CN111670769A

CN111670769A - Method for improving stress resistance of rice

Info

Publication number: CN111670769A
Application number: CN202010329661.4A
Authority: CN
Inventors: 朱咏华; 牛舒琪; 刘选明; 姚晴晴; 高艳; 资慧娴; 喻金兰
Original assignee: Hunan University
Current assignee: Hunan Xinchangshan Agricultural Development Co ltd
Priority date: 2020-04-23
Filing date: 2020-04-23
Publication date: 2020-09-18
Anticipated expiration: 2040-04-23
Also published as: CN111670769B

Abstract

The invention discloses a method for improving stress resistance (salt and drought) of rice, which adopts rice endophytic actinomycetesStreptomyces hydrogenansOsiLf-2, embedding the sterilized rice seeds with the spore suspension or spraying the spore suspension on the surfaces of rice leaves. Under the salt stress or drought stress, the method can effectively improve the salt-tolerant and drought-resistant capabilities of the rice, can obviously improve the biomass and the relative water content of rice seedlings, obviously promotes the root system development and finally improves the rice yield. Meanwhile, the strain is separated from rice, can form a mutual and beneficial symbiotic relationship with a host, and has the characteristics of no toxicity and harm to people and livestock, no environmental pollution, non-pathogenicity, strong stress resistance and the like. The method of the invention is simpleThe method can be widely applied to rice planting on the lands of mild and moderate saline-alkali soil and drought conditions, and has important economic, social and ecological values.

Description

Method for improving stress resistance of rice

Technical Field

The invention relates to the field of crop planting, in particular to a rice endophytic actinomycete and a method for improving stress resistance of rice by using the same.

Background

Rice is one of the main grain crops in the world, and the yield of the rice directly influences the world grain safety. Due to the complex and diverse environment, rice planting is under the threat of various abiotic adversities, and the problem of how to solve the drought and saline-alkali soil of rice and ensure rice production becomes a major challenge for all agricultural workers in this century. At present, the traditional genetic breeding method is adopted to cultivate a new salt-tolerant drought-resistant rice variety safely and efficiently, but the progress is slow; a new rice variety with salt and drought resistance is cultivated by adopting a genetic engineering breeding technology, but the stress-resistant molecular regulation network of the rice is complex, and related genes are difficult to excavate and take long time; the microbial improvement method can improve the ecological environment of plants, has the characteristics of good effect, strong persistence and environmental friendliness, and is receiving more and more attention. For example, Banaeiasl et al apply Pseudomonas fluorescens to rape roots, thereby increasing tolerance of rape to salt stress; the roots of the arabidopsis seedlings are soaked in the Sukweenadhi and the like by applying the Paenibacillus yongninensis DCY84 bacterial liquid, so that the drought resistance, aluminum stress resistance and salt stress resistance of the arabidopsis are improved; inoculation of Bacillus amyloliquefaciens SN13 to rice by Nautiyal et al can promote the growth of rice and improve the salt tolerance of rice.

Endophytes refer to those microorganisms that live within various tissues and organs of plants at some or all of their life history, and infected host plants (or temporarily) do not exhibit extrinsic conditions. Because the endophyte lives in the plant tissue for a long time, the endophyte can co-evolve with the plant to be more harmoniously and reciprocally symbiotic, such as promoting the growth of the plant and improving the stress resistance of the plant. For example, Win et al reported that endophytic bacteria (Pseudomonas spp.) were effective in ameliorating the adverse effects of salt stress on tomato seedlings; saravanakumar et al adopt plant endophytic bacterium Pseudomonas fluorescens Pf1 to inoculate mung beans, so that mung beans can better resist water stress; studies of Zhang wenying and the like find that endophytic fungi (Piriformospora indica) can induce and improve the drought resistance of corn, and also that Sun and the like also find that the endophytic fungi can improve the drought stress tolerance of a host plant Chinese cabbage; khan et al reported that the application of endophytic fungus (Penicillium respexanum LK6) with exogenous SA could alleviate the adverse effects of osmotic stress on pepper plants; nishimura and the like use endophytic actinomycetes Streptomyces AOK-30 to ensure that tissue culture seedlings of the laurel have the tolerance to high-concentration salt; yandigeri and other researches find that some drought-tolerant endophytic actinomycetes (Streptomyces sp) can promote wheat to resist water stress to a certain degree; jaemsaeng et al demonstrated that endogenetic actinomycete Streptomyces sp.GMKU336 can promote the growth of rice and improve its salt tolerance. Because of the limitations in techniques and methods for isolating endophytic actinomycetes and culture conditions, most endophytic actinomycetes in nature still have not been purely cultured, and thus reports of endophytic actinomycetes are still less compared with plant endophytic bacteria and fungi. The endophytic actinomycetes have rich metabolites and various interaction modes with plants, the research on the rice endophytic actinomycetes at present mainly focuses on the aspects of host disease resistance assisting and growth promoting performance, various active substances generated by the rice endophytic actinomycetes and the like, few reports about the stress resistance of the rice endophytic actinomycetes to hosts are provided, and the application of the rice endophytic actinomycetes to production is less.

