CN111466266B - Method for promoting rice growth and iron element absorption in rice iron-deficient environment - Google Patents

Abstract

The invention discloses a method that can promote the growth of rice in a normal growth environment and can promote the absorption of iron by rice in an environment lacking directly available iron. The method utilizes a rice endophytic actinomycete Streptomyces sp .OsiSh-2, using its spore suspension, the rice under normal circumstances can be treated with two different means: 1) spray the spore suspension on the surface of the rice leaf; 2) after its spore suspension is embedded and disinfected rice seeds. The method of the invention promotes the growth of rice, improves chlorophyll content and photosynthesis rate, and significantly increases rice biomass such as plant height, root length, fresh and dry weight of above-ground and underground parts; By burying the sterilized rice seeds, the iron content in the rice is significantly increased, which ensures the normal growth of the rice under iron deficiency stress.

Description

A method for promoting rice growth and promoting iron absorption in rice iron-deficient environment

technical field

The invention belongs to the technical field of biological fertilizers, and in particular relates to a method of utilizing a rice endophytic actinomycete to promote the growth of rice under normal growth conditions and to promote the absorption of iron by rice in an environment lacking directly available iron, so as to maintain the growth of rice. method.

Background technique

Rice is an important food crop in my country and even in the world, and its yield and quality are closely related to human health. At present, the means of promoting rice growth mainly include spraying chemical or biological fertilizers. With the formulation of the zero-growth strategy of chemical fertilizers in my country, more and more attention has been paid to the research on environmentally friendly biological fertilizers. For example, the Rhodopseudomonas swampis PSB06 inoculant in photosynthetic bacteria reported by Zeng Yibo et al. can promote the growth of rice seedling stage; the biofertilizer based on Trichoderma spp. reported by Doni Febri et al. can increase rice yield by 30%; Streptomyces sp. A20 reported by Suárez-Moreno Zulma Rocío et al. can promote rice growth.

Endophytes are a class of microorganisms that can stably colonize inside plant tissues without causing obvious plant diseases. Since endophytes live in plant tissues for a long time, they can co-evolve with plants, and the substances they secrete can be quickly recognized and utilized by plants to form a more coordinated mutualistic relationship. For example, endophytes can produce a series of growth-related Plant growth regulators such as IAA, ACC deaminase and siderophores promote the growth of the host. At the same time, the unique ecological niche of endophytes in plants has a survival advantage over rhizosphere microorganisms that are susceptible to environmental influence. Compared with fungi and bacteria, actinomycetes can produce abundant secondary metabolites, and their growth-promoting potential is better than other microorganisms under the same conditions. Based on this, endophytic actinomycetes are suitable for promotion as biological fertilizers. For example, Patel Janki K. et al. reported some endophytic actinomycetes Streptomyces spp., by adding actinomycete spore suspension to the rice hydroponic solution, the growth of the aerial part of rice can be promoted; Wang Wenfeng et al. Actinomycetes Streptomyces sp. A9 can promote the growth of rice seedlings by soaking rice seeds with spore liquid; in addition, Liotti Rhavena Graziela et al. It can promote the growth of the aerial part of pepper; Streptomyces spp. LuP30 and LuP47B reported by Le Xuyen H. et al. can increase the dry weight of the aerial part of alfalfa by embedding actinomycete spores in seeds; Ge Youyou et al. The nematode Streptomyces sp. SSD49 spore solution was directly irrigated in the soil where the plants were grown to promote the growth of soybean, sorghum and pepper. However, it is difficult to see the patents of biological fertilizers based on actinomycetes, especially endogenous defense bacteria. Different from the commonly reported methods, the method of the present invention is to treat the rice by embedding seeds or spraying spore liquid on the seedlings to ensure that the endophyte and the rice form a mutually beneficial symbiotic state, and finally promote the growth of the rice. .

Iron is an essential nutrient for all organisms, participates in a variety of biochemical reaction processes, and has important physiological and biochemical functions. Although the total content of iron in the soil is very rich, the effective content of iron in the soil is very low, especially in neutral and calcareous soils, where it mostly exists in the form of trivalent cations with extremely low solubility and is difficult to be absorbed and utilized by plants. Iron deficiency can lead to reduced chlorophyll synthesis in crops, reduced photosynthetic rate, and reduced biomass, which in turn will cause insufficient iron intake by animals and humans. At present, the treatment of iron deficiency in plants is still based on chemical fertilizers as the main means. The use of microorganisms to enhance the ability of plants to cope with iron deficiency stress has become an urgently needed new bioremediation technology. At present, there are few reports on endophytes enhancing the tolerance of plants to iron deficiency stress. Only Streptomyces sp. GMKU 3100 has been reported to point out that its siderophore can help plants maintain normal iron deficiency in an iron-deficient environment. grow. Different from the treatment method of soaking the roots of rice seedlings in the actinomycete spore suspension adopted in this report, the method of the present invention is to embed the rice seeds with the spore suspension of endophyte fungi, and this treatment method is beneficial to Rice is in contact with endophytes from the time of seed germination, so that rice can receive the beneficial effects of endophytes on its growth from the beginning of seed germination.

