CN111466266B

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

Info

Publication number: CN111466266B
Application number: CN202010061326.0A
Authority: CN
Inventors: 朱咏华; 高艳; 曹力丹; 刘选明; 喻金兰; 卜志刚; 姚晴晴
Original assignee: Hunan University
Current assignee: Hunan Xinchangshan Agricultural Development Co ltd
Priority date: 2020-01-19
Filing date: 2020-01-19
Publication date: 2022-06-17
Anticipated expiration: 2040-01-19
Also published as: CN111466266A

Abstract

The invention discloses a method for promoting the growth of rice in a normal growth environment and promoting the absorption of iron elements by the rice in an environment without directly utilizing iron, which utilizes a strain of rice endophytic actinomycetesStreptomycesOsish-2, with its spore suspension, rice under normal conditions can be treated by two different means: 1) spraying spore suspension on the surface of the rice leaf; 2) the sterilized rice seeds were embedded in their spore suspension. The method of the invention promotes the growth of rice, improves the chlorophyll content and the photosynthesis rate, and obviously increases the rice biomass such as the plant height, the root length, the fresh dry weight of the overground and underground parts and the like of the rice; under the adverse circumstances of iron deficiency, the sterilized rice seeds are embedded by the spore suspension, so that the iron content in the rice is obviously improved, and the normal growth of the rice under the stress of iron deficiency is ensured.

Description

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

Technical Field

The invention belongs to the technical field of biofertilizer, and particularly relates to a method for promoting rice growth under a normal growth condition and promoting the absorption of iron elements by rice in an environment without directly utilizing iron to maintain the growth of rice by utilizing a rice endophytic actinomycete.

Background

Rice is an important food crop in China and even all over the world, and the yield and the quality of the rice are closely related to the health of human beings. The means for promoting the growth of rice mainly comprises spraying chemical or biological fertilizer at present, and along with the formulation of the zero-growth strategy of chemical fertilizer in China, the research of environment-friendly biological fertilizer is more and more emphasized. For example, the Rhodopseudomonas palustris PSB06 microbial inoculum in photosynthetic bacteria reported by Zeng Yibo and the like can promote the growth of rice in seedling stage; doni Febri et al reportBased on TrichodermaTrichodermaThe spp biological fertilizer can improve the rice yield by 30%; su rez-Moreno Zulma Roc i o et alStreptomycessp. A20 can promote rice growth.

Endophytes are a class of microorganisms that can stably colonize plant tissues without causing significant disease symptoms in the plant. Because the endophyte lives inside plant tissues for a long time and can co-evolve with the plant, substances secreted by the endophyte can be quickly identified and utilized by the plant to form a more coordinated reciprocal symbiotic relationship, for example, the endophyte can generate a series of growth-related plant growth regulating substances, such as: IAA, ACC deaminase and siderophores etc. promote growth of the host. Meanwhile, the unique ecological niche of the endophyte in the plant body has survival advantage compared with rhizosphere microorganisms which are easily affected by the environment. Compared with fungi and bacteria, actinomycetes can produce abundant secondary metabolites, and have better growth promotion potential than other microorganisms under the same condition. Based on the method, the plant endophytic actinomycetes are suitable for being popularized as biological fertilizers. For example, Patel Janki K. et al report a number of endophytic actinomycetesStreptomycesspp, by adding actinomycete spore suspension into rice water culture solution, the growth of overground parts of rice can be promoted; wang Wenfeng et al report endogenetic actinomycetesStreptomycessp, A9, the method of soaking the rice seeds by the spore liquid can promote the growth of rice seedlings; in addition, Liotti Rhavena Graziela et al report an endophytic actinomyceteStreptomyces griseocarneusR132, spraying the bacterial liquid on the leaf surface to promote the growth of the overground part of the pepper; le Xuyen H. et al reportStreptomycesspp, LuP30 and LuP47B, which can improve the dry weight of the overground part of the alfalfa by embedding actinomycete spore liquid in seeds; ge Youyouu et al reported endophytic actinomycetesStreptomycessp, SSD49 spore liquid is directly irrigated in the soil for plant growth, which can promote the growth of soybean, sorghum and pepper. However, the current patents for biofertilizers based on actinomycetes, especially endophytic fungi, are basically difficult to see. Different from the commonly reported method, the method of the invention treats the rice in a mode of embedding seeds or spraying spore liquid on seedlings to protect the riceIt is proved that the endophyte and the rice form a mutual benefit symbiosis state, and finally the growth of the rice is promoted.