At present, related patents about the application of microbial improvement methods to improve and help plant stress resistance mostly focus on the production of biofertilizer and the excavation of microbial stress resistance genes, and the application of few endogenous actinomycetes is applied. For example, invar new patent (CN201110274941.0) about a bio-organic fertilizer for improving stress resistance and yield of rice and a preparation method thereof applies 5 beneficial bacteria such as bacillus subtilis, bacillus licheniformis, saccharomyces cerevisiae and the like, and the prepared bio-organic fertilizer can promote growth and fructification of crops and remarkably improve stress resistance and yield of the crops; zhangpeng et al, a patent of application of psychrophilic bacteria pseudoaltermonasp.S-5 and its fermentation product in plant stress resistance (CN200910256034.6), wherein the injury of freezing injury to crops is reduced by spraying the microbial fermentation product on the surfaces of crops; a patent for increasing the salt tolerance of plants is applied for a stage of stress resistance gene of the stenotrophomonas maltophilia by using Lumenzhu and the like (CN 201310086561.3); a patent (CN201110197581.9) about improving disease resistance and stress resistance of rice endogenetic azotobacter of Zhongjianhao et al uses a strain of rice endogenetic azotobacter-Burkholderia which has ACC deaminase activity and can effectively antagonize sclerotinia sclerotiorum.

Disclosure of Invention

In order to solve the influence of environmental stress on rice production, the invention provides a green and environment-friendly method for improving the stress resistance of rice and maintaining the growth and yield of the rice under the condition of drought or salt stress, which adopts the following technical scheme:

the method uses a strain of rice endophytic actinomycetes Streptomyces hygiens OsiLf-2 which is preserved in China general microbiological culture Collection center (CGMCC for short) in 11-16 th 2015 in 16 th month (the preservation address is No. 3 Siro. 1 of Beijing Kogyo north Chen), and the preservation number is CGMCC No. 11673.

The strain is found to be a rice endophytic actinomycete capable of efficiently antagonizing rice blast germs in earlier stage research, and the strain is further found to have the effect of improving the stress resistance of rice, so that the invention is completed.

The invention provides a method for improving stress resistance of rice, which adopts the endophytic actinomycete Streptomyces hydrogenens OsiLf-2, and adopts rice seeds embedded and disinfected by spore suspension and/or sprays the spore suspension on the surface of rice leaves.

The method is a method for improving the drought resistance or salt stress resistance of the rice, improves the rice tolerance, keeps the growth and maintains the yield under the condition of drought or salt stress.

The method, the final concentration of the spore suspension is 1 × 10⁷～1×10⁹CFU/mL, preferably at a final concentration of 1 × 10⁸CFU/mL。

The method comprises (1) embedding sterilized rice seeds in spore suspension at a ratio of seed/0.3% (v/v) xanthan gum/spore liquid of 1g/463 μ l/51 μ l to rice seedsThe spore suspension of the seed mixture is preferably at a final concentration of 1 × 10⁸CFU/mL, (2) spraying spore suspension (with the preferred concentration of 1 × 10) on the surface of rice leaf⁸CFU/mL), 0.2% (v/v) of Tween20 is added as a surfactant, the dosage is based on the condition that the whole rice leaf surface is contacted with the spore suspension, generally, 5 mL/rice is sprayed in the evening for the rice just entering the tillering stage, and the spraying is carried out for 1 time every two weeks.

The method is a method for promoting stress resistance of rice and keeping growth of the rice under the conditions of drought and salt stress, and the method is to embed and sterilize preferred rice seeds by using a spore suspension, wherein the using ratio of the spore suspension to seeds/0.3% (v/v) xanthan gum/spore liquid is 1g/463 mu l/51 mu l.

