CN109929777B - Halomonas strain H6, composition and application thereof in salt tolerance and growth promotion - Google Patents
Halomonas strain H6, composition and application thereof in salt tolerance and growth promotion Download PDFInfo
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Abstract
The invention aims to obtain a strain of halomonas (a) by separation and screeningHalomonas sp.) The new strain has the capacity of secreting indoleacetic acid, producing ACC deaminase and the like. Can grow well under the conditions of low temperature or high temperature and saline alkali. The microbial fertilizer prepared by the composition of the strain and the liquid fermentation technology relieves the salt-alkali stress of salt on crops and promotes the growth of the crops under the salt stress. The novel strain or the composition provided by the invention has the functions of being suitable for special geographical climate conditions, improving the physical and chemical properties and fertilizer supply capacity of soil, effectively utilizing environmental resources, reducing pesticide pollution and the like, has the characteristics of good effect, short period and good environmental compatibility, and has important practical value for solving the problem of soil salinization at present.
Description
The technical field is as follows:
the invention relates to the field of microorganisms, in particular to a novel halomonas strain with salt-tolerant growth-promoting effects, a composition and application thereof in improving salt stress tolerance of crops and promoting seedling growth of the crops.
Background art:
the problems of soil salinization and soil secondary salinization caused by irrigation are an important ecological environment problem, and generally occur in regions with dry climate, high soil evaporation intensity, high underground water level and more soluble salts. The problem of soil salinization now becomes an important factor influencing the agriculture, animal husbandry and economic sustainable development in arid and semiarid regions and threatening the ecological safety and stability.
Soil salinization is a major environmental risk caused by natural or human activities. According to incomplete statistics of the combined country's textbook organization and the grain and agricultural organization, the area of the saline-alkali soil is 9.5 hundred million hm2. The distribution range of the salinized soil in China is wide, the area is large, the types are many, and the total area is about 1 hundred million hm2Wherein the modern saline soil accounts for about 37 percent, the residual saline soil accounts for about 45 percent, and the potential saline soil accounts for about 18 percent, and mainly occurs in arid, semiarid and semihumid areas. The harm of the saline soil to the growth of plants is mainly reflected in the aspects of the salt damage of the plants and the salt resistance of the plants. Excessive salt in soil can cause that the water in the soil is difficult to be absorbed by plant root cells, even dehydration occurs, and meanwhile, the plants generate single salt poisoning effect to cause damage to normal physiological metabolism. After the plants are subjected to salt damage, the growth and development of the plants are influenced on a light basis, and the plants die on a heavy basis.
Xinjiang is the largest oasis farming area in China, and in recent years, with the increasing development intensity of agricultural land, unreasonable utilization of water resources and other factors, the problems of soil salinization and soil degradation gradually appear. All the materials have the characteristics of coexistence of salinization and desertification, stronger salt accumulation and surface aggregation, complex soil salt components and the like, thereby bringing a series of ecological environment problems and restricting the economic development of agricultural production and society. The saline soil in Xinjiang mainly utilizes a series of measures such as water-saving irrigation, physics, chemistry, biology and the like to comprehensively treat and improve. Through years of improvement, the saline-alkali soil in Xinjiang is improved to different degrees, but with the implementation of large-scale water-saving irrigation, the saline soil development presents new problems and rules, such as the problem of salt accumulation under the condition of drip irrigation under a film, the problem of salt discharge from a dry basin inland, and the like, the capital investment and the technical requirements are high, and the implementation of the remediation of the saline-alkali soil with a large area is difficult. Meanwhile, the problems of soil nutrient imbalance caused by blind wasteland reclamation, uneven land, single planted crop and the like, and the reduction of the salt resistance of the crop caused by the reduction of the application amount of the organic fertilizer are increasingly serious. As the most economic and effective measure for recovering the salinized land, the saline-alkali tolerant plant variety and the biological preparation are adopted to improve the saline-alkali tolerant characteristic of the plant, so that the biological improvement technology for overcoming the stress effect of the saline-alkali on the plant is one of the technologies which can be vigorously developed and utilized in Xinjiang.