Iron is an essential nutrient for all organisms, participates in various 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, and particularly in neutral and calcareous soil, the iron exists in a trivalent cation form, the solubility is extremely low, and the iron is difficult to absorb and utilize by plants. Iron deficiency results in reduced chlorophyll synthesis in crops, reduced photosynthetic rate, reduced biomass, and in turn, insufficient iron uptake by animals and humans. At present, the treatment of the iron deficiency of plants still uses chemical fertilizers as main means. The utilization of microorganisms to enhance the ability of plants to cope with iron deficiency stress has become an urgent need for a novel biological treatment technology. Few reports on endophyte strengthening plant tolerance to iron deficiency stress exist at present, and only the reports on endophyte strengthening plant tolerance to iron deficiency stress existStreptomycesGMKU 3100 is reported to help plants maintain normal growth in a iron deficient environment due to its produced siderophores. Different from the treatment method of soaking the roots of rice seedlings by actinomycete spore suspension adopted by the report, the method of the invention is to embed the rice seeds by the spore suspension of endophytic fungi, and the treatment method is favorable for the rice to contact with the endophytes from the seed germination period, so that the rice can receive the favorable influence of the endophytes on the growth of the rice from the seed germination period.

Disclosure of Invention

In order to solve the rice production requirements in the prior art, the invention provides a method for promoting rice growth and promoting the absorption of iron element in a rice iron-deficient environment, which adopts the following technical scheme:

the method of the invention uses a rice endophytic actinomyceteStreptomycessp, OsiSh-2, which is preserved in China general microbiological culture Collection center (CGMCC for short) with the preservation number of CGMCC number 8716.

The invention provides a method for promoting the growth of rice or/and promoting the absorption of iron element in the environment of iron deficiency of rice, which adopts the riceEndophytic actinomycetesStreptomycessp. OsiSh-2, spraying the spore suspension on the surface of rice leaves or embedding the sterilized rice seeds with the spore suspension.

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

The method is a method for promoting the growth of rice, and the method sprays rice endophytic actinomycetes on the surface of rice leavesStreptomycessp, OsiSh-2 spore suspension or rice endophytic actinomyceteStreptomycesEmbedding sterilized rice seeds with sp, OsiSh-2 spore suspension, or first using rice endophytic actinomycetesStreptomycesspraying rice endophytic actinomycetes on the surfaces of rice leaves on the rice seeds embedded and disinfected by the sp, OsiSh-2 spore suspensionStreptomycessp. OsiSh-2 spore suspension.

The method comprises the following steps of (1) spraying spore suspension on the surfaces of rice leaves, wherein 0.2% (v/v) of Tween 20 is added to serve as a surfactant, the dosage is based on the condition that the whole rice leaves are contacted with the spore suspension, and the dosage is generally 5 mL/rice for the rice in a three-leaf stage; (2) the sterilized rice seeds were embedded in a spore suspension at a seed/0.3% (v/v) xanthan gum/spore liquid =1g/463 μ l/51 μ l.

The above method, which is a method for promoting the absorption of iron element in an iron deficient environment of rice, comprises embedding sterilized rice seeds in a spore suspension, preferably, the spore suspension is used at a ratio of seeds/0.3% (v/v) xanthan gum/spore liquid =1g/463 μ l/51 μ l.

The invention uses the endophytic actinomycetesStreptomycessp, OsiSh-2 is prepared into spore suspension by the following method:

(1) the strain is inoculated on an ISP-2 solid culture medium and is statically cultured for 14 days in a constant temperature incubator at 30 ℃.

