CN116751714A - Rhizobium strain capable of inducing pigeon pea to have salt tolerance and promoting nodulation and nitrogen fixation and application thereof - Google Patents
Rhizobium strain capable of inducing pigeon pea to have salt tolerance and promoting nodulation and nitrogen fixation and application thereof Download PDFInfo
- Publication number
- CN116751714A CN116751714A CN202310692947.2A CN202310692947A CN116751714A CN 116751714 A CN116751714 A CN 116751714A CN 202310692947 A CN202310692947 A CN 202310692947A CN 116751714 A CN116751714 A CN 116751714A
- Authority
- CN
- China
- Prior art keywords
- hnmd5
- rhizobium
- pigeon pea
- strain
- nodulation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 235000010773 Cajanus indicus Nutrition 0.000 title claims abstract description 46
- 244000105627 Cajanus indicus Species 0.000 title claims abstract description 45
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 title claims abstract description 26
- 230000024121 nodulation Effects 0.000 title claims abstract description 16
- 241000589180 Rhizobium Species 0.000 title claims abstract description 13
- 230000015784 hyperosmotic salinity response Effects 0.000 title claims abstract description 13
- 229910052757 nitrogen Inorganic materials 0.000 title claims abstract description 13
- 230000001737 promoting effect Effects 0.000 title claims abstract description 7
- 230000001939 inductive effect Effects 0.000 title abstract description 4
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Classifications
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention discloses rhizobium strain capable of inducing pigeon pea to have salt tolerance and promoting nodulation and nitrogen fixation and application thereof. The preservation number of the rhizobium japonicum HNMD5-1 (Burkholderia sp.HNMD5-1) is as follows: GDMCC No.63247; the strain has good salt tolerance, can promote the growth of pigeon pea plants in a higher salt stress environment, obviously promote nodulation and nitrogen fixation of pigeon pea plants, and promote the growth and development of plants.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganisms, and particularly relates to rhizobium for inducing pigeon pea to have salt tolerance and promoting nodulation and nitrogen fixation and application thereof.
Background
According to incomplete statistics of textbook organizations and grain and agriculture organizations of united nations, the area of saline-alkali soil worldwide is 9.54 hundred million hectares, and the area of China is about 9900 ten thousand hectares. Saline-alkali soil is a part of important land resources in China. In the 21 st century of agricultural sustainable development in China, improvement and utilization of saline-alkali soil is still a problem. The rhizosphere microorganisms can effectively promote the stress resistance of plants, some rhizosphere beneficial microorganisms can obviously promote the growth of crops under salt stress, and the rhizosphere microorganisms have the potential of being developed into microbial fertilizers special for increasing the yield of saline-alkali soil, and provide a new strategy with low cost and environmental friendliness for improving the crop yield of saline-alkali cultivated lands by utilizing agricultural microbial technology.
The pigeon pea is a leguminous woody crop which is used as a feed for perennial grains and is also a sixth edible bean crop in the world. Is native to india and is an important cash crop in india, east africa and central america caribbean. The cultivation of pigeon pea in China is mainly distributed in southwest and south China provinces, such as Fujian, guangdong, guizhou and the like. The pigeon pea is drought-resistant, barren-resistant and rapid in growth, and is an excellent pioneer tree species for afforestation and water and soil conservation in the south mountain and island barren mountains; the pigeon pea grain has high protein content and important economic value; in addition, pigeon pea can form a symbiotic nitrogen fixation system with rhizobium, and has important significance in improving soil. However, most pigeon pea varieties have poor salt tolerance, so that screening of proper rhizobium has important significance in improving salt tolerance of pigeon pea-rhizobium symbiotic nitrogen fixation system, improving salinized soil, expanding pigeon pea cultivation range, improving pigeon pea industrialization and the like.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide rhizobium which induces pigeon pea to have salt tolerance and promote nodulation and nitrogen fixation and application thereof.
