CN109355235B

CN109355235B - Rhizobium FAMB126 and application thereof

Info

Publication number: CN109355235B
Application number: CN201811478026.1A
Authority: CN
Inventors: 廖红; 刘鹏; 钟永嘉; 田颖哲
Original assignee: Fujian Agriculture and Forestry University
Current assignee: Fujian Agriculture and Forestry University
Priority date: 2018-12-05
Filing date: 2018-12-05
Publication date: 2020-07-31
Anticipated expiration: 2038-12-05
Also published as: CN109355235A

Abstract

The invention discloses rhizobium FAMB126 and application thereof, wherein the strain is obtained by catching rhizobium in soil of Anxi area of Fujian province through Minhua No. 8 peanut variety, separating and purifying the rhizobium from fresh rhizobium, and carrying out strain treatment on the rhizobium16srDNA partial sequence was determined and determined to be of the genus Chronic rhizomatosis by phylogenetic analysisBradyrhizobium) The new strain of (1) and is named as Funong No. 2. The rhizobium FAMB126 disclosed by the invention is proved to have the characteristics of high-efficiency nodulation and strong nitrogen fixation capacity through a laboratory water culture tie-back test and a field tie-back test, can improve the biomass and the yield of peanuts by inoculating the rhizobium, is suitable for acid soil in south China, and is used as one of the conventional peanut cultivation measures.

Description

Rhizobium FAMB126 and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to rhizobium FAMB126 and application thereof.

Background

Rhizobia bacterium (A), (B), (C)rhizobium) Is a gram-negative bacterium widely distributed in soil, which can infect roots or stems of leguminous plants to form root nodules or stem nodules (stem nodules) and fix N in the air in a symbiotic manner₂Becoming NH which can be absorbed and utilized by plants₄ ⁺. The symbiont system between leguminous plants and rhizobia plays an extremely important role in nitrogen fixation of the whole organism. It is estimated that about 1 million tons of nitrogen are fixed from the air on the earth's surface every year, and about 5500 ten thousand tons of nitrogen are fixed by leguminous plants and rhizobia. The rhizobium and the leguminous plants form a symbiotic nitrogen fixation system, so that the nitrogen requirement of the plants can be met, and the redundant nitrogen can be provided for the non-leguminous plants which are mixed or intercropped with the rhizobium and the leguminous plants, such as the non-leguminous plants which are mixed and sowed with the gramineous plants, so that a nitrogen fertilizer can be applied to a small amount; by using¹⁵N tracing technology, researches show that 30% of nitrogen of gramineous plants sowed with leguminous plants in a mixed mode is derived from nitrogen fixed by the leguminous plants. The symbiotic nitrogen fixation system of the rhizobium and the leguminous plants has the advantages of strong nitrogen fixation capacity, large nitrogen fixation amount and strong stress resistance, and can improve soil fertility and provide protein nutrition for human and livestock. The research on the symbiotic nitrogen fixation of the rhizobium and the leguminous plants is beneficial to the realization of agriculture, environment and growthThe state can be continuously utilized and developed. Inoculation with rhizobia is an important measure to improve legume crop production. A large number of experiments at home and abroad prove that the inoculation of rhizobia can improve the yield and the quality of leguminous crops in most soils (Li Cao et al, 2000; Li Yoghuo et al, 2002). Bohlool, when summarizing the effect of many crops on many years of rhizobia inoculation, indicated that the yield of legume crops inoculated with rhizobia was 89.5% higher than when not inoculated with rhizobia (Bohlool et al, 1973).

Peanuts are important leguminous crops and also main oil crops in China. The rhizobia and the peanut root system can form root nodules to carry out symbiotic nitrogen fixation, and nitrogen is provided for peanut growth. Inoculating high-efficiency rhizobia is an important measure for promoting symbiosis and nitrogen fixation of peanuts and improving nitrogen nutrition of the peanuts. However, mature and effective peanut nitrogen-fixing rhizobium inoculant with high efficiency is still lacking at present. The research utilizes peanut varieties from different growing areas to carry out pot culture and field test, and separates and purifies the high-efficiency nitrogen-fixing rhizobia from rhizobia; then the purified rhizobia is identified by PCR technology and16ssequencing and identifying by rDNA technology; verifying host specificity and root nodule azotase activity by utilizing a tieback method in a greenhouse, finally screening a peanut high-efficiency rhizobium strain suitable for southern acid soil growth in a field test, and preparing a peanut high-efficiency rhizobium microbial inoculum according to soil nutrient characteristics. The research result provides practical and effective technical support for high-yield and high-efficiency production of peanuts.

