阅读说明：本技术 一种促进黑麦草耐盐性的方法 (Method for promoting salt tolerance of ryegrass ) 是由 张金林 何傲蕾 牛舒琪 赵祺 李惠茹 邵坤仲 于 2019-10-16 设计创作，主要内容包括：本发明公开了一种促进黑麦草耐盐性的方法,该发明涉及微生物技术领域,具体包括以下步骤：S1、黑麦草种子消毒处理后,播种在装有蛭石和沙子的花盆中；S2、枯草芽孢杆菌菌株WM13-24和大肠杆菌DH5ɑ均以1％接种量接种于LB液体培养基,分别在28℃和37℃和180r min<Sup>-1</Sup>下避光培养16h,待吸光值OD<Sub>600</Sub>＝0.9时备用,以等体积LB液体培养基为空白对照；S3、待幼苗出土后,将植物材料分为三组,分别接种WM13-24菌液、DH5ɑ菌液和LB液体培养基各600μl于幼苗茎部周围土壤,待幼苗生长一周后进行盐处理,设0、150、300mM NaCl三种盐梯度,两周后取样测定相关生长和生理指标。本发明通过将来自于梭梭根际的枯草芽孢杆菌菌株WM13-24接种于黑麦草根际土壤,能促进黑麦草的耐盐性。(The invention discloses a method for promoting salt tolerance of ryegrass, which relates to the technical field of microorganisms and specifically comprises the following steps: s1, after sterilizing ryegrass seeds, sowing the ryegrass seeds in a flowerpot filled with vermiculite and sand; s2, Bacillus subtilis WM13-24 and Escherichia coli DH5 alpha are inoculated in LB liquid medium at 1% inoculum size at 28 deg.C, 37 deg.C and 180r min respectively ‑1 Culturing in dark for 16h to obtain light absorption value OD 600 When the volume is 0.9, the blank is prepared by using an equal volume of LB liquid culture medium; s3, dividing the plant materials into three groups after the seedlings come out of the earth, respectively inoculating WM13-24 bacterial liquidAnd (2) respectively adding 600 mul of DH5 alpha bacterial liquid and LB liquid culture medium to soil around the stem of the seedling, performing salt treatment after the seedling grows for one week, setting three salt gradients of 0, 150 and 300mM NaCl, and sampling after two weeks to determine related growth and physiological indexes. The invention can promote the salt tolerance of ryegrass by inoculating the bacillus subtilis WM13-24 from the rhizosphere of the haloxylon to the rhizosphere soil of the ryegrass.)

1. A method for promoting salt tolerance of ryegrass specifically comprises the following steps:

s1, sterilizing ryegrass seeds: soaking ryegrass seeds in 70% ethanol for disinfection, continuously disinfecting with 2% NaClO, washing with distilled water for 7-8 times (3 min each time), stirring continuously during washing, and washing with ethanol and sodium hypochlorite on the surfaces of the seeds;

preparing S2, bacillus subtilis WM13-24 and escherichia coli DH5 alpha bacterial liquid;

s2.1, firstly preparing an LB liquid culture medium: 10g/L sodium chloride, 10g/L tryptone and 5g/L yeast powder, and the pH value is 7.0;

s2.2, sterilizing the culture medium at the high temperature of 121 ℃ for 20 minutes;

s2.3, after the temperature is cooled to room temperature, inoculating bacillus subtilis WM13-24 and escherichia coli DH5 alpha bacteria liquid into LB liquid culture medium with the inoculation amount of 1%, and respectively inoculating the bacillus subtilis WM13-24 and the escherichia coli DH5 alpha bacteria liquid into the culture medium at 28 ℃ and 37 ℃ in a constant temperature shaking table 180rmin^-1Culturing in dark for 16h, and determining the bacterial liquid concentration as OD₆₀₀When the value is 0.9, taking out for standby;

s3, planting by adopting a pot experiment;