However, how to improve the saline-alkali resistance and saline-alkali resistance of crops has become a major issue for researchers. The key to the problem of promoting the growth of plants in the halophytic environment is to improve the soil and improve the saline-alkali resistance of crops. Plant growth-promoting rhizobacteria (PGPR) is a kind of microorganism which can promote plant growth, control diseases and increase crop yield in plant rhizosphere soil. The application of PGPR is helpful for reducing the adverse effect of salt on plants, promoting the decomposition of soil organic matters, nutrient circulation and the utilization of plant nutrients, and further improving the biomass or yield of the plants, and the PGPR becomes an environment-friendly, economic and effective development strategy for promoting the growth of the plants in the salinized soil. Research shows that the rhizosphere growth-promoting bacteria promote the growth of plants in the saline soil through different mechanisms of non-symbiotic nitrogen fixation, inorganic phosphate dissolution, nutrient absorption improvement, plant hormone generation and the like, increase the crop yield, improve the accumulation of plant permeation regulating substances and increase K+Concentration and maintenance of high K+/Na+The ratio and the like reduce the influence of salt stress on crops, and rhizosphere bacteria produce ACC deaminase, induce system resistance, induce system tolerance and control on plant pathogenic microorganisms also help plants to relieve the salt stress.
Although a large number of plant growth-promoting bacteria resources have been found at home and abroad, in the microbial control industry, research and development of screening PGPR strains for promoting plants to grow under drought and salinity stress conditions and really applying the strains to fields are imperative. Due to the drought environment and the diversified salt and alkali environment in Xinjiang, the problems of poor adaptability and poor effect of related microorganisms exist. Therefore, the method screens and develops the novel microbial strain which is suitable for special geographical and climatic conditions of Xinjiang, has the effects of well relieving salt stress, promoting crop growth, improving soil physicochemical property and fertilizer supply capacity, effectively utilizing environmental resources, reducing pesticide pollution and the like on plants, has a good effect, a short period and good environmental compatibility, and has an important practical value for solving the current soil salinization.
The invention content is as follows:
the invention aims to obtain a new Halomonas (Halomonas sp) strain through separation and screening, the strain has the capabilities of secreting indoleacetic acid, producing ACC deaminase and the like, and the salt tolerance of plants can be obviously improved and the growth of crops can be promoted by using the composition of the strain and the microbial fertilizer prepared by using a liquid fermentation technology.
The purpose of the invention is realized by the following technical scheme:
the technical scheme of the invention is as follows: a new Halomonas sp strain H6 is obtained by separating and screening halophyte rhizosphere in Xinjiang and Shuo, and the salt-tolerant growth-promoting microbial fertilizer is prepared by utilizing a liquid fermentation technology to obtain a good effect. The invention comprehensively solves the adaptability of the microbial bacterial fertilizer in the saline-alkali soil and the growth promoting effect, and has simple manufacture, convenience and practicability.
Specifically, the invention provides a new strain Halomonas (Halomonas sp.) H6CGMCC No.15758 separated from the rhizosphere of halophytes in Xinjiang and major areas. The strain has the capabilities of salt tolerance, growth promotion, indoleacetic acid secretion, ACC deaminase production and the like, can be applied to improving the salt and alkali resistance of plants and promoting the growth, and has good application prospect of being developed into biological fertilizers.
The new strain Halomonas sp H6CGMCC No.15758 is obtained by separating, screening and domesticating plant rhizosphere in saline-alkali desert areas of Xinjiang and Shuo county, the strain number is H6, and the strain is determined to be a potential new species of the Halomonas sp by microbial taxonomic detection and identification. At present, this strain has been deposited in the international collection of microorganisms under the Budapest treaty before the filing date: china general microbiological culture Collection center (CGMCC). Address: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101, the preservation date is 2018, 05 and 14 days, and the preservation number is CGMCC No. 15758.
The new strain salt tolerant growth promoting bacteria Halomonas (Halomonas sp.) H6CGMCC No.15758 provided by the invention is gram positive, rod-shaped, 0.3-0.8 multiplied by 1.4-2.5 mu m, and does not form endospore. After 3-5 days of culture on TSA medium containing 2% NaCl, the colonies were circular, with neat edges, and slightly raised pale yellow colonies. The growth temperature range of the thalli is 10-45 ℃, and the optimal growth temperature is 28 ℃; the pH growth range is 5.0-10, the optimal pH value is 7.0, and the NaCl can be tolerated by 1-20%.