The rhizobium japonicum is separated from cayratia japonica, and is numbered HNMD5-1. Bacterial strain HNMD5-1 main morphological and biological characteristics: on YMB medium plates, colonies were yellowish in color, smooth in edge, and had protrusions. The strain HNMD5-1 can grow on a flat plate taking mannitol, inositol, sodium citrate, malonic acid, glucose, fructose, xylose, arabinose, lactose, sucrose and maltose as carbon sources, and the pH range suitable for growth is pH5-pH9, has the resistance to fosfomycin, tetracycline and ampicillin, has the capacity of generating alkali, decomposing organic phosphorus and inorganic phosphorus, does not have the capacity of decomposing potassium, can resist 4% NaCl at maximum, and can grow at 28 ℃ and 37 ℃. The morphological characteristics, physiological and biochemical characteristics of the control strain HNMD5-1 and the construction of phylogenetic tree analysis by the 16S rDNA gene sequence, identify that the strain HNMD5-1 belongs to the genus Burkholderia and is therefore named Burkholderia sp.HNMD5-1.
Accordingly, a first object of the present invention is to provide a Rhizobium cajanus HNMD5-1 having a accession number: GDMCC No.63247.
The second object of the present invention is to provide a microbial agent comprising said Rhizobium japonicum HNMD5-1 as an active ingredient.
The third object of the invention is to provide a biological bacterial fertilizer which contains the rhizobium japonicum HNMD5-1 and a fertilizer carrier.
A fourth object of the present invention is to provide the use of said cajanus cajan rhizobia HNMD5-1 in at least one of the following (1) - (3);
(1) Improving the salt tolerance of plants;
(2) Promoting plant nodulation and fixing nitrogen;
(3) Promote plant growth.
Preferably, the use comprises the step of inoculating said Cajanus cajan rhizobium HNMD5-1 to plant roots or plant rhizosphere soil.
Preferably, the rhizobium japonicum HNMD5-1 is used in the form of a rhizobium japonicum HNMD5-1 bacterial liquid, and the rhizobium japonicum HNMD5-1 bacterial liquid is prepared by adding the rhizobium japonicum HNMD5-1 into a pigeon pea nutrient solution and mixing until the bacterial liquid concentration OD600 = 0.01.
Preferably, the plant is pigeon pea.
The pigeonpea rhizobium HNMD5-1 has good salt tolerance, can promote pigeonpea plants to grow in a higher salt stress environment, and remarkably promote nodulation and nitrogen fixation of pigeonpea, thereby promoting the growth and development of plants.
Preservation description:
burkholderia sp.HNMD5-1 (Rhizobium japonicum HNMD 5-1) of the present invention was deposited at the Guangdong province microbiological bacterial collection center (GDMCC) at 04, 2023, accession number: GDMCC No.63247, deposit address: building 5, guangzhou city, first, middle road 100, university, 59, university of Guangdong, academy of sciences of China, and microbiological study.
Drawings
FIG. 1 shows colony morphology of Rhizobium japonicum HNMD5-1 on YMB medium.
FIG. 2 is a 16S rDNA phylogenetic tree of Cajanus cajan rhizobia HNMD5-1.
FIG. 3 is a basic biological characterization of Mucuna birdwoodiana HNMD5-1.
FIG. 4 shows phenotypes of Cajanus cajan (HNMD 5-1) and control Cajanus cajan (MD, rhizobium pakistanense strain) after tieba inoculation under normal conditions (CK, cajan nutrient solution) and NaCl (300 mM); wherein, figure a is the phenotype of pigeon pea plants after 5 weeks of inoculation with control bacteria MD; panel B shows the phenotype of the plants after 5-1 5 weeks of inoculation with HNMD; panel C is root length, plant height and root nodule count statistics of inoculated control bacterial MD and bacterial HNMD5-1 5 Zhou Houmu bean plants.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1
1. Isolation of Strain HNMD5-1
Taking root nodules of Hainan bean plants of coral island reefs, sterilizing with 75% alcohol for 30s, sterilizing with 2% NaClO water solution for 5min, rinsing with sterile water for 5-6 times, adding a proper amount of YMB culture medium, mashing the root nodules with sterile forceps, scribing and purifying in YMB flat plate, and culturing at constant temperature of 28deg.C for 2-3d.
