Bacteria of the genus ralstonia and their use in increasing iron deficiency tolerance in citrus

Abstract

The present application relates to a kind of Raoultella bacteria and its application in improving the iron deficiency tolerance of citrus, and the Raoultella bacteria is Raoultella ornithinolytica HZAU-P3, which was preserved in China Center for Type Culture Collection on January 9, 2023, with the preservation number CCTCC NO: M 2023048. Experiments have proved that applying HZAU-P3 to citrus and Arabidopsis thaliana root system can improve the iron deficiency tolerance of plants, mainly manifested as the increase of plant lateral roots and the promotion of plant growth under iron deficiency stress. The present application has important application value.

Description

Technical Field

[0001] This invention relates to the field of solutions for iron deficiency in plants, specifically to a bacterium of the genus *Lanola* and its application in improving the iron deficiency tolerance of citrus. Background Technology

[0002] Iron is an essential element for plant growth and development, participating in many physiological processes, including photosynthesis; however, plants have difficulty directly absorbing and utilizing Fe from the soil. 3+ Iron deficiency in plants leads to a decrease in chlorophyll content, resulting in yellowing symptoms. Prolonged iron deficiency damages the normal structure of chloroplasts, thus affecting photosynthetic efficiency. In many citrus-producing areas of western my country, the soil is primarily alkaline and calcareous, making iron deficiency and yellowing of trees widespread and severely impacting citrus yield and quality. Currently, iron deficiency and yellowing are common in citrus orchards, but the commonly used fertilization methods are not only ineffective but also pollute the environment and damage the rhizosphere microbial community. Therefore, identifying rhizosphere microorganisms that promote iron absorption is one of the most effective means to solve iron deficiency and yellowing in citrus. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a bacterium of the genus *Lanolarioides* and its application in improving the iron deficiency tolerance of citrus fruits, in order to address the above-mentioned shortcomings.

[0004] To solve the above technical problems, the present invention adopts the following technical solution:

[0005] A bacterium of the genus Raoultella, ornithine-lysinic Raoultella HZAU-P3, was deposited on January 9, 2023, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2023048.

[0006] Furthermore, the application of the aforementioned *Lanolarioides* bacteria in improving the iron deficiency tolerance of citrus fruits.

[0007] Furthermore, specifically, the plant roots are treated with an inoculum containing the aforementioned *Lanolarioides* bacteria.

[0008] Furthermore, OD containing Lanau's bacteria inoculants 600nm The value is 0.2.

[0009] The beneficial effects of this invention are as follows: Experiments have shown that the HZAU-P3 provided by this invention can promote the growth and development of Arabidopsis thaliana and citrus trifoliate orange under iron deficiency stress, and improve the iron deficiency tolerance of Arabidopsis thaliana and citrus. This invention has significant application value.

[0010] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0011] Figure 1 Schematic diagram of the effect of ornithine-degrading Raoulbacterium tumefaciens HZAU-P3 on Arabidopsis seedling growth in a completely iron-deficient medium;

[0012] Figure 2 A schematic diagram illustrating the effect of ornithine-degrading Raoulbacterium tumefaciens HZAU-P3 on the growth of Arabidopsis thaliana seedlings in alkaline iron-deficient medium;

[0013] Figure 3 A schematic diagram illustrating the effect of ornithine-releasing Raoulbacterium tumefaciens HZAU-P3 on Arabidopsis seedling growth in alkaline medium supplemented with an indicator;

[0014] Figure 4 and Figure 5 The results show the statistical effects of the number of lateral roots and the length of the taproot of Arabidopsis thaliana seedlings in different iron-deficient conditions on the effects of ornithine-releasing Raoulbacterium ravellae HZAU-P3 on the number of lateral roots and the length of the taproot of Arabidopsis thaliana seedlings in different iron-deficient conditions.

[0015] Figure 6 A comparison of the growth of trifoliate orange plants under iron deficiency stress after treatment with ornithine-releasing bacterium HZAU-P3 and the untreated control. Detailed Implementation

[0016] The following embodiments are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores. All quantitative experiments in the following embodiments were performed in triplicate, and the results were averaged.

[0017] LB liquid medium: Dissolve 10g of tryptone, 5g of yeast extract and 5g of sodium chloride in 1L of distilled water, then sterilize at 121℃ for 15min and use after cooling.

[0018] LB solid medium: Add agar to the LB liquid medium to a concentration of 15 g / L; then sterilize at 121°C for 15 min. Pour the liquid medium, cooled to approximately 55°C, into petri dishes and allow to cool naturally.

[0019] The wild-type Arabidopsis thaliana Columbia-0 subtype used will be referred to as Arabidopsis thaliana in the following text.

[0020] (1) Isolation, identification and preservation of Raoultella ornithinolytica HZAU-P3.