s3.1, sowing the disinfected ryegrass seeds in a flowerpot filled with vermiculite and sand, after the seedlings are exposed, respectively inoculating fresh bacillus subtilis WM13-24, escherichia coli DH5 alpha and LB culture media of 600 mul respectively around the stems of the seedlings, and planting 18 pots of seedlings in each treatment way;

s3.2, growth chamber conditions: the temperature is 28 ℃/23 ℃, the relative humidity is 70%, and the photoperiod is 16h/8 h;

s3.3, after the inoculated ryegrass seedlings grow for one week, performing salt treatment on the seedlings, and adding 0, 150 and 300mM NaCl into 1/2Hoagland nutrient solution respectively to perform salt treatment;

s3.4, sampling and measuring related physiological indexes after the seedlings under the salt treatment grow for two weeks.

2. The method of claim 1, wherein the salt tolerance of ryegrass is improved by: in step S1, the rye grass seeds are soaked in 70% ethanol for 10min, and then sterilized with 2% NaClO for 1 min.

3. The method of claim 1, wherein the salt tolerance of ryegrass is improved by: in the step S2.3, 100 mu L of Bacillus subtilis WM13-24 bacterial liquid is added into each 100mL of LB liquid culture medium.

4. The method of claim 1, wherein the salt tolerance of ryegrass is improved by: in step S2.3, the culture medium is cultured overnight on a constant temperature shaking table with the rotation speed of 180-.

5. The method of claim 1, wherein the salt tolerance of ryegrass is improved by: in the step S2, the bacillus subtilis WM13-24 strain is separated from the root of the desert plant haloxylon ammodendron in the desert of Mintallugegy in Gansu.

6. The method of claim 1, wherein the salt tolerance of ryegrass is improved by: in the step S3.1, the volume ratio of the fine sand to the vermiculite is 1: 1.

Technical Field

The invention relates to the technical field of biology, in particular to a method for promoting salt tolerance of ryegrass.

Background

Perennial ryegrass is a lawn grass and excellent forage grass which are most widely applied in China, has developed root system, strong tillering capability, high lawn forming speed, good wear resistance, high yield, tender stems and leaves and excellent quality, is a preferred variety for lawn planting and is also an ideal forage grass for herbivorous livestock. However, the soil saline-alkali soil and the area of the medium and low-yield field damaged by the saline-alkali soil in China are increased year by year, so that the growth conditions and the planting area of lawn grass and excellent pasture, namely ryegrass, are limited. The key technical problems to be solved by the invention are how to improve the salt tolerance of the ryegrass and relieve the harm of abiotic stress to the ryegrass.

The land improvement work of the saline-alkali land is a complex comprehensive project all the time, and in order to realize the saline-alkali land improvement in the real sense, comprehensive measures including engineering measures, biological measures, chemical measures and the like need to be taken, and the measures have the problems of high cost, complex method, poor effect continuity and the like.

Soil is the foundation of agricultural production and is an important life support system in the biosphere, and certain microorganisms in soil help the soil to function better, including nutrient circulation, energy movement, nutritional health of plants and soil animals, toxicity to degrade artificial fertilizers and biologicals remaining in the soil, etc. (Norris and Matthews,1994), and are called soil-beneficial microorganisms. However, with the development of agricultural modernization technologies (such as the application of chemical fertilizers), chemical fertilizers enter the biosphere and cause various adverse ecological reactions, causing environmental problems such as soil hardening, acidification, reduction of organic matter content and the like, breaking the balance between soil and plants in the agricultural ecosystem, and this artificial change destroys the natural balance of microbial communities, resulting in the loss of beneficial microorganisms and the entry of certain pathogens, thereby affecting agricultural production and reducing plant productivity (lujialong et al, 2001), and therefore, it is very necessary to develop and reasonably use novel fertilizers. The compound microbial fertilizer is a novel fertilizer, is formed by compounding specific beneficial microorganisms and nutrient substances, if the beneficial microorganisms enter a plant production system, the balance of a microbial community structure can be maintained, and the community structure balance has important significance for improving the productivity and stress resistance of plants. Therefore, in agricultural production, the selection of beneficial microorganisms, whether indigenous or not, to promote plant growth and improve the stress adaptability of plants is a very meaningful study (Antoun and Pr savost 2005).

