting bacterium JP-JH of the present invention on the growth of wheat (fresh weight on the ground).
For a person skilled in the art, other relevant figures can be obtained from the above figures without inventive effort.
Detailed Description
In order to make the technical solution of the present invention better understood, the technical solution of the present invention is further described below with reference to specific examples.
The first embodiment is as follows: separation and purification of salt-tolerant growth-promoting bacteria JP-JH
Sample source: suaeda glauca rhizosphere soil in saline-alkali soil of Haixing county of Cangzhou city in Hebei province of China.
LB medium (solid): 10g/L tryptone, 5g/L yeast extract, 50g/L sodium chloride and 20g/L agar, adjusting the pH value by adopting 5M/L NaOH, and then sterilizing by high-pressure steam at 121 ℃ for 30 min.
LB medium (liquid): 10g/L tryptone, 5g/L yeast extract and 50g/L sodium chloride, adjusting the pH value by adopting 5M/L NaOH, and then sterilizing by high-pressure steam at 121 ℃ for 30 min.
Separation and purification steps: 1) collecting suaeda salsa root systems in saline-alkali soil in Haixing county of Cangzhou city, Hebei province, shaking off the surface soil of the root systems, filling the root systems into a sterile 50mL centrifuge tube, adding sterile water, and performing vortex oscillation to obtain rhizosphere soil stock solution; 2) standing, collecting appropriate amount of supernatant, and sequentially diluting with sterile water to obtain 10-1、10-2And 10-3Respectively coating the soil suspension on solid LB culture medium containing 5% NaCl, and culturing in a constant temperature incubator at 28 deg.C; 3) and repeatedly picking single colonies with different forms and colors for plate streaking culture, and respectively subculturing for 4-5 generations to obtain pure single colonies with consistent forms.
Example two: identification of salt-tolerant growth-promoting bacteria JP-JH
1) Characterization of biological Properties
The colony of the salt-tolerant growth-promoting bacterium JP-JH on an LB culture medium is in an orange bulge shape, and the edge of the colony is in a slightly transparent shape (figure 1).
2) Systematic classification and identification
The invention adopts a Kit TIANAmp Bacteria DNA Kit (Tiangen, Beijing) and extracts the JP-JH genome DNA of the salt-tolerant growth-promoting Bacteria according to the operation steps. The strain JP-JH 16S rDNA sequence was amplified using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'). The PCR reaction system was 50. mu.L containing 10. mu.L EasyTaq buffer, 5. mu.L dNTPs, 0.5. mu.L primer 27F, 0.5. mu.L primer 1492R, 1.5. mu.L DNA template, 2. mu.L Easy Taq DNA polymerase, and finally sterile ultrapure water was added to 50. mu.L. The PCR reaction program is: (i) pre-denaturation at 94 ℃ for 5 min; (ii)35 cycles of denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, and extension at 72 ℃ for 1.5 min; (iii) the final extension conditions were 72 ℃ for 10 min. The PCR amplification product is submitted to the company of bioengineering (Shanghai) and sequenced, and the 16S rDNA of the strain JP-JH obtained by sequencing is described in the sequence table SEQ NO. 1.
Homology alignment of the sequenced sequences by NCBI (national Center for Biotechnology information) database showed that the 16S rDNA sequence of strain JP-JH has a sequence similarity of up to 99.86% to Kushneria indianinastrain G6. A phylogenetic tree (FIG. 2) was constructed using MAGA 4.0, and a stable evolutionary branch (bootstrap value 99) was constructed with the sequence of Kushneriai indianinastrain G6, and thus, it was named Kushneria indianina JP-JH.
Example three: evaluation of salt-tolerant growth-promoting bacteria JP-JH salt-tolerant and alkali-resistant capability
The single bacterial colony of the strain JP-JH on the solid LB culture medium is picked up and inoculated in 50mL of liquid LB culture medium, after the culture is carried out for 48h at 28 ℃ and 150r/min, the single bacterial colony is respectively inoculated on the LB solid culture medium containing NaCl solution with the concentration of 1%, 5%, 10%, 15% and 20% and combined with the pH value of 7.0, 8.0 and 9.0 in pairs for streak culture, and the result shows that the strain JP-JH can grow in the culture medium with the NaCl concentration of 1% -15% and the pH value of 7.0-9.0.
