Application of rhizobium in enhancing aluminum resistance of plants

Abstract

The invention provides application of rhizobium in enhancing aluminum resistance of plants, and belongs to the technical field of microorganisms. The invention provides an application of a strain of rhizobium Paraburkholderia strydomiana HM3 in enhancing the aluminum resistance of plants, and particularly provides an application of the strain of the rhizobium Paraburkholderia strydomiana HM3 in enhancing the aluminum resistance of the plants. The rhizobium liquid is inoculated to the roots of red bean seedlings in a root irrigation mode, the seedling height, ground diameter and biomass of the red bean seedlings under the aluminum stress condition can be effectively increased, the malondialdehyde content is reduced, the activity of peroxidase and catalase is enhanced, the chlorophyll and proline content and the root carbon and nitrogen content, the stem potassium content, the leaf potassium content and the phosphorus content are increased, and the yield of the red bean seedlings is increased. Therefore, the aluminum tolerance of the ormosia hosiei is enhanced, and an effective way is provided for cultivation of high-quality nursery stocks of the ormosia hosiei resistant to aluminum and acid and cultivation of the ormosia hosiei in southern acid-aluminum stress soil.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to the application of a strain of rhizobia in enhancing plant aluminum tolerance. Background Art

[0002] Aluminum is the most abundant metallic element in the lithosphere. In neutral or alkaline soils, aluminum usually exists in the form of insoluble or poorly soluble minerals such as silicates, phosphates, sulfides, and oxides, causing low or no toxicity to plants. However, under acidic soil (pH < 5.5) conditions, the solubility of aluminum increases, and aluminum ions are released under the action of H + ions, thus causing toxicity to plants.

[0003] Aluminum ions inhibit the growth of plant roots and shoots, interrupt the absorption of plant water and nutrients, and hinder the physiological, biochemical, and metabolic processes of plants, thereby affecting plant growth and development and leading to a decline in plant quality, yield, and soil fertility (Rahman et al., 2024). Aluminum is not an essential element for plant growth and development. For most plants, even micromolar (10 μmol) Al 3+ can rapidly inhibit root growth (Eriko, et al., 2018; He et al., 2019; Vance et al., 2021). However, for a few plants, low concentrations of aluminum can promote plant growth. For example, for tea plants, when the aluminum concentration is less than 2 mM, it can promote their growth. In addition, aluminum toxicity causes excessive production of reactive oxygen species at the plant root tip, leading to oxidative burst, and lipid peroxidation causes protein and DNA denaturation, thereby affecting the physiological characteristics, photosynthetic characteristics, and mineral nutrition metabolism of plants (Guo et al., 2018; Rahman et al., 2024).

[0004] Ormosia hosiei Hensl. et Wils., belonging to the genus Ormosia of the family Fabaceae, is a species endemic to China, a national second-class protected and endangered plant, an evergreen or deciduous tree, which can be used as a garden tree and a scenic tree, and is also an excellent material for wood carving and high-grade furniture. It mainly grows on acidic soils in the south and is listed as one of the important tree species in the national precious tree species list and the national reserve forest construction.

[0005] Plants of the Fabaceae family are the third largest angiosperm family after the Asteraceae and Orchidaceae families. They have a vast root system, grow rapidly, and have extremely strong ecological adaptability, showing good development prospects and application values. Rhizobia symbiotically fix nitrogen with Fabaceae plants, and their nitrogen fixation can improve soil structure, increase the organic matter content of the soil, enhance soil fertility, reduce the input of nitrogen fertilizers, and thus improve the soil ecological environment (Zhao Yezhou et al., 2013; Chen Wenxin and Wang Entao, 2011). Therefore, screening a rhizobial strain that can enhance the aluminum tolerance of Ormosia hosiei to improve the acid and aluminum tolerance of Ormosia hosiei seedlings, enhance its adaptability to acid-aluminum stress and special environments, and lay a foundation for improving the artificial forest cultivation technology of Ormosia hosiei in acid-aluminum tolerant environments, increasing the reserve resources of precious timber of Ormosia hosiei, and promoting the ecological protection and restoration of aluminum mining areas. Summary of the Invention

[0006] The purpose of the present invention is to provide an application of a rhizobial strain in enhancing the aluminum tolerance of plants, improve the aluminum tolerance of Ormosia hosiei, guide its cultivation in acid-aluminum tolerant environments, protect rare species, and promote the ecological protection and restoration of aluminum mining areas.