2. Basic biological characterization of Strain HNMD5-1
2.1 analysis of NaCl tolerance characteristics of Strain HNMD5-1
(1) YMB medium (normal YMB; YMB solid medium supplemented with 1%, 2%, 3%, 4%, 5% NaCl by mass) was prepared one day in advance. Weighing the components according to the formula of Table 1, mixing, adding water to a constant volume of 1L, adjusting pH to 7.0 with 5% NaOH solution or 5% HCl solution, and sterilizing at 121deg.C for 30min; and then split into YMB medium plates.
Table 1 YMB medium formulation (1L)
| Component (A) | Dosage of |
| Mannitol (mannitol) | 10.0g |
| Yeast extract powder | 3.0g |
| Dibasic potassium phosphate trihydrate | 0.1g |
| Anhydrous magnesium sulfate | 0.1g |
| Sodium chloride | 0.1g |
| Calcium chloride hexahydrate | 0.05g |
| Congo red | 0.2g |
| Agar powder | 15g |
(2) Respectively diluting bacterial solutions from different sources 10 4 Later (1 mL YMB was added to a 2mL sterilized centrifuge tube, and 10. Mu.L of the corresponding rhizobia was added thereto, and the mixture was homogenized and handled by an alcohol burner).
(3) And (3) sucking 5 mu L of diluted bacterial liquid on a flat plate, writing a strain number and a date on the flat plate, placing the flat plate in a 28 ℃ incubator for culture, and observing the growth vigor of bacteria under different salt concentrations by comparing the growth vigor on normal YMB after 3 days, recording and photographing.
2.2pH experiments
YMB medium (medium of pH3, pH5, pH7, pH9, pH11 and pH 14) was prepared, 5. Mu.L of the diluted bacterial liquid was pipetted onto plates of different pH, and after 3 days, the growth was observed and photographed.
2.3 temperature tolerance
Preparing YMB medium, sucking 5 μl of diluted bacterial liquid, plating on YMB plate, culturing in incubator at different temperature (16deg.C, 28deg.C, 37deg.C and 45deg.C), observing growth condition after 3d, and photographing.
2.4 carbon source utilization and antibiotics
Different carbon sources of YMB (mannitol is replaced by different carbon sources, different treatments are respectively 0 carbon source or 10g/L mannitol, inositol, glycerol, sodium citrate, malonic acid, sodium oxalate, starch, glucose, fructose, D-xylose, arabinose, lactose, sucrose or maltose) and different antibiotics of YMB (antibiotics are all filter sterilized, cooled to 60 ℃ after medium sterilization, 200 mu L erythromycin, 100 mu L chloramphenicol, 50 mu L fosfomycin, 100 mu L gentamicin, 100 mu L rifampicin, 100 mu L kanamycin, 25 mu L aureomycin, 100 mu L streptomycin, 20 mu L tetracycline, 100 mu L ampicillin, 100 mu L spectinomycin or 0 added antibiotics) are added, the different carbon sources/antibiotics of YMB are respectively added to 100mL YMB medium after the medium is sterilized, the medium is placed in a 24-well sterilization plate, and the corresponding carbon sources/antibiotics at different positions are recorded.
Then, 5. Mu.L of the diluted bacterial liquid was pipetted into the center of the medium in the well, left to stand for several minutes (to prevent flow), then covered with a cap and sealed with a sealing film, and incubated at 28℃for 3 days, and then photographed by observing the growth.
2.5 phosphate and Potassium dissolving capability
Preparing Meng Jinna organic phosphorus (lecithin) and calcium phosphate culture medium plates, dividing each plate into 6 areas, sequentially sucking 5 mu L of bacterial liquid spots at the center of each area (sampling ensures accuracy and does not flow bacterial liquid), culturing for 3 days in a 28 ℃ incubator, and observing whether the strain grows and forms a phosphate solubilizing ring.
The formula of the culture medium is as follows: (1) meng Jinna organophosphorus (lecithin) bacterial medium (1L): glucose 10.0g, (NH) 4 ) 2 SO 4 0.5g，NaCl 0.3g，MgSO 4 ·7H 2 O 0.3g，FeSO 4 0.03g，MnSO 4 ·H 2 O 0.03g，KCl 0.3g，CaCO 3
1.0g, lecithin 0.3g, agar 20g, pH 7.0. Wherein lecithin is dissolved in 75% ethanol under heating, sterilized alone, mixed with sterilized and cooled to 60deg.C, and poured into a plate.