Disclosure of Invention

The invention aims to provide a peanut rhizobium strain which is a new strain of bradyrhizobium and has the characteristics of high nodulation rate and strong nitrogen fixation capacity.

The other purpose of the invention is to utilize the rhizobium strain to apply the rhizobium strain to the agricultural production of leguminous plant peanuts, and promote the growth of the peanuts by improving the biological nitrogen fixation capacity of the peanuts so as to achieve the purposes of weight reduction and efficiency improvement in the peanut production.

The technical purpose of the invention is realized by the following technical scheme:

a rhizobium FAMB126 strain (Funong No. 2) is preserved in China center for type culture Collection (CCTCC M2018218) in 2018, 4 and 19 months, and the address of the China center for type culture Collection is China, Wuhan and Wuhan university.

It is composed of16sThe rDNA sequence is shown in SEQ ID NO. 1.

The rhizobium FAMB126 strain obtained by separation is slow-growing in colony morphology, small in circle, milky white, translucent and more in mucilage. The diameter of the YMA plate after 5 to 7 days of growth is 0.8 to 2.0 mm.

Furthermore, the separated rhizobium FAMB126 strain has an obvious promotion effect on nodulation and nitrogen fixation of peanuts, and can be applied to the improvement of peanut yield.

The invention has the advantages that:

(1) the rhizobium has the characteristics of high nodulation rate and strong nitrogen fixation capacity, and the inoculation of the rhizobium can improve the fresh weight and the dry weight of peanut plants and can be used for actual production compared with the inoculation without the rhizobium;

(2) inoculating peanut with the rhizobium of the invention can obviously improve the total nitrogen content of plants (a)P<0.01), thereby achieving the purposes of promoting the growth of peanut plants and improving the yield of peanuts;

(3) the rhizobia provided by the invention is suitable for acid soil areas in south China and can be used as one of important measures for conventional cultivation of peanuts.

Drawings

FIG. 1 shows the colony morphology of Rhizobium FAMB126 on YMA medium.

FIG. 2 is a drawing of Rhizobium FAMB12616srDNA partial sequence analysis phylogenetic tree (Rhizobium FAMB126 in box).

FIG. 3 is a graph showing the effect of hydroponic tieback of Rhizobium FAMB 126.

FIG. 4 shows the nitrogenase activity and the number of nodules.

FIG. 5 is a graph showing the effect of hydroponic inoculation of Rhizobium FAMB126 on the growth of flowers.

FIG. 6 shows the effect of rhizobium FAMB126 inoculated in a field on the growth and yield of a peanut.

FIG. 7 is a graph showing the effect of rhizobium FAMB126 inoculated in a field on the growth and yield of a peanut.

Detailed Description

Example 1 isolation and purification of Rhizobium FAMB126

(1) In the flowering period of normal growth of the peanuts, taking the fresh whole peanut root, taking the fresh, mature and large and full root nodule of the peanuts at the root, washing the root with water, and sucking the surface water with filter paper.

(2) Firstly, the mixture is put into ethanol with the volume fraction of 95 percent for treatment for 3 to 5 minutes, taken out and washed by sterile water for 5 to 6 times, and then 1 g/L HgCl is put into the mixture₂Sterilizing for 3-5 minutes, taking out and washing with sterile water for 5-6 times.

(3) The sections were cut in half on a flame-sterilized slide glass, and the half-cell was held with sterile forceps, and the cut was streaked to the surface of YMA (Table 1) medium, which was then inverted and cultured at 28 ℃.

(4) After the thalli grow out, scraping a small amount of rhizobium colonies from the flat plate by using a sterilized toothpick, diluting the rhizobium colonies with sterile water, performing streak culture on the flat plate again, observing the colony condition after 3 days, and observing for 15 days (the slow rhizobium colonies need 7-15 days to appear). If no single colony appears, the streaking purification on the plate needs to be repeated until the single colony appears.