Example four: qualitative determination of Indole Acetic Acid (IAA) secretion capability of salt tolerant growth-promoting bacterium JP-JH
Preparation of Salkowski colorimetric solution: 49mL of 35% HClO was taken4(70% of HClO was taken425mL of water to 50mL) and 1mL of 0.5mol/L FeCl3(0.811 g of FeCl was taken3Dissolved in 10mL of pure water) and mixed well.
The strain JP-JH is respectively inoculated in an LB liquid culture medium (code C0) containing 1% NaCl without L-tryptophan and an LB liquid culture medium containing 1% NaCl with final concentration of 0.25mg/mL, 0.50mg/mL and 0.75mg/mL L-tryptophan (code C1, C2 and C3 respectively), after culturing for 1d at 28 ℃ and 160r/min, a proper amount of bacterial suspension is taken out of a sterile transparent 2.0mL centrifuge tube, an equal volume of Salkowski solution is added into the centrifuge tube, a mixed solution of the LB liquid culture medium containing 1% NaCl and the equal volume colorimetric solution is used as a blank control (code CK), and the condition of color change is observed after the mixture is placed for 30min at room temperature. The results show that the blank CK did not show red color, and that strain JP-JH showed a distinct red color (FIG. 3) with and without L-tryptophan induction (C0, C1, C2 and C3), indicating that strain JP-JH is capable of secreting IAA and that IAA is secreted without L-tryptophan induction.
Example five: influence of salt-tolerant growth-promoting bacteria JP-JH on wheat growth
Negative control was set up for the experiment (in H)2O), positive control strain JP-JH inactivated body (expressed as JHD; 121 ℃ C., 30min autoclaving) and experimental group strain JP-JH Living body (JHL), NaCl solution concentrations were set to 0mM, 100mM and 200mM, respectively, for a total of 9 treatments, each of which was repeated 3 times (FIG. 4).
Preparing bacterial liquid: a strain JP-JH single colony on a solid LB culture medium is selected and inoculated in 50mL of liquid LB culture medium, after the culture is carried out for 48h at 28 ℃ and 150r/min (the OD600 of the strain is measured and recorded at the same time), the strain is inoculated in 200mL of liquid LB culture medium according to the inoculum size of 2 percent, after the culture is carried out for 48h at 28 ℃ and 150r/min, the strain is centrifugally collected at 8000rpm and 15min, after the strain is washed for 2 times by using sterile water, and the OD600 of the strain is adjusted to 0.6-0.8 by using the sterile water. The obtained bacterial liquid is uniformly distributed into 2 triangular flasks, one of which is placed in a refrigerator at 4 ℃ for standby (JHL), and the other is subjected to high-pressure steam sterilization at 121 ℃ for 30min for standby (JHD).