The endophytic actinomycete Streptomyces maritlf-2 used by the invention is prepared into spore suspension by the following method:

(1) inoculating the strain to a PDA solid culture medium, and standing and culturing for 4 days in a constant-temperature incubator at 30 ℃;

(2) collecting the thalli matured in the step (1) by using an inoculating loop, transferring the thalli to a triangular flask containing sterile distilled water and glass beads, violently shaking the triangular flask to break up spores and mycelia, and filtering by using four layers of gauze to obtain a spore suspension;

(3) counting the spore suspension obtained in the step (2) by using a blood counting plate, and adjusting the concentration to be 1 × 10⁷～1×10⁹CFU/mL。

The invention utilizes a final concentration of 1 × 10⁷～1×10⁹CFU/mL (preferably 1 × 10)⁸CFU/mL) of Streptomyces grilf-2 spores, and the rice is treated by two different modes to relieve the damage of salt stress or drought stress to the rice, thereby maintaining the growth purpose:

(1) embedding sterilized rice seeds in a spore suspension, wherein the use ratio of the seeds to 0.3% (v/v) xanthan gum to spore liquid is 1g/463 mu l/51 mu l;

(2) spraying spore suspension on the surface of rice leaves, wherein 0.2% (v/v) Tween20 is added as a surfactant to improve the adhesion capacity on the rice, the dosage is based on the condition that the whole rice leaf surface is contacted with the spore suspension, for example, for the rice which just enters the tillering stage, 5mL of the spore suspension is sprayed per rice plant at the evening, and the spraying is carried out for 1 time every two weeks.

Compared with the existing method for relieving the damage of adversity stress to rice and ensuring the growth of plants, the method has the following obvious advantages:

1. the rice endophytic actinomycetes Streptomyces hygienics OsiLf-2 can generate rich active substances, for example, the generated plant growth regulating substances can effectively promote the growth and development of rice, and compared with chemical fertilizers, the rice endophytic actinomycetes Streptomyces hygienics OsiLf-2 is more stable, safer and more environment-friendly, and ensures the production and eating safety of rice under adversity stress.

2. The method for improving the stress resistance of the rice comprises the steps of embedding rice seeds by using a strain spore suspension, or spraying the strain spore suspension on the surfaces of leaves in the seedling stage of the rice, wherein the strain can not damage the growth and development of plants when entering the rice for colonization, can effectively enhance the stress resistance of the rice, and ensures the growth and development of a host, thereby reducing the yield reduction loss of the rice caused by drought or salinization.

3. The endophyte used in the invention is separated from the rice body, and is safer for the environment compared with a method for dealing with drought and salinization environment problems by physical chemistry or a method for obtaining the stress-resistant plant by modifying in a gene engineering mode, and does not influence the agricultural ecological environment and the human body health.

4. The treatment of the endophytic actinomycetes is convenient, simple and diversified, and compared with other environmental management methods, the method is time-saving, trouble-saving, free from the limitation of regions and manpower, and suitable for wide popularization.

The endophytic actinomycetes used by the invention can be colonized in the rice body, and the salt-tolerant and drought-resistant capability of the rice is effectively improved. Under the salt stress or drought stress, the method can obviously improve the biomass, the relative water content and the plant height of rice seedlings, obviously promote the root system development and finally improve the rice yield. Meanwhile, the strain is separated from rice, can form a mutual and beneficial symbiotic relationship with a host, and has the characteristics of no toxicity and harm to people and livestock, no environmental pollution, non-pathogenicity, strong stress resistance and the like. The method is simple, can be widely applied to rice planting on the slight and medium saline-alkali soil and the arid land, and has important economic, social and ecological values. The method of the invention is to embed the rice seeds with the spore suspension of the endophytic actinomycetes or spray the spore suspension on the rice leaves, and the processing method is simple and convenient to operate and has the effect of lasting the whole rice growth period. In conclusion, the method for improving the stress resistance of the host by using the Streptomyces hygrogenans OsiLf-2 as the rice endophytic actinomycetes belongs to innovative research and has a great application prospect.

Drawings

FIG. 1: effect of embedding Streptomyces hydrogenes OsiLf-2 spore suspension on growth of rice seedlings under drought and salt stress conditions (E-: untreated rice, E +: OsiLf-2 treated rice).

FIG. 2: effect of embedding Streptomyces hygrogens OsiLf-2 spore suspension on root growth of rice seedlings under drought and salt stress conditions (E-: untreated rice, E +: OsiLf-2 treated rice).

FIG. 3: the influence of Streptomyces hydrogenes OsiLf-2 spore suspension on the growth of rice under drought and salt stress conditions (E-: untreated rice, E +: OsiLf-2 treated rice; after OsiLf-2 leaves are sprayed, the growth condition of rice in the booting stage after natural drought and 1% NaCl stress for 2 weeks; the influence of effective ear length and ear bearing of rice).

Detailed Description

The technical solution of the invention is further illustrated below with reference to specific examples, which are not to be construed as limiting the technical solution.