Amplifying a 16S rDNA sequence of a new strain Halomonas (Halomonas sp.) H6CGMCC No.15758 strain by taking the total genomic DNA of the strain as a template, sequencing to obtain the 16S rDNA sequence with the length of 1431bp, and performing homology comparison analysis on the sequence and related strains recorded in a GenBank database to determine that the strain belongs to the genus Halomonas and the standard model strain Halomonas titanice BH1TAnd Halomonas neptunia Epume 1THomology is 98.6% and 98.3%, respectively, and no formal taxonomical nomenclature of the strain has been carried out so far, with the provisional strain name Halomonas (Halomonas sp.) H6.
Meanwhile, the present invention provides a culture using the aforementioned Halomonas (Halomonas sp.) H6 strain.
The present invention further provides compositions utilizing a culture of the Halomonas (Halomonas sp.) H6 strain described above, in liquid, frozen or dried powder form.
In the present invention, the above composition contains an auxiliary.
In the present invention, when the composition is in the form of a frozen or dried powder, it further comprises a solid carrier.
In the invention, the solid carrier comprises one or more of peat, turf, talc, lignite, pyrophyllite, montmorillonite, alginate, filter pressing slurry, sawdust, perlite, mica, silica, quartz powder, calcium bentonite, vermiculite, kaolin, light calcium carbonate, diatomite, medical stone, calcite, zeolite, white carbon black, fine sand and clay.
In the invention, the auxiliary agent comprises one or more of sodium dodecyl benzene sulfonate, sodium butyl naphthalene sulfonate, trehalose, glycerol, sodium lignin sulfonate, sodium alkyl naphthalene sulfonate polycondensate, nicotinic acid, alcohol, buffer salt, sodium chloride, amino acid, vitamins, protein, polypeptide, polysaccharide or monosaccharide, yeast paste, white carbon black, tea saponin and skim milk.
In the present invention, the above-provided series of compositions is utilized, further comprising an agriculturally effective amount of a compound or composition selected from the group consisting of: nutrients, fertilizers and microbial fertilizers.
In the invention, the salt monad (Halomonas sp.) H6 or the composition provided by the invention is used for improving the salt stress tolerance of cotton and promoting the seedling growth of crops.
Specifically, the new strain Halomonas (Halomonas sp.) H6CGMCC No.15758 strain is characterized by being capable of growing well at low temperature or high temperature and under the saline-alkali condition, obviously relieving the salt-alkali stress of salt on economic crops mainly comprising cotton, and particularly promoting the growth of the cotton under the salt stress by secreting auxin through growth metabolism.
In the invention, the salt monad (Halomonas sp.) H6 or the composition provided by the invention is used for improving the salt stress tolerance of cotton or wheat and promoting the seedling growth of crops.
The invention has the following beneficial technical effects:
(1) the new strain Halomonas (Halomonas sp.) H6CGMCC No.15758 strain provided by the invention has the characteristics of low-temperature and high-temperature growth, and is resistant to high salt and high pH, so that the salt and alkali stress can be obviously relieved, and the growth of crops is promoted. And has the functions of synthesizing indoleacetic acid, producing ACC deaminase and promoting plant growth.
(2) The new strain Halomonas (Halomonas sp.) H6CGMCC No.15758 provided by the invention can obviously promote the growth of cotton seeds soaked in the bacterial liquid diluent for 15 days. Compared with a control group, the root length and the plant weight of the treatment group using the microbial inoculum are respectively and remarkably increased by 11.54 percent and 15.38 percent; after the wheat is soaked in diluent of halomonas H6 for 15 days, the root length and fresh weight are obviously promoted, and are respectively and obviously increased by 23.08% and 41.67%. Proves that the growth of cotton flowers under salt stress can be obviously improved by using a fungicide Halomonas sp H6, and the fungicide Halomonas sp H6 has obvious salt-tolerant growth-promoting effect.
(3) The application of the provided Halomonas (Halomonas sp) H6 or the composition in improving the salt stress tolerance of cotton and wheat and promoting the seedling growth of crops has obvious salt tolerance and growth promotion effects, so that the consumption of chemical fertilizers can be reduced, the expenditure of farmers can be saved, the economic income can be increased, and good ecological benefits and social benefits can be achieved.