SUMMARY OF THE INVENTION

In order to solve the rice production demand existing in the prior art, the present invention provides a method for promoting the growth of rice and promoting the absorption of iron in the iron-deficient environment of rice. The present invention adopts the following technical solutions:

The method of the present invention uses a rice endophytic actinomycetes Streptomyces sp. OsiSh-2, which is preserved in the General Microorganism Center of the China Microorganism Culture Collection Management Committee (CGMCC for short), and the preservation number is CGMCC No.8716.

The present invention provides a method for promoting the growth of rice or/and promoting the absorption of iron in the iron-deficient environment of rice. The method adopts the above-mentioned endophytic actinomycetes Streptomyces sp. OsiSh-2, and sprays its spore suspension on the surface of rice leaves. The sterilized rice seeds are embedded in liquid or its spore suspension.

In the method, the concentration of the spore suspension is 1×10 ⁷ to 1×10 ⁹ CFU/mL, and the preferred concentration is 1×10 ⁸ CFU/mL.

The above-mentioned method, the method is a method for promoting the growth of rice, and this method sprays the rice endophytic actinomycete Streptomyces sp . 2. The sterilized rice seeds are embedded in the spore suspension, or the sterilized rice seeds are first embedded in the spore suspension of Streptomyces sp. OsiSh-2, and then the surface of the rice leaves is sprayed with rice endophytic actinomycetes. Streptomyces sp. OsiSh-2 spore suspension.

According to the method, (1) spraying the spore suspension on the surface of the rice leaves, wherein 0.2% (v/v) of Tween 20 is added as a surfactant, and the dosage is used to meet the requirement that the entire rice leaf surface is contacted with the spore suspension. Generally, for rice in the three-leaf stage, it is 5mL/plant; (2) The rice seeds after embedding and sterilization in spore suspension are used in the ratio of seeds/0.3% (v/v) xanthan gum/spore solution =1g / 463 μl / 51 μl.

The above-mentioned method, the method is to promote the absorption of iron in the iron-deficient environment of rice, and the method uses the spore suspension to embed the sterilized rice seeds, preferably, the spore suspension is used in a ratio of seed/0.3% (v/ v) Xanthan gum/spore solution = 1 g / 463 μl / 51 μl.

The present invention prepares the used endophytic actinomycetes Streptomyces sp. OsiSh-2 into its spore suspension, and the method is as follows:

(1) The strains were inoculated on ISP-2 solid medium and cultured in a constant temperature incubator at 30°C for 14 days.

(2) Collect the mature cells in step 1) with an inoculating loop, transfer them to a conical flask containing sterile distilled water and glass beads, shake vigorously and filter to obtain a spore suspension.

(3) Count the spore suspension obtained in step (2) with a hemocytometer to adjust its concentration to be 1×10 ⁷ -1×10 ⁹ CFU/mL.

The spore suspension of Streptomyces sp. OsiSh-2 with a concentration of 1×10 ⁸ CFU/mL used in the present invention grows under normal conditions, and can treat rice in two different ways to achieve the purpose of promoting growth:

(1) Spray the spore suspension on the surface of rice leaves, in which 0.2% (v/v) Tween 20 is added as a surfactant to improve the adhesion on rice, and the dose is used to meet the spores on the entire rice leaf surface. The suspension shall prevail, for example, for rice at the three-leaf stage, it is 5mL/plant of rice;

(2) Embed the sterilized rice seeds in a spore suspension, using a ratio of seeds/0.3% (v/v) xanthan gum/spore fluid = 1 g / 463 μl / 51 μl.

Growing under iron-deficiency conditions, rice can be treated in one way to promote iron absorption: the sterilized rice seeds are embedded in spore suspension, using a ratio of seeds/0.3% (v/v) xanthan gum/ Spore fluid = 1 g / 463 μl / 51 μl.