(2) Meng Jinna inorganic phosphorus (tricalcium phosphate) bacterial medium (1L): glucose (C) 6 H 12 O 6 ) 10g of ammonium sulfate [ (NH) 4 ) 2 SO 4 ]0.5g, sodium chloride (NaCl) 0.3g, magnesium sulfate (MgSO 4 ·7H 2 O) 0.3g of ferrous sulfate (FeSO) 4 ·7H 2 O) 0.03g, manganese sulfate (MnSO 4 ·H 2 O) 0.03g of calcium carbonate (CaCO) 3 ) 5.0g, potassium chloride (KCl) 0.3g, tricalcium phosphate [ Ca ] 3 (PO 4 ) 2 ]5.0g, 20g of agar and pH 7.0-7.5.
(3) Potassium-decomposing Activity assay Medium (1L): sucrose 2.0g, (NH) 4 ) 2 SO 4 0.5g，Na 2 HPO 4 1.5g，MgSO 4 ·7H 2 0.5g of O, 5.0g of potassium feldspar powder and pH7.2.
Detection of acid-producing or alkali-producing ability on culture medium of 2.6HNMD5-1 strain
Preparing an acid-base (bromophenol blue) culture medium. The components were weighed according to the formulation of Table 2 and mixed, water was added to a constant volume of 1L, pH was adjusted to 7.0 with 5% NaOH solution or 5% HCl solution, and sterilization was performed at 121℃for 30min. And then split-charging the plates.
TABLE 2 acid-base culture medium recipe (1L)
| Component (A) | Dosage of |
| Mannitol (mannitol) | 10.0g |
| Yeast extract powder | 3.0g |
| Dibasic potassium phosphate trihydrate | 0.1g |
| Anhydrous magnesium sulfate | 0.1g |
| Sodium chloride | 0.1g |
| Calcium chloride hexahydrate | 0.05g |
| Bromophenol blue | 0.2g |
| Agar powder | 15g |
After sterilization, 5 mu L of azotobacter isolated by the experiment is inoculated, and the culture is carried out for 3 to 5 days at the temperature of 28 ℃ to observe the color of the culture medium. The culture medium turns blue and represents azotobacter to produce alkali; the culture medium turns yellow to represent the acid production of azotobacter; the culture medium is not discolored, is green, and represents no acid or alkali production.
The results showed that strain HNMD5-1 had smooth colony edges, raised colony and yellowish colony color after incubation for 3 days at 28℃in YMB plate (FIG. 1). The strain HNMD5-1 can grow on a flat plate taking mannitol, inositol, sodium citrate, malonic acid, glucose, fructose, xylose, arabinose, lactose, sucrose and maltose as carbon sources, and the pH range suitable for growth is pH5-pH9, has the resistance to fosfomycin, tetracycline and ampicillin, has the capacity of generating alkali, decomposing organic phosphorus and inorganic phosphorus, does not have the capacity of decomposing potassium, can resist 4% NaCl at the maximum, and can grow at 28 ℃ and 37 ℃.
3. Molecular biological identification of Strain HNMD5-1
The HNMD5-1 strain is identified by 16S rDNA, PCR amplification is carried out on the genome DNA of the HNMD5-1 strain by using a primer (F: AGAGTTTGATCCGGCTCAG; R: TACGGCTACCTTGTTACGACTT), a fragment with the size of about 1.4kb (the nucleotide sequence is shown as SEQ ID NO.1, 1433 bp) is amplified, the sequence is subjected to sequence alignment by DNAMAN software, SNAPGENE software is subjected to sequence splicing, and homology alignment is carried out in NCBI (https:// blast. NCBI. Nih. Gov /) gene library, so that the HNMD5-1 strain belongs to the genus Burkholderia. A phylogenetic tree was constructed by the adjacency method (Neighbor-Joining) using MEGA5.0 software, and phylogenetic analysis was performed, and HNMD5-1 was closest to Burkholderia sp.HNMD5-1 (FIG. 2), so that the strain was named Burkholderia sp.HNMD5-1.