Wheat culture: collecting non-saline-alkali soil, sieving with 2mm sieve, and mixingThe fresh soil is subpackaged according to 1.20 kg/pot, the water content is measured at the same time, and the dry soil weight is calculated to be about 1.10 kg/pot. After base fertilizer is applied according to the quick-acting nitrogen of 70mg/kg, the quick-acting phosphorus of 30mg/kg and the quick-acting potassium of 90mg/kg and the soil is thoroughly poured, salt-tolerant wheat is planted in the small weir 60. 12 seeds were planted in each pot (day 1), and thinning was started after the seedlings emerged regularly (day 5), so that 4 wheat seedlings were retained in each pot. The culture conditions are 25 deg.C, 16h light/8 h dark (light intensity 100 μmol. m)-2·s-1). When wheat seedlings are healthy and strong (day 7), salt stress treatment is carried out by respectively using 100mM NaCl solution and 200mM NaCl solution, comparison treatment is carried out by using sterile water (0mM NaCl solution), irrigation is carried out once every 2-4 days, irrigation is continuously carried out for 3-5 times to enable the NaCl content of soil to reach about 5.5g/kg and 11g/kg respectively, then the irrigation with the NaCl solution is stopped, and post-irrigation is carried out by using sterile water. Simultaneously, inoculating 30mL of strain JP-JH living body (JHL) and strain JP-JH inactivated living body (JHD) to the rhizosphere soil of the wheat for promoting growth in each irrigation, and inoculating sterile water (H)2O) control treatment. The culture conditions are 25 + -5 deg.C in daytime, 20 + -5 deg.C in nighttime, 16h light/8 h dark (light intensity 100 μmol. m)-2·s-1)。
After 56 days of culture, observation, measurement and data statistics analysis results: 1) the growth of wheat was observed: the comparison (H) of the wheat added with the live microbial inoculum (JHL) and the inactivated microbial inoculum (JHD) is found2O) and green leaf color (fig. 4); 2) measuring the growth of wheat: measuring and counting indexes such as the length and the root length of the wheat leaves, the fresh weight and the dry weight on the ground and the like, and finding that under the conditions of no salt stress (0mM) and salt stress (100mM and 200mM), the growth of the wheat is promoted by adding the living microbial inoculum (JHL) and the inactivated microbial inoculum (JHD), which are mainly expressed as the increase of the length and the fresh weight on the ground of the wheat leaves (figures 5 and 6); 3) statistically analyzing the growth of wheat: based on Duncan's method (P) using SPSS software<0.05 represents significant difference) and the measurement results are statistically analyzed, under the condition of 100mM salt stress, the addition of the live microbial inoculum (JHL) has significant promotion effect on the length of the wheat leaves and the fresh weight on the ground, and the length of the wheat leaves and the fresh weight on the ground are respectively increased by 10.00 percent and 12.67 percent (fig. 5 and 6); adding live bacteriaThe agent (JHD) has a remarkable promoting effect on the overground fresh weight of wheat, and the overground fresh weight of wheat is increased by 19.53 percent (figure 6). Meanwhile, under the condition of no salt stress (0mM), the addition of the living microbial inoculum (JHL) has a remarkable promoting effect on the overground fresh weight of the wheat, and the overground fresh weight of the wheat is increased by 21.48 percent (figure 6). In conclusion, the salt-tolerant growth-promoting bacteria JP-JH disclosed by the invention can play a remarkable role in promoting the growth of wheat in a living state, an inactivated state, a salt-free stress condition and a salt stress condition, and the promoting roles are mainly represented by increasing the biomass of the overground part of the wheat and relieving the damage of the salt stress to the wheat.
The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.
SEQUENCE LISTING
<110> research center of agricultural resources of institute of genetics and developmental biology of Chinese academy of sciences