Description of the drawings:
FIG. 1 shows a developmental tree of Halomonas sp H6CGMCC No.15758 system.
FIG. 2 shows the colony morphology of Halomonas sp H6CGMCC No. 15758.
FIG. 3 shows the effect of different NaCl concentrations on the growth of Halomonas (Halomonas sp.) H6CGMCC No. 15758.
FIG. 4 shows the effect of different pH values on the growth of Halomonas H6CGMCC No. 15758.
FIG. 5 shows the growth curve of Halomonas sp H6CGMCC No.15758 in fermentation medium.
FIG. 6 shows the growth promoting effect of Halomonas sp H6CGMCC No.15758 on cotton seedlings.
FIG. 7 shows the growth promoting effect of Halomonas sp H6CGMCC No.15758 on wheat seedlings.
The specific implementation mode is as follows:
in order to better explain the invention, the following further illustrate the main content of the invention in connection with specific examples, but the content of the invention is not limited to the following examples. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, the raw materials used are all commercially available products and can be purchased through public channels, and the equipment and instruments used in the process are all common equipment in the art.
2% NaCl TSA medium: tryptone 15g, soybean peptone 5g, NaCl 20g, agar 15g, H2O 1000mL，pH 7.3。
Fermentation medium composition (g/L): glucose 3.0, peptone 5.0, yeast extract 2.0, K2HPO4 3.0，NaCl 20.0，pH7.0-7.2。
DF Medium (g/L): KH (Perkin Elmer)2PO4 4.0，Na2HPO4 6.0，MgSO4·7H2O0.2, glucose 2, sodium gluconate 2, citric acid 2, (NH)4)2SO4 2.0，FeSO4·7H2O0.001, 0.1mL each of the first and second trace element solutions, adding the sample one by one, dissolving completely, and autoclaving at 121 deg.C for 20 min. Solution of trace elements: the component one: h3BO3 10mg、MnSO4·H2O 11.19mg、ZnSO4·7H2O 124.6mg、CuSO4·5H2O 78.22mg、MoO3 10mg、 H2O100 mL, dissolved in 100mL sterile distilled water. And (2) component two: 100mg of FeSO4·7H2O was dissolved in 100mL of sterile distilled water.
The first embodiment is as follows: isolation, screening and characterization of Halomonas sp H6
(I) separating and screening:
collecting plant roots in saline-alkali desert regions of Xinjiang and Shuo countyAnd (3) separating and purifying the microorganism by a gradient dilution method. Weighing 10g of soil sample, putting the soil sample into 100mL of sterile water, adding sterilized glass beads, rotating at 150rpm and 30 ℃, and shaking for 20min to fully disperse the sample. Sucking supernatant 100 μ L, adding into sterile water 900 μ L, and diluting with sterile water to obtain 10-2-10-5A dilution of concentration. Taking 100 mu L of each concentration diluent, coating the diluent on a TSA solid culture medium plate containing 2% NaCl by adopting a conventional coating method, placing the TSA solid culture medium plate in a constant-temperature incubator at 30 ℃ for culturing for 48 hours, and picking single colonies on the plate for purification culture after the colonies grow on the plate until no impurity colonies exist. A strain with the number of H6 is preferably selected from the strain, and the strain is transferred to a culture medium containing 2% NaCl TSA to be stored for standby.
(II) classification and identification:
1. sequencing and analysis of 16S rDNA of Halomonas sp H6:
(1) extraction of PCR template DNA:
inoculating the purified strain H6 into a TSA culture medium with 2% NaCl, culturing for 2 days at 30 ℃ by a shaking bed, collecting thalli, and extracting total genomic DNA by adopting a DNA extraction kit.
(2) PCR amplification
Specific primers are adopted:
27F:5'-AGAGTTTGATCCTGGCTCAG-3'，
1492R:5'-GGTTACCTTGTTACGACTT-3'；
the total volume of the PCR reaction system is 25 mu L, and the PCR amplification condition is 94 ℃ for 5 min; 94 ℃ for 45s, 56 ℃ for 45s, 72 ℃ for 45s, 30 cycles; 10min at 72 ℃.