[0021] I. Isolation of Raoultella ornithinolytica HZAU-P3

[0022] 1. Add 5g of rhizosphere soil sample (collected from the rhizosphere of citrus trees at Menghu Farm, Quanzhou, Fujian) to 45mL of phosphate buffer, stir for 15min, let stand for 10min, then take 1mL of the supernatant and add it to a sterile test tube containing 9mL of phosphate buffer and mix thoroughly (the dilution at this point is recorded as 10). -1 Then, take 1 mL from the test tube and add it to another sterile test tube containing 9 mL of phosphate buffer, mix thoroughly, and so on to prepare 10 mL of phosphate buffer. -2 10 -3 10 -4 10 -5 Different dilutions of bacterial suspension. Take 0.1 mL of each dilution gradient and spread it evenly on LB solid medium. Incubate at 28°C for 2-3 days.

[0023] 2. After completing step 1, pick a single colony from the LB solid medium and purify it repeatedly at least 3 times. Name the screened bacteria HZAU-P3.

[0024] II. Identification of bacteria HZAU-P3

[0025] 16SRNA sequence homology analysis

[0026] The 16SRNA of bacteria HZAU-P3 was compared with the sequence in NCBI. The results showed that bacteria HZAU-P3 had the highest homology with the Raoultella ornithinolytica strain, reaching 99.87%.

[0027] III. Preservation

[0028] Based on the above 16SRNA sequence homology analysis results, the bacteria HZAU-P3 obtained from the isolation and purification in step one was identified as belonging to the genus *Lanolario*.

[0029] Bacterial culture preservation: A single colony of *Rauvolfia ornithine-releasing* HZAU-P3 was inoculated onto LB liquid medium and cultured at 28°C and 200 rpm for 8 h to obtain a bacterial culture. One volume of the bacterial culture was mixed with one volume of 60% (v / v) glycerol and stored at -80°C.

[0030] (2) Preparation of Ornithine-Rauvolfia HZAU-P3 bacterial agent

[0031] 1. Activate the bacterial culture stored at -80℃ in Example 1 on LB solid medium.

[0032] 2. Pick a single colony from LB solid medium and inoculate it into a sterilized Erlenmeyer flask (250 mL) containing 100 mL of LB liquid medium. Incubate at 28°C and 200 rpm for 8 hours. The resulting bacterial culture will have an OD value of [missing value]. 600nm The value was adjusted to 0.2, and the bacterial solution was the prepared ornithine-releasing Raoulbacterium tumefaciens HZAU-P3 bacterial agent.

[0033] (3) The role of Ornithine-Rauvolfia HZAU-P3 in improving iron deficiency tolerance in plants. I. Preparation of culture medium

[0034] Petri dish dimensions: 10cm × 10cm

[0035] Completely iron-deficient 1 / 2MS solid medium: Dissolve 2.23g of customized iron-free 1 / 2MS basic medium powder (Coolaber) and 20g of agar in distilled water, then bring the volume to 1L with distilled water, adjust the pH to 5.8, and sterilize at 121°C for 15 minutes. Pour the liquid medium, which has cooled to about 55°C, into petri dishes and allow it to cool naturally before use.

[0036] Alkaline iron-deficient 1 / 2MS solid medium: Dissolve 2.23g of 1 / 2MS basic medium powder (Coolaber) and 20g of agar in distilled water, then bring the volume to 1L with distilled water, adjust the pH to 8, and then sterilize at 121℃ for 15 minutes. Pour the liquid medium cooled to about 55℃ into petri dishes and allow it to cool naturally before use.

[0037] Alkaline iron-deficient 1 / 2MS solid medium with added indicator: Dissolve 2.23g of 1 / 2MS basic medium powder (Coolaber) and 20g of agar in distilled water, add 25mg of bromocresol purple indicator, then bring the volume to 1L with distilled water, adjust the pH to 8, and then sterilize at 121℃ for 15 minutes. Pour the liquid medium cooled to about 55℃ into petri dishes and allow it to cool naturally before use.

[0038] II. Effects of Ornithine-Rauvolfia HZAU-P3 inoculant on iron deficiency tolerance in plants

[0039] The cultivation conditions were: 22℃; 14h light / 10h dark; light intensity of 5000Lx.

[0040] 1. Take Arabidopsis thaliana seeds, treat them with 75% alcohol solution for 30 seconds, then wash them three times with sterile water, then treat them with 2.6% (v / v) sodium hypochlorite aqueous solution for 6 minutes, and finally wash them three times with sterile water.

[0041] 2. After completing step 1, Arabidopsis seeds were sown on normal 1 / 2 MS solid medium and vernalized at 4℃ for two days. Then, they were placed in a constant temperature incubator at 22℃ with 14h light / 10h darkness and a light intensity of 5000Lx for 7 days.