Example 1: embedding and applying spore suspension of Streptomyces hydrogenens OsiLf-2 to rice seeds to improve stress resistance of rice

The planting mode is as follows: the rice is planted in a greenhouse constant-temperature illumination incubator, the photoperiod is 16 hours of illumination/8 hours of darkness, the culture temperature is 28-30 ℃, and the air humidity is 60-70%.

And (3) test crops: rice (indica rice 93-11)

Experimental treatment: according to the above methodPreparing spore suspension of Streptomyces hygrogenans OsiLf-2, embedding sterilized rice seeds at ratio of rice seeds/0.3% (v/v) xanthan gum/spore suspension of 1g/463 μ l/51 μ l to obtain final spore concentration of 1 × 10⁸CFU/mL, rice seed/0.3% (v/v) xanthan gum/sterile water ratio of 1 g/463. mu.l/51. mu.l, as a control. Each treatment was repeated in 3 groups, each group comprising 48 rice seedlings. The specific method for preparing the spore suspension is as follows:

(1) inoculating the strain on PDA solid culture medium, and standing and culturing in 30 deg.C constant temperature incubator for 4 days;

(2) collecting the thallus matured in the step (1) by using an inoculating loop, transferring the thallus into a triangular flask containing sterile distilled water and glass beads, and filtering after violently shaking to obtain a spore suspension;

(3) counting the spore suspension obtained in the step (2) by using a blood counting plate to adjust the concentration of the spore suspension

1×10⁷～1×10⁹CFU/mL。

Observing the growth condition of rice after germination, and culturing in water culture nutrient solution (0.3mM KH)₂PO₄，0.35mMK₂SO₄，1mM MgSO₄.7H₂O，0.5mM Na₂SiO₃.9H₂O，1mM CaCl₂.2H₂O，9μM MnCl₂.4H₂O，20μM H₃BO₃，0.77μM ZnSO₄.7H₂O，0.32μM CuSO₄.5H₂O，20μM NaFeEDTA，0.39μM Na₂MoO₄.2H₂O, pH 5.8) were cultured in an incubator, and the hydroponic medium was changed every 2 days. The specific culture conditions are as follows: fluorescent lamp illumination (300-^-2·s^-1) (ii) a Long day conditions (16h light/8 h dark); the temperature is 30 ℃; the relative humidity is 60-70%. At 2 weeks of seedling growth, the following treatment groups were set: e- (untreated rice), E + (OsiLf-2 treated rice), (E-) + 20% PEG6000, (E +) + 20% PEG6000, (E-) +150mmol NaCl, (E +) +150mmol NaCl. Treatment with 20% PEG6000 for 7 days and 150mM NaCl for 10 days, followed by 7 days of recovery, respectively. And measuring the relevant physiological indexes.

And (3) effect statistics: and (4) counting the biomass, the relative water content, the plant height and the root form indexes of the seedlings after drought stress treatment and salt stress treatment respectively.

Results of the experiments are shown in Table 1 and FIG. 1. spore suspension (final concentration: 1 × 10) of Streptomyces hydrogenes OsiLf-2, which is an endophytic actinomycete strain of rice of this example 1, was seed-embedded⁸CFU/mL), the growth condition of the rice can be obviously improved compared with that of a control group under the condition of salt stress or simulated drought stress.

TABLE 1 Effect of embedding Streptomyces hygrogenans OsiLf-2 spore suspension on Rice seedling growth under drought and salt stress conditions

(10 replicates per treatment different letters indicate significant differences between differently treated seedlings (P <0.05))

The rice seeds are embedded with Streptomyces hydrogenogens OsiLf-2 spore suspension, the suspension is cultured for 2 weeks under normal conditions, 20% of PEG6000 simulated drought and 150mM NaCl salt stress treatment are respectively carried out, the drought stress lasts for 24 hours, and the fresh weight of the upper part of the E + group rice seedling, the fresh weight of the underground part, the dry weight of the upper part of the ground and the dry weight of the underground part are respectively and obviously increased by 47.78%, 47.58%, 58.39% and 43.16% compared with the E-group rice; the fresh weight of the overground part, the fresh weight of the underground part, the dry weight of the overground part and the dry weight of the underground part of the E + group rice seedlings are obviously improved by 45.57%, 58.60%, 52.89% and 68.06% respectively compared with those of E-group rice seedlings after salt stress is carried out for 24 hours. Under the conditions of drought and salt stress, the relative water content of the E-group rice is respectively reduced by 4.5 percent and 6.07 percent, while the relative water content of the E + group rice is respectively reduced by only 2.01 percent and 4.00 percent.