Whether rhizobia is considered can be preliminarily judged according to the following method:

colony morphology: the rhizobium colonies are round, milky white, translucent, neat in edge and much in mucilage. The diameter of the bacterial colony reaches 2-4 mm after 3-5 days of culture, and the diameter of the bacterial colony reaches 1 mm after 7-10 days of culture, and the bacterial colony is the rhizobium bradyrhizobium.

The strain FAMB126 is a slow-growing rhizobium, and is small in circle, milky white, translucent and more in mucilage in colony morphology; the diameter of the YMA plate after 5-7 days of growth is 0.8-2.0 mm (figure 1); the optimal growth conditions are as follows: the pH is 7.0, the temperature is 28 ℃, the rotating speed is 180 r/min, and a wide range of carbon sources and nitrogen sources can be utilized.

By applying the separation and purification method, repeated purification is carried out for a plurality of generations, streaking culture is carried out in YMA solid culture medium (table 1), a plurality of pure strains are obtained, the nodulation and nitrogen fixation capability of the pure strains is verified by a water culture tieback test, and the strain FAMB126 with high nodulation rate and strong nitrogen fixation capability is separated and purified in the embodiment.

TABLE 1 YMA (Yeast Mannitol agar) Medium formulation

Example 2 of Rhizobium16srDNA sequence sequencing

Carrying out PCR specific amplification on the rhizobium monoclonal bacteria liquid, wherein primers are V3-V4-F and V4-V5-R respectively, forward primers V3-V4-F are 5 '-GWATTACCGCGGCKGCTG-3', reverse primers V4-V5-R are 5 '-CCGTCAATTCMTTTRAGTTT-3', and an enzyme is detected by using a 2 × star mix PCR amplification product as an electrophoresis imaging technology to observe whether the product has a strip or not, and the rest PCR amplification product is used for sequence determination, wherein a sequencing result is shown as SEQ ID NO.1, and a PCR reaction system is as follows:

TABLE 216s rDNA2 × starMix enzyme reaction system

Obtaining 12 reference strain sequences from NCBI (GenBank) database, and separating and reference strains by using software BioEdit and MEGA616srDNA partial sequence was analyzed to construct phylogenetic trees of the isolate and the reference strains (FIG. 2). Thereby determining that the Rhizobium FAMB126 is of the genus bradyrhizobium (II)Bradyrhizobium) The new strain of (1) and is named as Funong No. 2.

Example 3 tieback test

1. Strain culture:

transferring the test strain stored at-80 deg.C to YMA liquid culture, culturing to logarithmic phase, detecting the growth condition of the strain with nucleic acid protein analyzer, and calculating the bacteria content with OD value. The formula of YMA (Yeast Manninitol agar) liquid culture medium is as follows: weighing 10 g of mannitol and MgSO₄∙7H₂0.2g of O, 0.1 g of NaCl, 3 g of yeast powder and K₂HPO₄0.25g，KH₂PO₄0.25 g，CaCO₃3 g (added during storage), 15 g of agar was dissolved in 1L pure water.

2. And (3) sterilization treatment:

packaging 2.5L plastic bucket, beaker, forceps, culture dish, filter paper and glass rod required by test, packaging vermiculite with fresh-keeping bag, sterilizing at 121 deg.C for 30min, packaging YMA liquid culture medium and distilled water with bottle, and sterilizing at 121 deg.C for 20 min.

Putting peanut seeds into 1 g/L HgCl₂Sterilizing epidermis for 3min, washing with clear water, placing into 2.5L plastic bucket containing vermiculite, germinating in artificial growth chamber for 7 days, and inoculating water culture when radicle grows out 3-5 cm.