[0042] 3. Select Arabidopsis seedlings from step 2 and transplant them into three media: completely iron-deficient 1 / 2 MS solid medium, alkaline iron-deficient 1 / 2 MS solid medium, and alkaline iron-deficient 1 / 2 MS solid medium with added indicator. Place 5 seedlings in each medium. Divide the seedlings into a control group and a treatment group. The control group received 2 μL of sterile water inoculated at the root tip of each Arabidopsis seedling; the treatment group received 2 μL of inoculant inoculated at the root tip of each Arabidopsis seedling. The treatment method is described in [link to treatment instructions]. Figure 1-3 After subsequent vertical cultivation, the roots of Arabidopsis thaliana grew to the culture medium inoculated with the inoculum (this experiment studied the effect of the inoculum's secretions on the root growth of Arabidopsis thaliana).

[0043] 4. After completing step 3, culture the completely iron-deficient 1 / 2 MS solid medium, the alkaline iron-deficient 1 / 2 MS solid medium, and the alkaline iron-deficient 1 / 2 MS solid medium with added indicator horizontally for 14 days and observe the growth of Arabidopsis thaliana.

[0044] 5. The growth status of Arabidopsis thaliana is as follows: Figure 1-3 (Left side is control group, right side is ornithine-free Raouli HZAU-P3 treatment).

[0045] 6. Observe the growth and development phenotypes of Arabidopsis thaliana among different treatment groups, and count the root length and number of lateral roots of the plants, as follows: Figure 4-5 .

[0046] The results showed that under the inoculated conditions, Arabidopsis thaliana grown on the culture medium grew well, with normal leaf condition and a significantly increased number of lateral roots. In contrast, the Arabidopsis thaliana in the control group exhibited severe leaf yellowing, poor root development, and was severely affected by iron deficiency stress. This indicates that *Ornithine Raoultella* HZAU-P3 plays an important role in promoting plant salt and alkali tolerance.

[0047] (4) Application of Ornithine-Rauvolfia HZAU-P3 in improving iron deficiency tolerance of Citrus aurantium

[0048] The cultivation conditions were: 25℃; 14h light / 10h dark; light intensity of 5000Lx.

[0049] 1. Take citrus trifoliate orange seeds, treat them with 75% alcohol solution for 30 seconds, then wash them three times with sterile water, then treat them with 2.6% (v / v) sodium hypochlorite aqueous solution for 6 minutes, and finally wash them three times with sterile water.

[0050] 2. After completing step 1, sow the trifoliate orange seeds in vermiculite and place them in a constant temperature incubator at 25℃ with 14h light / 10h darkness and a light intensity of 5000Lx. Transplant the seeds after they germinate.

[0051] 3. Transplant the seedlings of the trifoliate orange after completing step 2 into pots containing alkaline substrate soil mixed with quicklime, 2 seedlings per pot, to obtain 18 pots of trifoliate orange seedlings, for a total of 36 seedlings.

[0052] 4. Take 18 trifoliate orange trees and inoculate the OD (dry seed) using the root drenching method. 600nm Inoculate each seedling with 5 mL of *Raoultella ornithine-releasing* inoculum with a concentration of 0.2. Take another 18 *Citrus trifoliata* seedlings and inoculate each seedling with 5 mL of liquid LB medium.

[0053] 5. Place the trifoliate oranges that have completed step 4 into a constant temperature incubator with 14 hours of light and 10 hours of darkness and a light intensity of 5000 Lx for 30 days, and observe the growth of the trifoliate oranges.

[0054] 6. By Figure 6 It can be seen that, compared with the control, HZAU-P3 can enhance the iron deficiency tolerance of trifoliate orange.

[0055] The above description provides examples of the preferred embodiments of the present invention. Parts not detailed herein are common knowledge to those skilled in the art. The scope of protection of the present invention is determined by the claims. Any equivalent modifications based on the technical teachings of the present invention are also within the scope of protection of the present invention.

Claims

1. Bacteria of the genus *Lanola*, characterized by: Raoultella ornithinolytica HZAU-P3 was deposited at the China Center for Type Culture Collection on January 9, 2023, with accession number CCTCC NO: M2023048.

2. The application of the *Lanolarioides* bacteria as described in claim 1 in improving the iron deficiency tolerance of citrus fruits.

3. A method for improving the iron deficiency tolerance of citrus fruits, characterized in that, Specifically, the plant roots are treated with an inoculum containing the Lanorbacterium bacteria described in claim 1.

4. The method for improving the iron deficiency tolerance of citrus fruits according to claim 3, characterized in that, OD containing Lanau's bacteria 600nm The value is 0.2.

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