TABLE 2 influence of Streptomyces hygrogenans OsiLf-2 spore suspension on root growth of rice seedlings under drought and salt stress conditions

Under drought stress, the root system development of the E + group rice seedlings is superior to that of the E-control group (table 2), and the total root length, the root projection area, the root surface area, the root volume and the root branch number of the seedlings are respectively and obviously improved by 35.84%, 31.97%, 30.51%, 15.38% and 42.25% compared with those of the E-group seedlings when the seedlings are stressed for 7 days; under the condition of salt stress, the root system development of the E + group rice seedlings is superior to that of the E-control group, and the total root length, the root projection area, the root surface area, the root volume and the root branch number of the seedlings are obviously improved by 81.76%, 60.93%, 62.39%, 66.67% and 109.09% respectively compared with those of the E-control group seedlings when the E + group rice seedlings are stressed for 7 days. The method is shown to relieve the damage of the stress on the rice and ensure the growth of the rice (see figure 1 and figure 2).

Example 2: application of Streptomyces hydrogenens OsiLf-2 in greenhouse experiments to improve stress resistance of rice

And (3) test crops: rice (indica 93-11).

The planting mode is as follows: rice was grown in greenhouse PVP pots at the university of hunan.

Test methods A spore suspension of Streptomyces hydrogens OsiLf-2 was prepared according to the method of example 1 to a final concentration of 1 × 10⁸CFU/mL, adding 0.2% Tween20 for treatment; water containing 0.2% Tween20 was used as a control.

Each pot was planted with 3 seedlings of rice of similar growth, which was repeated 10 pots per treatment. And spraying for treating the rice at the evening when the rice just enters the tillering stage, and spraying for 1 time after 2 weeks. The dosage of each spraying is 5mL per rice plant. And (3) stopping watering when the rice grows to the booting stage to be used as drought stress, treating for 14 days, irrigating with 1% NaCl solution to be used as salt stress, and treating for 14 days. After the stress treatment, normal watering was resumed.

And (3) test results: the spore suspension of Streptomyces hydrogenens OsiLf-2 is sprayed on the leaf surfaces of rice, and the growth condition of the rice can be obviously improved under the adversity stress compared with that of untreated control. Under drought stress, compared with a control group, the spike length, the single spike seed setting rate and the net photosynthetic rate are respectively and obviously improved by 13.80 percent, 8.62 percent and 12.53 percent; under the salt stress, compared with a control group, the spike length, the single spike seed setting rate and the net photosynthetic rate are respectively and obviously improved by 9.38 percent, 12.90 percent and 35.15 percent (see figure 3).

TABLE 3 influence of Streptomyces hygrogenans OsiLf-2 spore suspension on drought and salt stress during rice reproductive growth

(10 replicates per treatment different letters indicate significant differences between treated seedlings (P <0.05)

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for improving stress resistance of rice is characterized by comprising the following steps: the method adopts a rice endophytic actinomyceteStreptomyces hydrogenansOsiLf-2, embedding the sterilized rice seeds with the spore suspension or spraying the spore suspension on the surfaces of rice leaves.

2. The method of claim 1, wherein: the rice endophytic actinomycetesStreptomyces hydrogenansOsiLf-2, the preservation number is CGMCC number 11673.

3. The method of claim 1, wherein the spore suspension has a concentration of 1 × 10⁷~1×10⁹CFU/mL。

4. The method of claim 3, wherein the spore suspension has a concentration of 1 × 10⁸CFU/mL。

5. The method according to any one of claims 1-4, wherein: the method for improving the stress resistance of the rice is to improve the drought resistance or salt stress resistance of the rice, improve the tolerance of the rice under the condition of drought or salt stress and promote the growth of the rice.

6. The method of claim 5, wherein:

the rice seeds after spore suspension embedding and disinfection are added with xanthan gum as an adhesive to improve the adhesive capacity on the rice seeds, and the preferable proportion is as follows: seeds/0.3% (v/v) xanthan gum/spore liquid = 1g/463 μ l/51 μ l, and after the treatment, the seeds were blow-dried for pregermination.

7. The method of claim 5, wherein: spraying spore suspension on the surface of rice leaves, wherein 0.2% (v/v) Tween20 is added as a surfactant, and the dosage is based on the condition that the whole rice leaf surfaces are contacted with the spore suspension.

8. The method of claim 7, wherein: for rice just entering the tillering stage, the dose was 5 mL/rice plant/time.

9. The method of claim 8, wherein: spraying leaves in the evening, and spraying for 1 time every two weeks for 2 times.