Example 2: strain HNMD5-1 induces pigeon pea to have salt tolerance, promote nodulation and fix nitrogen
1. Method for inoculating pigeonpea and treating pigeonpea with salt
1. Seed germination
The filled pigeon pea seeds were selected and immersed overnight in sterile water (seed: water mass ratio about 1:2-4) in a can. Then put the pigeon pea seeds into a culture dish, slightly wet and spread the filter paper under the culture dish, and spread the seeds in the culture dish at certain intervals. After the seeds are swelled (about 2 d), the seeds are planted into the soil, a part of the seeds are kept to be exposed out of the soil, the seeds are sealed by a preservative film, and the preservative film is removed after 3-4 d.
2. Soil mixing
The soil is vermiculite to be sterilized: ceramsite with volume ratio of 1:1, uniformly mixing to obtain the product. The experiment used control Cajanus rhizobia (MD, rhizobium pakistanense strain) and strain HNMD5-1 for the inoculation of Cajanus. Wherein the control bacterium MD is another rhizobium strain separated from pigeon pea rhizomes, and has closest relationship with a host plant pigeon pea; the inventor performs a comparison experiment in the earlier stage, and the result shows that under the normal condition or the NaCl treatment condition, the pigeonpea inoculant MD and the pigeonpea inoculant MD which are not inoculated with any bacteria (as comparison) have no obvious difference in the aspects of nodulation number, plant root length and plant height; therefore, bacteria MD was selected as a control bacteria. Bacterial MD or bacterial HNMD5-1 were added to 300mM NaCl solution or pigeon pea nutrient solution (125. Mu.L of A, B, C, D mother liquor in Table 1 per L of water) respectively, followed by a total of 4 treatments: the mixed bacterial solution is added with bacterial solution with the concentration OD600 = 0.01, the bacterial solution is used for mixing soil, and part of bacterial solution is sprayed on the soil after seeds are buried in the soil. Watering with pigeonpea nutrient solution and filtered sterile water in turn, observing pigeonpea nodulation phenotype after 4-5 weeks, and measuring various physiological indexes. The formula of the mother liquor of the pigeon pea nutrient solution is shown in table 3.
Table 3 mother liquor formulation of pigeon pea nutrient solution
2. Results
The results of fig. 4 show that, under normal conditions, the pigeon pea plants treated with HNMD5-1 strain showed significant nodulation, the number of nodulation was significantly greater than the pigeon pea plants treated with bacterial MD; and under NaCl treatment, the pigeonpea plants treated by HNMD5-1 strain also have more nodulation compared with bacterial MD treatment. Both bacterial MD and bacterial HNMD5-1 had longer root length under NaCl treatment than under normal conditions, and had shorter plant height than under normal conditions. The plant height and root length of the strain HNMD5-1 treated are higher than those of the control strain MD treated under NaCl treatment and normal conditions, so that the HNMD5-1 is proved to improve the salt tolerance of the plant and promote the nodulation, nitrogen fixation and plant growth of the plant.
Claims (7)
1. A strain of rhizobium japonicum HNMD5-1, characterized by a deposit number of: GDMCC No.63247.
2. A microbial agent comprising the Rhizobium cajan HNMD5-1 as an active ingredient.
3. A biological bacterial fertilizer, characterized by comprising the rhizobium japonicum HNMD5-1 and a fertilizer carrier.
4. Use of the rhizobium japonicum HNMD5-1 of claim 1 in at least one of the following (1) - (3);
(1) Improving the salt tolerance of plants;
(2) Promoting plant nodulation and fixing nitrogen;
(3) Promote plant growth.
5. The use according to claim 4, comprising the step of inoculating the cajanus cajan rhizobium HNMD5-1 of claim 1 to plant roots or plant rhizosphere soil.
6. The use according to claim 5, wherein said rhizobium japonicum HNMD5-1 is used in the form of a rhizobium japonicum HNMD5-1 bacterial solution, said rhizobium japonicum HNMD5-1 bacterial solution is prepared by adding rhizobium japonicum HNMD5-1 to a nutrient solution of pigeon pea and mixing until the bacterial solution concentration od600=0.01.
7. The use according to claim 4, wherein the plant is pigeon pea.
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