[0007] To achieve the above-mentioned invention purposes, the present invention provides the following technical solutions:

[0008] The present invention provides an application of a rhizobial strain in enhancing the aluminum tolerance of plants, and the rhizobial strain is Paraburkholderia strydomiana HM3.

[0009] Preferably, the usage method is to inoculate the bacterial liquid of the rhizobial strain onto the roots of plants by the root irrigation method.

[0010] Preferably, the OD 600 of the bacterial liquid of the rhizobial strain is 0.6 - 0.8, and the dosage is 100 - 120 ml / pot.

[0011] Preferably, the plant is Ormosia hosiei of the Fabaceae family.

[0012] The rhizobial strain provided by the present invention has excellent aluminum tolerance. Inoculating this strain can increase the seedling height, ground diameter, and biomass of Ormosia hosiei seedlings under aluminum stress conditions, reduce the malondialdehyde content, enhance the activities of peroxidase and catalase, increase the chlorophyll and proline contents, as well as the nitrogen content in roots, potassium content in stems, potassium content in leaves, and phosphorus content, thereby enhancing the aluminum tolerance of Ormosia hosiei and providing an effective way for the cultivation of Ormosia hosiei on acid-aluminum stressed soils in the south. Brief Description of the Drawings

[0013] Figure 1Growth results of strains No. 15 (black line), No. 16 (red line), and No. 17 (blue line) in YMA liquid medium with different concentrations of AlCl3 in Example 1 (a - d: AlCl3 concentrations are 100, 200, 300, and 400 μmol / L respectively).

[0014] Figure 2 Growth morphology and growth curve of strain No. 16 on YMA agar medium in Example 1.

[0015] Figure 3 Phylogenetic tree of strain No. 16 and other related strains in Example 1.

[0016] Figure 4 Measurement results of seedling height and ground diameter of ormosia seedlings in Example 2.

[0017] Figure 5 Measurement results of dry weights of roots, stems, and leaves of ormosia seedlings in Example 2.

[0018] Figure 6 Measurement results of SS, SP, and PRO of ormosia seedlings in Example 2.

[0019] Figure 7 Measurement results of MDA content, SOD activity, POD activity, and CAT activity of ormosia seedlings in Example 2.

[0020] Figure 8 Measurement results of chlorophyll content (chla, chlb, chlc) of ormosia seedlings in Example 2.

[0021] Figure 9 Measurement results of C and N contents in roots, stems, and leaves of ormosia seedlings in Example 2.

[0022] Figure 10 Measurement results of P and K contents in roots, stems, and leaves of ormosia seedlings in Example 2. Specific implementation mode

[0023] The technical solutions provided by the present invention are described in detail below in combination with the examples, but they cannot be understood as limiting the protection scope of the present invention.

[0024] Example 1 Screening of aluminum - tolerant rhizobia

[0025] Activated culture: Among the 31 rhizobia isolated from the root nodules of Ormosia hosiei collected from 18 townships in 3 provinces of Guizhou, Sichuan and Fujian, 10 rhizobial strains with better acid tolerance (Table 1) were selected and inoculated onto YMA agar medium for activation, and cultured in an inverted position at 28 °C for 5 d. YMA agar medium formula: mannitol 10 g; K2HPO4 0.5 g; MgSO4·7H2O 0.2 g; NaCl 0.2 g; yeast extract 0.8 g; 0.5% Na2MoO4 4 ml; H3BO3 4 ml; agar 18 - 20 g; sterile water 1 L, pH 7.0.

[0026] Table 1 Test strains

[0027]

[0028] Plate culture detection: Set YMA agar medium plates with AlCl3 concentrations of 0, 100, 200, 300 and 400 μmol / L respectively. Use a sterilized toothpick to pick single colonies and spot-inoculate them on the aluminum-containing plates, with 3 spots on each plate. Put the plates into an incubator and culture at 28 °C for 5 d, and observe the growth of the strains on the plates.

[0029] Growth curve determination: Inoculate the activated strains into YMA liquid medium, and culture them in a shaker at 28 °C and 120 r / min until OD 600 is about 2.4. Divide the YMA liquid medium with AlCl3 concentrations of 0, 100, 200, 300 and 400 μmol / L into conical flasks at 50 ml / bottle, with 3 replicates for each treatment. Respectively pipette 100 μl of the bacterial solution and inoculate it into the conical flasks, and culture it in a shaker at 28 °C and 120 r / min. Measure the OD value every 12 h for a total of 5 times, and draw a growth curve based on the OD value.