Plant Growth Promoting Rhizobacteria (PGPR) belongs to beneficial soil microorganisms, and the microorganisms play an important role in promoting Plant growth and improving the stress resistance of plants. In recent decade, the sources for separating PGPR in China are more and more abundant, wherein Pseudomonas, Bacillus and Enterobacter are the dominant flora surrounding the PGPR in the roots of tobacco, alfalfa and wheat, and the strains possibly have stronger capability of adapting to the adverse environment and unique physiological and ecological functions. The bacillus pumilus, the bacillus and the enterobacter remarkably improve the growth and development of cherry roots and the biomass of overground parts, the simple bacillus, the flavobacterium, the psychrophile and the bacillus have low-temperature growth-adaptive characteristics on rape, rice and corn, and promote the germination of corn seeds and the growth of arabidopsis, and under the conditions of salt stress, drought and low temperature, the PGPR can more effectively promote the normal growth and development of plants and improve the capability of the plants in adapting to adverse environments. Therefore, PGPR has wide application prospect in solving some problems in crop production and promoting sustainable development of agriculture and animal husbandry.

The Bacillus subtilis is widely distributed in nature, is one of the dominant strains of PGPR, has the effects of antagonizing pathogenic bacteria and promoting plant growth, and has the functions of producing bacteriostatic metabolic products such as siderophores, antibiotics, extracellular hydrolase, HCN and the like, and effectively protecting plant roots from being invaded by pathogenic microorganisms. Therefore, the bacillus subtilis can be used as an excellent strain for preparing the composite microbial fertilizer.

In conclusion, in order to better improve the saline-alkali soil resistance of plants and guarantee the development of agricultural production, the research and development of the compound microbial fertilizer become one of the current innovative methods. In the research, rhizosphere growth-promoting bacteria bacillus subtilis WM13-24 is separated from the rhizosphere of the haloxylon and is inoculated to ryegrass, and the rhizosphere growth-promoting bacteria bacillus WM13-24 is found to have a good effect of improving the salt tolerance of the ryegrass.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a method for promoting the salt tolerance of ryegrass, which can relieve the damage of plants caused by salt stress by inoculating bacillus subtilis WM13-24 into saline-alkali soil.

In order to achieve the purpose, the invention provides the following technical scheme: a method for promoting salt tolerance of ryegrass specifically comprises the following steps:

s1, sterilizing ryegrass seeds: soaking rye grass seed in 70% ethanol (10min) for sterilization, continuously sterilizing with 2% NaClO (1min), washing with distilled water for 7-8 times (3 min each time), and cleaning with ethanol and sodium hypochlorite;

preparing S2, bacillus subtilis WM13-24 and escherichia coli DH5 alpha bacterial liquid;

s2.1, firstly preparing an LB liquid culture medium: sodium chloride (10g/L), tryptone (10g/L) and yeast powder (5g/L), pH 7.0;

s2.2, sterilizing the culture medium at the high temperature of 121 ℃ for 20 minutes;

s2.3, after the temperature is cooled to room temperature, inoculating bacillus subtilis WM13-24 and escherichia coli DH5 alpha bacteria liquid into LB liquid culture medium with the inoculation amount of 1%, and respectively inoculating the bacillus subtilis WM13-24 and the escherichia coli DH5 alpha bacteria liquid into the culture medium at 28 ℃ and 37 ℃ in a constant temperature shaking table 180rmin^-1Culturing in dark for 16h, and determining the bacterial liquid concentration as OD₆₀₀When the value is 0.9, taking out for standby;

s3, planting by adopting a pot experiment;