(3) Sequence determination
The PCR amplification product is subjected to electrophoresis detection and purification and then sequenced to obtain a 16S rDNA sequence with the length of 1431bp, the sequence is shown in SEQ ID NO 1 provided after the sequence is attached, the obtained sequence is subjected to comparative analysis through a common NCBI website, a phylogenetic tree is constructed, the phylogenetic tree of the strain is shown in the attached figure 1, and the strain H6 and the standard model strain Halomonas titanica BH1 are found through the comparative analysisTAnd Halomonas neptunia Epume 1TMost closely related, 16S rDNA sequences and bothHomology is respectively 98.6% and 98.3%, a phylogenetic tree is established by utilizing MEGA 5.0 software commonly adopted in the field through a Neighbor-Joining method, and the results are compared and analyzed to find that the strain H6 and the Halomonas titanica BH1TClustering on one branch, wherein the confidence values are 86% respectively, which shows that the strain has extremely high performance as a new strain, excellent stability and extremely high support rate are shown in an evolutionary tree, the strain H6 can be determined as a new Halomonas (Halomonas sp.) species through the molecular level identification of series strains, the strain has the typical characteristics of the new strain, and the strain is temporarily named as Halomonas sp H6 from the taxonomic angle.
2. Physiological and biochemical assays
(1) The results of the growth condition studies showed that Halomonas sp H6, gram-positive, rod-shaped, 0.3-0.8X 1.4-2.5. mu.m, did not form endospores. After growing and culturing for 3-5d on the TSA culture medium containing 2% NaCl, the colony is a round, neat-edged and slightly convex yellowish colony, and the colony morphology is shown in the attached figure 2. The growth temperature of the thalli is 10-45 ℃, and the optimal growth temperature is 28 ℃; the pH growth range is 5.0-10, the optimal pH value is 7.0, and the NaCl can be tolerated by 1-20%.
(2) Catalase and oxidase activities were positive and casamino acid saponins were hydrolyzed.
(3) The carbon source that Halomonas sp H6 can utilize was determined to be a-D-glucose, D-sorbitol, pectin, rho-hydroxy-phenylacetic acid, D-mannitol, D-galacturonic acid, gamma-amino-butyric acid, D-fructose, D-arabitol, L-alanine, L-galacturonic acid lactone, D-methyl lactate, D-trehalose, D-galactose, inositol, L-arginine, D-gluconic acid, L-lactose, beta-hydroxy-D, L-butyric acid, D-cellobiose, 3-formylglucose, glycerol, L-aspartic acid, D-glucuronic acid, citric acid, etc., using a Biolog strain identifier GNIII test plate, D-fructose, D-glucose-6-phosphate, L-glutamic acid, alpha-ketoglutaric acid, acetoacetate, sucrose, N-acetyl-beta-D-mannosamine, L-fructose, D-malic acid, propionic acid, D-turanose, quinic acid, L-malic acid, acetic acid, inosine, and L-serine.
The results of 16S rDNA sequence analysis, phylogenetic analysis and microbiological characteristic analysis show that the Halomonas H6 provided by the invention is a typical new bacterial strain of the Halomonas, and is tentatively named as Halomonas (Halomonas sp.) H6. The strain is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 05 and 14 months, and the address is as follows: the collection number of the strain is CGMCC No.15758, No. 3 of Xilu No.1 of Beijing, Chaoyang, and institute of microbiology of Chinese academy of sciences.
Example two: growth promoting ability test of Halomonas sp H6 strain
(1) Ability to secrete indoleacetic acid (IAA)
Inoculating Halomonas H6 into TSA liquid culture medium containing 100mg/L L-tryptophan and 2% NaCl, culturing at 30 deg.C for 1d with a shaking table at 180r/min, dripping 50 μ L of the bacterial suspension onto a white ceramic plate, and simultaneously adding equal volume of Salkowski colorimetric solution, wherein the Salkowski colorimetric solution is 50mL of 35% HClO4And 1mL of 0.5mol/L FeCl3Mixing was performed, and a mixed solution of 50. mu.L of a 2% TSA liquid medium without inoculation and an equal volume of colorimetric solution was added as a control. The white ceramic plate is placed in the dark at room temperature for 30min, and 50 mu L of halomonas H6 bacterial suspension is added to the white ceramic plate to change the color into red.