[0030] As can be seen from Table 1, among the 10 selected rhizobia, strains 16, 18 and 19 can grow in an environment with pH 4.0 and have the strongest acid tolerance; strains 2, 15, 16, 17 and 29 can all grow on YMA agar plates with 100 - 200 μmol / L AlCl3, but the strains that can grow on YMA agar medium with 300 - 400 μmol / L AlCl3 are strains 15, 16 and 17; in YMA liquid medium, the strains with the fastest growth are strains 15, 16 and 17, and strain 16 grows better than strains 15 and 17 in YMA liquid medium with different concentrations of AlCl3 (100, 200, 300, 400 μmol / L) ( Figure 1 ). Therefore, through comprehensive analysis, strain 16 was screened out as the aluminum-tolerant rhizobium. This strain forms round, neat-edged, convex, viscous and milky white colonies on YMA agar medium ( Figure 2 a), and the growth curve is asFigure 2 b. The 16S rDNA sequencing of this strain was carried out, and the obtained 1228 bp sequence is shown as SEQ ID NO:1 below:

[0031] SEQ ID NO:1:

[0032]

[0033] As Figure 3 can be seen, through comparison, it is determined that this strain is Paraburkholderia strydomiana HM3 (NCBI accession number: OQ076255.1).

[0034] Example 2 Pot experiment

[0035] Germination acceleration: Select plump Ormosia hosiei seeds, disinfect the surface with 0.5% potassium permanganate solution for 30 min, rinse with sterile water 5 times. After piercing the seed coat with a small knife (without piercing the endosperm), soak in clean water for 4 d, changing the water once a day. After the seeds imbibe water, place them in a sterile germination box to germinate.

[0036] Seedling raising: When the germinated seedlings grow to about 4 cm, transplant them into 17 cm×15 cm×12 cm flower pots, 3 plants per pot. The substrate is peat soil:vermiculite:perlite = 1:1:1. Before transplantation, sterilize and disinfect the substrate with carbendazim and 0.1% potassium permanganate.

[0037] Inoculation: One month after transplanting the Ormosia hosiei seedlings, inoculate the selected aluminum-tolerant strain HM3 into YMA liquid medium (pH 7.0), shake culture on a shaker at 120 r / min and 28 °C until the OD 600 value is 0.8. Then inoculate the Ormosia hosiei seedlings by root irrigation method, 100 ml per pot, inoculate once a month for a total of 3 times.

[0038] Aluminum stress: After randomly checking the formation of root nodules, select healthy Ormosia hosiei plants with consistent growth from the inoculated group and the non-inoculated group for the acid aluminum stress test. The AlCl3 concentration is set to 0 and 1.2 mmol / L, pH 4.5, and the treatment lasts for 8 weeks.

[0039] Measure the growth, physiological indexes and nutrient elements of the Ormosia hosiei after the above AlCl3 treatment

[0040] 1. Measurement of growth indexes:

[0041] Seedling height and ground diameter: Measure the seedling height with a ruler and the ground diameter with a vernier caliper;

[0042] Biomass: After blanching the seedlings, dry them in an oven at 80 °C until constant weight, weigh them with an analytical balance, and record the dry matter weights of roots, stems and leaves. The results are as Figure 4 and Figure 5 shown.

[0043] From Figure 4 and Figure 5It can be seen that without aluminum stress (AlCl3 concentration is 0 mmol / L), the seedling height, ground diameter, root dry weight, and stem dry weight of Ormosia hosiei seedlings inoculated with HM3 can reach 15.52 cm, 4.13 mm, 1.58 g, and 0.75 g respectively, which are increased by 15.36%, 8.03%, 10.96%, and 17.27% respectively compared with non-inoculated Ormosia hosiei, and all show significant differences; under aluminum stress (AlCl3 concentration is 1.2 mmol / L), the seedling height, ground diameter, root dry weight, stem dry weight, and leaf dry weight of Ormosia hosiei seedlings inoculated with HM3 can reach 18.15 cm, 4.34 mm, 1.67 g, 0.94 g, and 0.94 g respectively, which are increased by 9.00%, 14.91%, 11.69%, 17.88%, and 16.02% respectively compared with non-inoculated Ormosia hosiei, and all show significant differences.