Compared with the existing methods for promoting the growth of rice and promoting the absorption of iron by rice in an iron-deficiency stress environment, the present invention has the following obvious advantages:

1. The rice endophytic actinomycetes Streptomyces sp. OsiSh-2 can produce abundant active substances, such as siderophores and plant growth regulators, which can effectively promote the growth and development of rice, making rice stable and high yield; using its own secretion of iron reductase, Phenolic substances and siderophores can promote plants to absorb iron elements that are difficult to use in the environment, increase the iron content in plants, and increase the nutritional value of rice.

2. The method is to encapsulate the rice seeds with endophytes when the rice is still in the seed stage, or spray the rice in the seedling stage. It will cause rice diseases, and ensure that it can play its growth-promoting and iron-absorbing functions from the early stage of rice growth.

3. Rice endophytes are isolated from the body of rice, which is safer for the environment than chemical fertilizers and will not affect the agricultural ecological environment and human health.

4. The treatment of rice endophytes is more convenient, concise and diversified. Compared with other land management methods, it saves time and trouble, and is not limited by geographical and manpower, so it is suitable for widespread promotion.

Description of drawings

Figure 1: Growth of rice after foliar spraying of Streptomyces sp. OsiSh-2 spore suspension under normal growth conditions;

Figure 2: Growth of rice embedded with Streptomyces sp. OsiSh-2 spore suspension under normal conditions;

Figure 3: Growth of rice embedded with Streptomyces sp. OsiSh-2 spore suspension under iron-deficiency stress.

Detailed ways

The technical solutions of the present invention are further described below in conjunction with specific embodiments.

Example 1: Under normal circumstances, foliar spraying of Streptomyces sp. OsiSh-2 promotes the growth of rice

Experimental site: greenhouse soil culture

Test crop: Rice (Indica 93-11).

Experimental treatment group: Streptomyces sp. OsiSh-2 spore suspension was prepared at a concentration of 1×10 ⁸ CFUmL ^-1 , and 0.2% Tween 20 was added for treatment; water containing 0.2% Tween 20 was used as a control. The specific method for preparing the spore suspension is as follows:

(1) The strains were inoculated on ISP-2 solid medium and cultured in a constant temperature incubator at 30°C for 14 days.

(2) Collect the mature cells in step 1) with an inoculating loop, transfer them to a conical flask containing sterile distilled water and glass beads, shake vigorously and filter to obtain a spore suspension.

(3) Count the spore suspension obtained in step (2) with a hemocytometer to adjust its concentration to be 1×10 ⁷ -1×10 ⁹ CFU/mL.

Four rice seedlings of similar vigour were planted in each pot with three replicates per treatment. When the rice grows to the tillering stage, the rice is sprayed in the evening for two consecutive days, and the total dose is 5 mL/plant of rice. Plants were tested for agronomic traits up to one month after spraying the spore suspension.

The test results are shown in Table 1: the spore suspension of Streptomyces sp. OsiSh-2 was sprayed on the leaves of rice, which can significantly improve the growth of rice. Compared with the control, rice plant height, tiller number, net photosynthetic rate and chlorophyll content increased by 12.6%, 25.1%, 18.5% and 23.5%, respectively (see Figure 1).

Table 1

Plant height/cm Number of tillers/piece Net photosynthetic rate Chlorophyll content/SPAD Control 59.91±0.306 7.86±0.340 15.77±0.294 33.53±0.273 OsiSh-2 67.46±0.838** 9.71±0.454** 19.73±0.414** 39.73±0.186**

In the table **P≤0.01 is the significant level, the same below.

Example 2: Under normal circumstances, Streptomyces sp. OsiSh-2 embeds rice seeds to promote rice growth

Experimental conditions: Rice was grown hydroponically in a constant temperature light incubator in a greenhouse with a photoperiod of 16 hours of light/8 hours of darkness, a culture temperature of 28-30°C, and an air humidity of 60-70%.

Test crop: Rice (Indica 93-11).

Experimental treatment group: The spore suspension of Streptomyces sp. OsiSh-2 was prepared according to the method of Example 1, the concentration was 1×10 ⁸ CFU mL ^-1 , and the suspension was with rice seeds/0.3% (v/v) xanthan gum/spores The ratio of the liquid is 1g / 463 μl / 51 μl. The sterilized rice seeds are embedded in the ratio of rice seeds / 0.3% (v / v) xanthan gum / sterile water is 1 g / 463 μl / 51 μl, as a control. Each treatment was replicated three times, and each group contained 48 rice seedlings. Three weeks after germination, the growth of rice was observed and its agronomic traits were measured.