(2) Test for producing ACC deaminase
The halomonas H6 is inoculated in a DF solid culture medium containing 3mM ACC, after passage for 3 times, the growth of the halomonas H6 on the culture medium with the ACC as the only nitrogen source is good, and the grown strain is a deaminase-producing positive strain.
The research shows that: halomonas sp H6 has the ability to secrete indole acetic acid and ACC deaminase.
Example three: salt tolerance detection of Halomonas (Halomonas sp.) H6 strain
Inoculating Halomonas sp.H6CGMCC No.15758 into a TSA liquid culture medium, culturing at 30 ℃ and 180rpm for 24h, inoculating the bacterial suspension into the TSA liquid culture medium with 1%, 5%, 10%, 15% and 20% NaCl concentration, performing shaking culture at 30 ℃ and 180rpm for 24h, measuring the OD value at 600nm, and observing the growth condition of Halomonas sp.H6 to determine the salt tolerance.
As shown in figure 3, the Halomonas sp.H6 can grow in the NaCl concentration range of 1-20%, but the growth amount of the Halomonas sp.H6 is in a remarkable descending trend along with the increase of the salt concentration,
example four: detection of pH resistance of Halomonas sp H6 strain
Halomonas sp.H6CGMCC No.15758 is inoculated in a TSA liquid culture medium, the culture is carried out for 24h at 30 ℃ and 180rpm, the bacterial suspension is respectively inoculated in the TSA liquid culture medium with the pH values of 3, 5, 7 and 9, the shaking culture is carried out for 24h at 30 ℃ and 180rpm, the OD value at 600nm is measured, and the growth condition of Halomonas sp.H6 is observed to determine the pH resistance capability of the Halomonas sp.H6.
The research result is shown in figure 4, Halomonas sp.H6 of Halomonas is more inclined to grow under weak alkaline condition, the Halomonas sp.H6 grows well within the pH value range of 7-9, and the optimal growth pH value is 7. When the pH is 5 or less, the amount of growth is remarkably reduced.
Example five: growth curve of Halomonas (Halomonas sp.) H6 and preparation of microbial inoculum
Halomonas sp.H6CGMCC No.15758 is inoculated on a TSA solid culture medium containing 2% NaCl for activation, then is transferred into a TSA liquid culture medium (100 mL in a 500mL triangular flask), and is cultured for 18h at 30 ℃ and 180rpm to obtain seed liquid. Inoculating the seed liquid into fermentation culture medium at a ratio of 1:100, culturing at 30 deg.C and 200rpm for 24 hr until the maximum concentration of microbial inoculum reaches 6 × 109To obtain the microbial agent. In actual use, the fermentation inoculum can be diluted by a certain multiple for use according to requirements. See FIG. 5 for a plot of growth of the bacteria in fermentation medium (10-fold dilution).
Example six: growth promoting effect of Halomonas (Halomonas sp.) H6 on cotton seedlings
Selecting cotton seeds with full grains and no obvious damage, sterilizing for 5min by using 0.1% mercuric chloride, cleaning with sterile water, and soaking the seeds for 4h by using bacterial liquid with 1/100 concentration; the control treated seeds without inoculation were immersed in sterile distilled water.
Adopting a hole planting pot with 4 multiplied by 5 holes, washing with water, drying with yellow sand, and then using the washed yellow sand as hole planting soil. After the soil is soaked by a proper amount of 5% NaCl solution, overnight added, placed with tap water and mixed uniformly, so that the water content of the soil is up to 20% and the salt content is up to 0.75%. Experimental design each 10 wells were treated, and each cotton seed was spotted into a well-planting pot, 4 grains per well, approximately 1.5cm deep, covered with a plastic film, and cultured in a climatic chamber. Culturing at 20 deg.C for 8h without illumination; irradiating at 22 deg.C and 30% for 2 hr; irradiating at 25 deg.C under 100% light for 12 hr; and (5) illuminating for 2h at 30%. After germination of the seeds, the film was removed and the seeds were irrigated once every 5 days (about 10mL per hole). After germination for 15 days, the root length and the plant weight of the cotton plants of the test group and the control group are measured.