[0044] 2. Determination of physiological indexes

[0045] The content of malondialdehyde (MDA) was determined by the thiobarbituric acid (TBA) method (Wang Xuekui and Huang Jianliang, 2015); the activity of superoxide dismutase (SOD) was determined by the nitroblue tetrazolium (NBT) photoreduction method (Chen Jianxun and Wang Xiaofeng, 2002); the activity of peroxidase (POD) was determined by the guaiacol colorimetric method (Lu Wenjing and Li Yisong, 2012); the activity of catalase (CAT) was determined by the ultraviolet absorption method (Li Hesheng, 2000); the content of soluble sugar (SS) was determined by the anthrone colorimetric method (Wang Xuekui and Huang Jianliang, 2015); the content of soluble protein (SP) was determined by the bicinchoninic acid (BCA) method (Wang Xuekui and Huang Jianliang, 2015); the content of proline (PRO) was determined by the acidic ninhydrin staining method (Li Hesheng, 2000); chlorophyll (chl) was extracted and determined with 80% acetone. The results are as Figures 6 - 8 shown.

[0046] It can be Figures 6 - 8 seen that without aluminum stress (AlCl3 concentration is 0 mmol / L), the POD activity, SS, and Pro content of Ormosia hosiei inoculated with HM3 can reach 644.58 U·g -1 FW, 16.73 mg·g -1 FW, 26.15 μg·g -1 FW respectively, which are increased by 39.48%, 12.38%, and 30.55% respectively compared with non-inoculated Ormosia hosiei, and all show significant differences; under aluminum stress, the MDA of Ormosia hosiei inoculated with HM3 is 67.30 nmol·g -1 FW, which is decreased by 10.93% compared with non-inoculated Ormosia hosiei, showing a significant difference; at the same time, under aluminum stress (AlCl3 concentration is 1.2 mmol / L), the POD and CAT activities of Ormosia hosiei inoculated with HM3 are 590.91 and 37.48 U·g -1FW, the contents of chla, chlb, chlc and Pro were 20.36, 11.97 and 2.41 mg·cm -1 , compared with the non-inoculated Ormosia hosiei, they were increased by 39.51%, 34.90%, 12.99%, 7.91% and 18.12% respectively, and all the differences were significant.

[0047] 3. Determination of nutrient element contents

[0048] After the roots, stems and leaves of the plants were dried to a constant total weight, all the root, stem and leaf samples of each treatment were mixed evenly, ground with a pulverizer, and then sieved through a sieve with a diameter of 0.45 mm for the determination of nitrogen (N), phosphorus (P) and potassium (K) in the plants. After digestion with H2SO4-H2O, nitrogen (N) and phosphorus (P) were determined by an automatic chemical analyzer, and potassium (K) was determined by the flame photometer method; carbon (C) was determined by the potassium dichromate-sulfuric acid oxidation method. The results are as Figure 9 and Figure 10 shown.

[0049] It can be seen from Figures 9 - 10 that without aluminum stress (AlCl3 concentration was 0 mmol / L), the contents of N, P and K in the roots of Ormosia hosiei inoculated with HM3 could reach 18.87, 1.98 and 30.73 g·kg -1 , the contents of P and K in the stems could reach 1.84 and 28.67 g·kg -1 , and the contents of P and K in the leaves could reach 1.25 and 38.49 g·kg -1 , compared with the non-inoculated Ormosia hosiei, they were increased by 25.96%, 60.50% and 18.91%, 3.97% and 13.92%, 11.83% and 17.86% respectively, and all the differences were significant; under aluminum stress (AlCl3 concentration was 1.2 mmol / L), the C contents in the roots and stems of Ormosia hosiei inoculated with HM3 could reach 664 and 745 g·kg -1 , compared with the non-inoculated Ormosia hosiei, they were increased by 21.01% and 7.85% respectively, and all the differences were significant; the contents of K in the stems, K and P in the leaves could reach 25.07, 41.38 and 0.87 g·kg -1 , compared with the non-inoculated Ormosia hosiei, they were increased by 8.65%, 24.14% and 12.05% respectively, and all the differences were significant.

[0050] In summary, inoculating with the aluminum-tolerant rhizobium HM3 significantly increased the seedling height, ground diameter and biomass of Ormosia hosiei, while significantly reducing the malondialdehyde content, significantly increasing the activities of peroxidase and catalase, the contents of chlorophyll and proline, and the contents of root carbon and nitrogen, stem potassium, leaf potassium and phosphorus, thus enhancing the aluminum tolerance of Ormosia hosiei.

[0051] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a rhizobium strain in enhancing aluminum tolerance of plants, characterized in that: The rhizobium is Paraburkholderia strydomiana HM3.

2. The use according to claim 1, characterized in that The method of use is to inoculate the rhizobium solution into the roots of plants using the root irrigation method.

3. The use according to claim 2, characterized in that The OD of the bacterial solution of the rhizobium 600 =0.6~0.8, dosage is 100~120ml / pot.

4. The use according to claim 3, characterized in that The plant is the orchid tree of the leguminous family.

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