<120> salt-tolerant growth-promoting bacterium JP-JH for relieving salt damage of plants and application thereof
<130>1
<160>1
<170>PatentIn version 3.5
<210>1
<211>1442
<212>DNA
<213>Kushneria indalinina
<400>1
ggcatggcgc agctacacat gcagtcgagc ggtaacaggg gtagcttgct acccgctgac 60
gagcggcgga cgggtgagta atgcatgggt atctgcccgg tagtggggga taactcgggg 120
aaacccgagc taataccgca tacgtcctac gggagaaagc aggggatctt cggaccttgc 180
gctatcggat gagcccatgt cggattagct tgttggtgag gtaacggctc accaaggcga 240
cgatccgtag ctggtctgag aggatgatca gccacaccgg gactgagaca cggcccggac 300
tcctacggga ggcagcagtg gggaatattg gacaatgggg ggaaccctga tccagccatg 360
ccgcgtgtgt gaagaaggcc ttcgggttgt aaagcacttt cagtggggaa gaaggcatga 420
tgattaatac tcgtcatgaa ggacatcacc cacagaagaa gcaccggcta actccgtgcc 480
agcagccgcg gtaatacgga gggtgcaagc gttaatcgga attactgggc gtaaagggcg 540
cgtaggcggc ttgccaagcc ggatgtgaaa gccccgggct caacccggga acggcattcg 600
gaactggtag gctagagtgc aggagaggaa ggtggaattc ccggtgtagc ggtgaaatgc 660
gtagagatcg ggaggaatac cagtggcgaa ggcggccttc tggactgaca ctgacgctga 720
ggcgcgaaag cgtgggtagc aaacaggatt agataccctg gtagtccacg ccgtaaacga 780
tgtcgaccag ccgttggacc ccttgagggt ttggtggcgc agttaacgca ataagtcgac 840
cgcctgggga gtacggccgc aaggctaaaa ctcaaatgaa ttgacggggg cccgcacaag 900
cggtggagca tgtggtttaa ttcgatgcaa cgcgaagaac cttacctgct cttgacatcc 960
tgcgaattcc tcagagatga ggaagtgcct tcgggaacgc agagacaggt gctgcatggc 1020
tgtcgtcagc tcgtgttgtg aaatgttggg ttaagtcccg taacgagcgc aacccctatc 1080
cctttttgcc agcgcgtaat ggcgggaact tcagggagac tgccggtgac aaaccggagg 1140
aaggtgggga cgacgtcaag tcatcatggc ccttacgagc agggctacac acgtgctaca 1200
atggccggta caaagggttg cgaagcggcg acgtgaagcc aatcccagaa agccggcctc 1260
agtccggatc ggagtctgca actcgactcc gtgaagtcgg aatcgctagt aatcgtggat 1320
cagaatgcca cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccatggga 1380
gtggactgca ccagaagtgg ttagcttaac cttcgggaga gcgatcacca cggtgtcgtt 1440
gg 1442
Claims (7)
1. A salt-tolerant growth-promoting bacterium JP-JH for relieving salt damage of plants is characterized in that: the strain belongs to Kushneiaindalinina, is preserved in China center for type culture Collection with the preservation number of CCTCC M20191039 and the preservation date of 12 months and 12 months in 2019.
2. The salt-tolerant growth-promoting bacterium of claim 1, wherein: the strain has strong salt and alkali resistance, the growth of the strain can tolerate NaCl concentration of 1-15% (w/v), and the pH value of the strain can be 7.0-9.0.
3. The salt-tolerant growth-promoting bacterium of claim 1, wherein: the strain has the capability of secreting IAA, and can secrete IAA without L-tryptophan induction.
4. A microbial agent capable of relieving salt damage of plants is characterized in that: the microbial inoculum is prepared by the salt-tolerant growth-promoting bacteria of claim 1.
5. A microbial agent capable of promoting plant growth, characterized in that: the microbial inoculum is prepared by the salt-tolerant growth-promoting bacteria of claim 1.
6. Use of the salt tolerant growth-promoting bacteria of claim 1, or the microbial inoculant of claim 4 or 5, for the preparation of a biofertilizer.
7. Use of the salt tolerant growth-promoting bacteria of claim 1, or the microbial inoculant of claim 4 or 5, or the biofertilizer of claim 6 for alleviating salt damage or promoting plant growth.
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| CN113913327A (en) * | 2021-09-30 | 2022-01-11 | 中国科学院东北地理与农业生态研究所农业技术中心 | Saline-alkali-tolerant growth-promoting bacterium YS-AT1 capable of secreting IAA and application thereof |
| CN113913327B (en) * | 2021-09-30 | 2024-04-16 | 中国科学院东北地理与农业生态研究所农业技术中心 | Saline-alkali-resistant growth-promoting bacterium YS-AT1 capable of secreting IAA and application thereof |
| CN115786178A (en) * | 2022-10-10 | 2023-03-14 | 中国科学院东北地理与农业生态研究所农业技术中心 | Saline-alkali-tolerant citrobacter and application thereof in promoting plant growth |
| CN115786178B (en) * | 2022-10-10 | 2023-08-29 | 中国科学院东北地理与农业生态研究所农业技术中心 | Saline-alkali resistant Citrobacter and application thereof in promoting plant growth |
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