The test results are shown in Table 2: the Streptomyces sp. OsiSh-2 spore suspension of the present embodiment was embedded in the rice seeds, and cultivated under normal conditions. Compared with the unembedded rice, some fresh dry weights increased by 15.1%, 12.7%, 50%, 35.7%, 26.7% and 30.8%, respectively; the chlorophyll content and photosynthesis rate of rice also increased significantly, by 15.6% and 15.6%, respectively. 35.5%, indicating that the method achieved the effect of promoting rice growth (see Figure 2).

Table 2

Plant height/cm Total root length/cm Fresh weight of aerial parts/g Fresh weight of underground part/g Aboveground dry weight/g Dry weight of underground part/g Chlorophyll content/SPAD Net photosynthetic rate Control 19.24±0.159 67.66±0.618 0.08±0.002 0.14±0.003 0.015±0.0001 0.013±0.0003 32.54±0.672 2.846±0.011 OsiSh-2 22.14±0.276** 76.27±1.425** 0.12±0.003** 0.19±0.002** 0.019±0.0013** 0.017±0.0004** 37.61±0.410** 3.857±0.011**

Example 3: Streptomyces sp. OsiSh-2 Embeds Rice Seeds to Promote Rice Growth and Iron Absorption under Iron Deficiency

Experimental conditions: Rice was grown hydroponically in a constant temperature light incubator in a greenhouse with a photoperiod of 16 hours of light/8 hours of darkness, a culture temperature of 28-30°C, and an air humidity of 60-70%.

Test crop: Rice (Indica 93-11).

Experimental treatment group: The spore suspension of Streptomyces sp. OsiSh-2 was prepared according to the method of Example 1, the concentration was 1×10 ⁸ CFU mL ^-1 , and the suspension was with rice seeds/0.3% (v/v) xanthan gum/spores The ratio of the liquid is 1g / 463 μl / 51 μl. The sterilized rice seeds are embedded in the ratio of rice seeds / 0.3% (v / v) xanthan gum / sterile water is 1 g / 463 μl / 51 μl, as a control, each treatment was repeated three times, and each group contained 48 rice seedlings. After germination, they were planted for one week under normal conditions, and then cultured in iron-deficient hydroponic solution for two weeks to observe the growth of rice, and to measure its agronomic traits and iron content in different parts of the rice.

The test results are shown in Table 3 and Table 4: the rice embedded with the Streptomyces sp. OsiSh-2 spore suspension of the present embodiment can be cultured under iron-deficient conditions, and has the effect of promoting the growth of rice. The plant height, root length, fresh and dry weight of the aerial part and fresh and dry weight of the underground part of rice were shown in Table 3, which increased by 18.6%, 36.7%, 37.5%, 37.5%, 50.0% and 25.0%, respectively. The chlorophyll content of rice and photosynthesis (see Table 3) were also significantly improved by 26.1% and 92.8%, respectively (see Figure 3). The iron ion content of the aerial part and the underground part of rice is shown in Table 4, both of which were significantly increased, by 26.6% and 20.7%, respectively.

table 3

Plant height/cm Total root length/cm Fresh weight of aerial parts/g Fresh weight of underground part/g Aboveground dry weight/g Dry weight of underground part/g Chlorophyll content/SPAD Net photosynthetic rate Control 16.86±0.302 89.31±2.951 0.08±0.002 0.16±0.003 0.012±0.0003 0.016±0.0003 25.27±0.912 1.39±0.031 OsiSh-2 20.00±0.347** 122.06±1.865** 0.11±0.003** 0.22±0.003** 0.018±0.0030** 0.020±0.0004** 31.86±0.834** 2.68±0.011**

Table 4

Iron content of aerial parts (mg/Kg) Iron content in underground part (mg/Kg) Control 1.90±0.026 8.38±0.117 OsiSh-2 2.91±0.017** 12.72±0.127**

The specific embodiments of the present invention have been described above in detail, but they are only used as examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be included within the scope of the present invention.

Claims (4)

1. A method for promoting the absorption of iron element in the iron-deficient environment of rice to promote the growth of the rice is characterized in that: the method adopts a rice endophytic actinomyceteStreptomycessp, OsiSh-2, embedding the disinfected rice seeds by using the spore suspension, and promoting the absorption of the rice to iron elements in an iron-deficient environment by using the endophytic actinomycetes so as to be beneficial to the growth and development of the rice; the rice endophytic actinomycetesStreptomycesOsiSh-2, the preservation number is CGMCC number 8716.

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

3. The method of claim 2, wherein: the spore suspension has a concentration of 1 × 10⁸ CFU/mL。

4. The method of claim 3, wherein: spore suspensions were used at a ratio of seed/0.3% (v/v) xanthan gum/spore liquid =1g/463 μ L/51 μ L.

Images