As shown in the attached figure 6, the experimental result shows that compared with the control treatment, after the cotton seeds are soaked by the halomonas H61/100-concentration microbial inoculum, the root length and the plant weight of the cotton seeds after 15 days of growth are respectively and obviously increased by 11.54 percent and 15.38 percent, which indicates that the halomonas microbial inoculum diluent has obvious promotion effect on the cotton seeds after being soaked.
Example seven: growth promoting effect of Halomonas sp H6 on wheat
Selecting wheat seeds with full grains and no obvious damage, sterilizing for 5min by using 0.1% mercuric chloride, cleaning by using sterile water, and soaking the seeds for 4h by using bacterial liquid with the concentration of 1/100; control treated seeds without inoculation were immersed in sterile distilled water.
Adopting a hole planting pot with 4 multiplied by 5 holes, washing with water, drying with yellow sand, and then using the washed yellow sand as hole planting soil. After the soil is soaked by a proper amount of 5% NaCl solution, overnight added, placed with tap water and mixed uniformly, so that the water content of the soil is up to 20% and the salt content is up to 0.75%. Experimental design each 10 pits were treated, each treated wheat seed was spotted into a pit pot, 4 grains per pit, about 1.5cm deep, covered with a plastic film, and cultured in a phytotron. Culturing at 20 deg.C for 8h without illumination; irradiating at 22 deg.C and 30% for 2 hr; irradiating at 25 deg.C under 100% light for 12 hr; and (5) illuminating for 2h at 30%. After germination of the seeds, the film was removed and the seeds were irrigated once every 5 days (about 10mL per hole). After germination for 15 days, various morphological parameters of the wheat plants of the test group and the control group are measured, including plant height, root length and fresh weight.
As shown in the attached figure 7, the experimental result shows that, compared with the control treatment, after the wheat seeds soaked by the halomonas H61/100-concentration microbial inoculum grow for 15 days, the promotion effect of the plant height is relatively weak, and is only increased by 3.06%, but the promotion effects on the root length and the fresh weight are obvious, and are respectively increased by 23.08% and 41.67%, so that the halomonas microbial inoculum diluent has an obvious growth promotion effect on wheat.
Example eight: a composition comprising a Halomonas sp H6 strain
On the basis of the above examples, a culture of the Halomonas (Halomonas sp.) H6 strain provided in the first example above was used.
A composition of a culture of the Halomonas (Halomonas sp.) H6 strain, said composition being in the form of a liquid, frozen or dried powder.
The composition comprises an auxiliary agent.
When the composition is in the form of a frozen or dried powder, it further comprises a solid carrier.
The solid carrier comprises one or more of peat, turf, talc, lignite, pyrophyllite, montmorillonite, alginate, filter-pressing slurry, sawdust, perlite, mica, silica, quartz powder, calcium bentonite, vermiculite, kaolin, light calcium carbonate, diatomite, medical stone, calcite, zeolite, white carbon black, fine sand and clay.
The auxiliary agent comprises one or more of sodium dodecyl benzene sulfonate, sodium butyl naphthalene sulfonate, trehalose, glycerol, sodium lignin sulfonate, sodium alkyl naphthalene sulfonate polycondensate, nicotinic acid, alcohol, buffer salt, sodium chloride, amino acid, vitamins, protein, polypeptide, polysaccharide or monosaccharide, yeast extract, white carbon black, tea saponin and skimmed milk.
Utilizing the above provided series of compositions, further comprising an agriculturally effective amount of a compound or composition selected from the group consisting of: nutrients, fertilizers and microbial fertilizers.
As described above, the present invention can be preferably implemented, and the above-mentioned embodiments are merely descriptions of preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the present invention.