(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 step (2) by using a blood counting plate to adjust the concentration to 1 × 10⁷~1×10⁹CFU/mL。

The concentration utilized by the invention is 1 x 10⁸CFU/mL ofStreptomycessp, OsiSh-2 spore suspension, growing under normal conditions, can treat rice in two different ways to achieve the purpose of growth promotion:

(1) spraying spore suspension on the surfaces of rice leaves, wherein 0.2% (v/v) Tween 20 is added as a surfactant to improve the adhesion capacity on the rice, and the dosage is based on the condition that the whole rice leaf surfaces are contacted with the spore suspension, for example, 5 mL/rice is used for the rice in the three-leaf stage;

(2) the sterilized rice seeds were embedded in a spore suspension using a ratio of seeds/0.3% (v/v) xanthan gum/spore liquid =1g/463 μ l/51 μ l.

The rice can be treated in a way to promote the absorption of iron element when growing under the condition of iron deficiency: the sterilized rice seeds were embedded in a spore suspension using a ratio of seeds/0.3% (v/v) xanthan gum/spore liquid =1g/463 μ l/51 μ l.

Compared with the prior method for promoting the growth of rice and promoting the absorption of iron elements in the iron-deficiency stress environment, the method has the following obvious advantages:

1. endophytic actinomycetes of riceStreptomycessp, OsiSh-2 can produce rich active substances, such as siderophores and plant growth regulating substances, can effectively promote the growth and development of rice, and ensures stable and high yield of the rice; the self-secreted iron reductase, the phenolic substances and the iron carrier can promote plants to absorb iron elements which are difficult to be obtained in the environment, improve the iron content in the plants and increase the nutritional value of rice.

2. According to the method, rice seeds are embedded with endophytes when the rice is still in a seed stage, or the rice can be sprayed in a seedling stage, rice diseases cannot be caused when the rice endophytes are applied by external sources and a host reestablishes a symbiotic relationship, and the functions of promoting growth and promoting iron element absorption of the rice can be exerted from the early growth stage of the rice.

3. The rice endophyte is separated from the rice body, is safer to the environment compared with chemical fertilizers, and does not influence the agricultural ecological environment and the human health.

4. The treatment of the rice endophyte is more convenient, simpler and diversified, and compared with other land treatment methods, the method saves time and labor, is not limited by regions and manpower, and is suitable for wide popularization.

Drawings

FIG. 1: foliage spraying under normal growth environmentStreptomycesGrowth of rice after sp. OsiSh-2 spore suspension;

FIG. 2: normally usingStreptomycesGrowth of rice embedded in sp, OsiSh-2 spore suspension;

FIG. 3: used under the condition of iron deficiency and stressStreptomycesGrowth of rice embedded in sp. OsiSH-2 spore suspension.

Detailed Description

The technical scheme of the invention is further explained by combining specific examples.

Example 1: under normal conditions, spraying on leaf surfacesStreptomycessp, OsiSh-2, promoting rice growth

An experimental field: greenhouse soil culture

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

Experimental treatment group: preparation ofStreptomycesspore suspension of sp. OsiSh-2 at a concentration of 1X 10⁸CFU mL^-1Adding 0.2% Tween 20 for treatment; water containing 0.2% Tween 20 was used as a control. The specific method for preparing the spore suspension is as follows:

(3) Suspending the spores obtained in the step (2) by using a blood counting plateThe concentration of the suspension is adjusted to 1 × 10 by counting⁷~1×10⁹CFU/mL。

Four rice seedlings of similar growth were planted in each pot, three replicates per treatment. When the rice grows to the tillering stage, spraying the rice for two days continuously, wherein the total dose is 5mL per plant of rice. And detecting the agronomic characters of the plants after spraying the spore suspension for one month.

The test results are shown in Table 1: spray on rice foliageStreptomycesThe spore suspension of sp, OsiSh-2 can obviously improve the growth condition of rice. Compared with the control, the plant height, tillering number, net photosynthetic rate and chlorophyll content of the rice are respectively increased by 12.6%, 25.1%, 18.5% and 23.5% (see figure 1).