3. Hydroponic and inoculation

(1) Placing the germinated peanut in culture dish, adding purified rhizobia (figure 4), and completely immersing peanut root in rhizobia for 30min with bacteria content of 10⁹∙mL^-1。

(2) Transplanting the rejoined peanuts into a 2.5L plastic pot, adding 2L plant low-nitrogen nutrient solution, and culturing in an artificial growth chamber (photoperiod: day/night = 16h/8h, temperature: day/night = 26 ℃/24 ℃), wherein the plant low-nitrogen nutrient solution is prepared from Ca (NO)₃)₂∙4H₂O 0.03 g，MgSO₄·7H₂O 0.12 g, CaCl₂·2H₂O0.10 g，KH₂PO₄0.10 g，Na₂HPO₄·12H₂O 0.15 g，C₆H₅O₇Fe·5H₂0.005 g of O, 1 m L of trace element, 1000 m L of distilled water, pH 5.8-6.0, trace element formula (g ∙ L)^-1): H₂MoO₄0.02 g，MnSO₄1.81 g，H₃BO₃2.86 g，CuSO₄·5H₂O 0.8 g，ZnSO₄·7H₂O 0.22 g。

And (3) repeating the steps by taking non-inoculation as a control, harvesting the rhizobium after 30 days of inoculation, and taking the fresh weight, the dry weight, the nodulation number, the nitrogen content of the plant and the SPAD value of the plant as judgment indexes for measuring the binding property and the nitrogen fixing capacity of the rhizobium.

The results show that the blank Control (CK) has no nodulation, and the tieback treatment can nodulate an average of 11.33 nodules per peanut. The inside of the root nodule was found to be red by cutting the root nodule, and the test strain FAMB126 was basically judged to be a root nodule bacterium having nitrogen-fixing efficiency (FIG. 4), and its nitrogen-fixing enzyme activity was measured to be 0.75 umol ∙ (g ∙ h)^-1。

As can be seen from FIGS. 3 and 5, the inoculation of Rhizobium Fujianum, FAMB126, in Minhua No. 8, significantly increased the fresh weight, dry weight, SPAD value and nitrogen content of the plant, as compared to the absence of inoculation of Rhizobium (see (a))P<0.01). The rhizobium FAMB126 is high in binding property and nitrogen fixation efficiency.

Example 4 field inoculation test

1. Strain culture:

transferring a test strain stored at-80 ℃ to YMA liquid for culture until logarithmic phase, detecting the growth condition of the strain by using a nucleic acid protein detector, and calculating the bacteria content by using OD value; YMA (Yeast Manninitol agar) liquid culture medium comprises the following components: weighing 10 g of mannitol and MgSO₄∙7H₂0.2g of O, 0.1 g of NaCl, 3 g of yeast powder and K₂HPO₄0.25g，KH₂PO₄0.25 g，CaCO₃3 g (added during storage), 15 g of agar was dissolved in 1L pure water.

2. And (3) field test implementation:

the test is carried out in a tea garden of Yangyuan tea industry cooperative society of Yanyuan village and Yanyuan village in Anxi county, Fujian province, the sowing period is 2018, 3 and 28 days.

The field survey is carried out in the flowering period (26 days 6 and 26 months in 2018) and the fructification period (26 days 7 and 26 months in 2018) of peanuts, and the nodulation number, biomass, SPAD value, nitrogen content of plants and yield of individual plants are surveyed (the result is shown in figure 6).

As can be seen from FIG. 6, inoculation with Rhizobium FAMB126 can promote peanut nodulation. The number of nodules in the inoculation treatment and the non-inoculation treatment are different significantly. The biomass of the inoculated and treated single plant is obviously different from that of the non-inoculated and treated single plant, and the inoculation of rhizobium is beneficial to promoting the growth of peanuts (as shown in figure 7). The above-ground dry weight of the inoculated peanuts was significantly higher than that of the non-inoculated treatment, with an increase of 37.7%. Inoculation with rhizobium FAMB126 had a significant effect on peanut pod count, with an increase of 89.6%. Inoculation with rhizobium FAMB126 had a significant effect on peanut yield, with an increase of 74.2%.

The experimental research shows that under the condition of not applying nitrogen fertilizer, the rhizobium FAMB126 is used alone to have obvious yield increasing effect, so that the rhizobium FAMB126 developed by the research laboratory can be applied to large-area popularization and application.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

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Claims

1. A rhizobium bacterium (A)Rhizobiumsp.) strain FAMB126, which has been deposited in the China center for type culture Collection in 2018, 4 and 19 months, with the deposit number of CCTCC NO: m2018218.

2. The use of the rhizobium strain FAMB126 as claimed in claim 1 for increasing peanut yield.