Sequence listing
<110> institute of microorganism application of Sinkiang academy of agricultural sciences (Xinjiang-Yameiya bioengineering research and development center, China)
<120> halomonas H6, composition and application thereof in salt tolerance growth promotion
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<170> SIPOSequenceListing 1.0
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<213> Halomonas sp
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attcacgatt actagcgatt ccgacttcac ggagtcgagt tgcagactcc gatccggact 180
gagaccggct ttaagggatt cgctgactct cgcgagctcg cagccctttg taccggccat 240
tgtagcacgt gtgtagccct acccgtaagg gccatgatga cttgacgtcg tccccacctt 300
cctccggttt gtcaccggca gtctccttag agttcccgac attactcgct ggcaaataag 360
gacaagggtt gcgctcgtta cgggacttaa cccaacattt cacaacacga gctgacgaca 420
gccatgcagc acctgtctta cagttcccga aggcacacca gaatctcttc cggcttctgt 480
agatgtcaag ggtaggtaag gttcttcgcg ttgcatcgaa ttaaaccaca tgctccaccg 540
cttgtgcggg cccccgtcaa ttcatttgag ttttaacctt gcggccgtac tccccaggcg 600
gtcgacttat cgcgttaact tcgccacaaa gtgctctagg cacccaacgg ctggtcgaca 660
tcgtttacgg cgtggactac cagggtatct aatcctgttt gctacccacg ctttcgcacc 720
tcagtgtcag tgtcagtcca gaaggccgcc ttcgccactg gtattcctcc cgatctctac 780
gcatttcacc gctacaccgg gaattctacc ttcctctcct gcactctagc ctgacagttc 840
cggatgccgt tcccaggttg agcccggggc tttcacaacc ggcttatcaa gccacctacg 900
cgcgctttac gcccagtaat tccgattaac gcttgcaccc tccgtattac cgcggctgct 960
ggcacggagt tagccggtgc ttcttctgcg agtgatgtct ttcctaccgg gtattaaccg 1020
ataggcgttc ttcctcgctg aaagtgcttt acaacccgag ggccttcttc acacacgcgg 1080
catggctgga tcagggttgc ccccattgtc caatattccc cactgctgcc tcccgtagga 1140
gttcgggccg tgtctcagtc ccgatgtggc tgatcatcct ctcagaccag ctacggatcg 1200
ttgccttggt aagccattac cttaccaact agctaatccg acataggctc atccaatagc 1260
gggagccgga gccccctttc tcccgtagga cgtatgcggt attagcctgg gtttccccag 1320
gttatccccc actatcgggc agattcctat gcattactca cccgtccgcc gctcgtcagc 1380
atgtagcaag ctagccctgt taccgctcga cttgcatgtg gttaggcccc g 1431
Claims (7)
1. Halomonas (a)Halomonas sp.) H6, characterized in that (I) is Halomonas sp. (Halomonas sp.) H6 with the preservation number of CGMCC No.15758, halomonas(s) ((Halomonas sp.) The 16S rDNA sequence of H6 is shown in SEQ ID NO 1.
2. The halomonas of claim 1 (c: (a: (b))Halomonas sp.) H6, characterized in that said strain is tolerant to 1-20% NaCl.
3. Halomonas containing（Halomonas sp.) Composition of H6, characterized in that it is in the form of a liquid, frozen or dried powder comprising the bacterium Halomonas (Halomonas: (Halomonas))Halomonas sp.) Cultures and adjuvants of strain H6; when the composition is in the form of a frozen or dried powder, the composition further comprises a solid carrier; the salt monad (A), (B), (C)Halomonas sp.) The preservation number of the strain of H6 is CGMCC No. 15758.
4. The halomonas (Halomonas) of claim 3Halomonas sp.) The composition of H6 is characterized in that the auxiliary agent comprises one or more of sodium dodecyl benzene sulfonate, sodium butyl naphthalene sulfonate, trehalose, glycerol, sodium lignin sulfonate, sodium alkyl naphthalene sulfonate polycondensate, buffer salt, sodium chloride, amino acid, protein, monosaccharide, yeast extract, white carbon black, tea saponin and skimmed milk.
5. The halomonas (Halomonas) of claim 3Halomonas sp.) H6 composition, wherein the solid carrier comprises one or more of peat, turf, talc, lignite, pyrophyllite, montmorillonite, alginate, filter press mud, sawdust, perlite, mica, silica, quartz powder, calcium bentonite, vermiculite, kaolin, precipitated calcium carbonate, diatomaceous earth, medical stone, calcite, zeolite, white carbon, fine sand and clay.
6. The halomonas (Halomonas) of claim 3Halomonas sp.) A composition of H6, characterized in that the composition further comprises an agriculturally effective amount of a compound or composition comprising one or both of nutrients and microbial manure.
7. The halomonas (Halomonas) of any one of claims 3-6Halomonas sp.) Application of the H6 composition in improving salt stress tolerance of cotton or wheat and promoting seedling growth of crops.
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