s3.1, sowing (1.0 g/pot) the sterilized ryegrass seeds in a flowerpot (the diameter is 20cm, the depth is 15cm) filled with vermiculite and sand (1: 1), respectively inoculating fresh bacillus subtilis WM13-24, escherichia coli DH5 alpha and LB culture media of 600 mul respectively around the stems of seedlings after the seedlings are exposed to soil, and planting 18 pots in each treatment way;

s3.2, growth chamber conditions: the temperature is 28 ℃/23 ℃ (day/night), the relative humidity is 70%, and the light cycle is 16h/8h (day/night);

s3.3, after the inoculated ryegrass seedlings grow for one week, performing salt treatment on the seedlings, and adding 0, 150 and 300mM NaCl into 1/2Hoagland nutrient solution respectively to perform salt treatment;

s3.4, sampling and measuring related physiological indexes after the seedlings under the salt treatment grow for two weeks.

The method of claim 1, wherein the salt tolerance of ryegrass is improved by: in step S1, the rye grass seeds are soaked in 70% ethanol for 10min, and then sterilized with 2% NaClO for 1 min.

In a preferred embodiment, in the step S2.3, 100. mu.L of Bacillus subtilis WM13-24 bacterial liquid is added to 100mL of LB liquid medium.

The method of claim 1, wherein the salt tolerance of ryegrass is improved by: in step S2.3, the culture medium is cultured overnight on a constant temperature shaking table with the rotation speed of 180-.

The method of claim 1, wherein the salt tolerance of ryegrass is improved by: in the step S2, the bacillus subtilis WM13-24 strain is separated from the root of the desert plant haloxylon ammodendron in the desert of Mintallugegy in Gansu.

The method of claim 1, wherein the salt tolerance of ryegrass is improved by: in the step S3.1, the volume ratio of the fine sand to the vermiculite is 1: 1.

the invention has the technical effects and advantages that:

1. according to the method, the bacillus subtilis WM13-24 separated from the root of the haloxylon ammodendron in the desert plant is inoculated to the salt-treated ryegrass seedlings, so that the damage caused by the salt stress of the ryegrass can be relieved, and the biomass and the salt tolerance of the ryegrass are improved;

2. the invention relates to a bacillus subtilis strain WM13-24 separated from the rhizosphere of haloxylon, which is inoculated on the rhizosphere of a forage grass ornamental plant ryegrass seedling, the change of related physiological indexes of the ryegrass seedling under salt stress is observed, and a blank LB liquid medium with the same inoculation volume and an Escherichia coli DH5 alpha bacteria liquid are used as a control. It was found that under salt stress, the chlorophyll content, root length and root volume of Secale cereale and K⁺/Na⁺The content gradually decreased with increasing salt concentration, but the physiological index associated with ryegrass seedlings inoculated with WM13-24 was significantly higher than that of the two controls; the improvement of the salt tolerance of the ryegrass seedlings is benefited by inoculating the haloxylon rhizosphere bacillus subtilis WM13-24, so that the damage of salt stress to the ryegrass seedlings is relieved, the biomass of the ryegrass seedlings is improved, and the plants grow better.

Drawings

FIG. 1 shows the results of the present invention under different salt stresses (from top to bottom: 0, 150 and 300 mmol. multidot.L, respectively)^-1NaCl), inoculated with Bacillus subtilis WM13-24, Escherichia coli DH5 alpha and blank LB culture medium, and grown for two weeks.

FIG. 2 is a graph showing the effect of Bacillus subtilis WM13-24 on the increase of the fresh and dry weight of rye grass leaves.

FIG. 3 is a graph showing the effect of Bacillus subtilis WM13-24 on the increase of chlorophyll content in Secale cereale L.

FIG. 4 is a graph showing the effect of Bacillus subtilis WM13-24 on the root length and root volume improvement of ryegrass.

FIG. 5 shows the bacillus subtilis WM13-24 of the present invention for Lolium perenne K⁺/Na⁺The effect graph of content promotion.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.