TABLE 1

	Plant height/cm	Tillering number/number	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.ltoreq.0.01 is the significance level, as follows.

Example 2: in the normal case of the operation of the device,Streptomycesembedding rice seeds in sp, OsiSh-2 to promote rice growth

The experimental conditions are as follows: the rice is cultivated in greenhouse constant temperature illumination incubator with 16 hr illumination/8 hr darkness photoperiod, cultivation temperature of 28-30 deg.C, and air humidity of 60-70%

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

Experimental treatment group: prepared according to the method of example 1Streptomycesspore suspension of sp. OsiSh-2 at a concentration of 1X 10⁸ CFU mL^-1The sterilized rice seeds were embedded at a ratio of rice seeds/0.3% (v/v) xanthan gum/spore suspension of 1 g/463. mu.l/51. mu.l, and the rice seeds/0.3% (v/v) xanthan gum/sterile water of 1 g/463. mu.l/51. mu.l were used as a control. Each treatment was repeated three times, each group containing 48 rice seedlings. And (4) observing the growth condition of the rice after three weeks of planting after pregermination, and measuring the agronomic characters of the rice.

The test results are shown in Table 2: the rice seeds were embedded with the rice seeds of the present exampleStreptomycesCulturing the sp, OsiSh-2 spore suspension under normal conditions, wherein the plant height, the root length, the fresh dry weight of the overground part and the fresh dry weight of the underground part of the rice are respectively increased by 15.1%, 12.7%, 50%, 35.7%, 26.7% and 30.8% compared with the rice which is not embedded; the chlorophyll content and the photosynthesis rate of the rice are also obviously improved to be 15.6 percent and 35.5 percent respectively, which shows that the method achieves the effect of promoting the growth of the rice (see figure 2).

TABLE 2

Plant height/cm

Total root length/cm

Fresh weight of aerial parts/g

Fresh weight of underground part/g

Dry weight of aerial parts/g

Dry weight/g of underground part

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: in the case of iron deficiency, the iron-rich alloy,Streptomycesembedding rice seeds in sp, OsiSh-2, promoting rice growth and iron absorption

Test crops: rice (indica 93-11).

Experimental treatment group: prepared according to the method of example 1Streptomycesspore suspension of sp. OsiSh-2 at a concentration of 1X 10⁸ CFU mL^-1The embedding treatment was carried out on sterilized rice seeds at a ratio of rice seeds/0.3% (v/v) xanthan gum/spore suspension of 1 g/463. mu.l/51. mu.l, and the embedding treatment was carried out on sterilized rice seeds at a ratio of rice seeds/0.3% (v/v) xanthan gum/sterile water of 1 g/463. mu.l/51. mu.l, as a control, in which each treatment was repeated three times, each group containing 48 rice seedlings. After germination accelerating, the rice is planted for one week under normal conditions, then the iron-deficient water culture solution is cultured for two weeks, the growth condition of the rice is observed, and the agronomic characters and the iron contents of different parts of the rice are measured.

The test results are shown in tables 3 and 4: embedding the present embodimentStreptomycesThe rice with sp, OsiSh-2 spore suspension is cultured under the condition of iron deficiency, and has the function of promoting the growth of rice. Wherein the plant height, root length, fresh dry weight of the overground part and fresh dry weight of the underground part of the rice are shown in table 3, which are respectively improved by 18.6%, 36.7%, 37.5%, 50.0% and 25.0%, and the chlorophyll content and photosynthesis (shown in table 3) of the rice are respectively and obviously improved by 26.1% and 92.8% (see fig. 3). The overground part and the underground part of riceThe iron ion contents are shown in table 4, and are improved obviously, and are respectively 26.6% and 20.7%.

TABLE 3

Plant height/cm

Total root length/cm

Fresh weight of aerial parts/g

Fresh weight of underground part/g

Dry aerial parts per gram

Underground part dry weight/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 of underground part (mg/Kg)
			Control	1.90±0.026	8.38±0.117
OsiSh-2	2.91±0.017**	12.72±0.127**

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

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.