A rhizosphere iron mobilizing bacterial strain for alleviating iron deficiency chlorosis of litsea cubeba and application thereof

Abstract

This invention discloses a rhizosphere iron-promoting bacterial strain that alleviates iron deficiency-induced premature aging in Litsea cubeba and its applications. It belongs to the field of agricultural microbiology and plant mineral nutrition regulation technology. This invention provides related products and specific applications. The strain of this invention possesses both ferric iron (Fe3+) reduction ability and siderophore production ability. On the one hand, it can promote the conversion of ferric iron to ferrous iron (Fe2+); on the other hand, it can chelate and activate iron in the rhizosphere environment through siderophores, thereby increasing the supply of active iron in the rhizosphere of Litsea cubeba. After the Litsea cubeba rhizosphere iron-reducing bacterium LcB6 is reintroduced, it can increase the chlorophyll content of Litsea cubeba leaves and the soluble iron content of the plant, increase the content of ferrous iron and available iron in the rhizosphere soil, and improve iron deficiency-induced premature aging phenotypes such as leaf yellowing and decreased plant growth vigor. This provides a new bacterial resource and application pathway for the regulation of rhizosphere iron nutrition in Litsea cubeba.

Description

Technical Field

[0001] This invention relates to the fields of agricultural microorganisms and plant mineral nutrition regulation technology, and more specifically to a rhizosphere iron-promoting strain that alleviates iron deficiency-induced premature aging in Litsea cubeba and its application. Background Technology

[0002] Litsea cubeba ( Litsea cubeba Litsea cubeba is an important woody spice and medicinal plant belonging to the genus Litsea in the Lauraceae family, widely distributed in subtropical regions of my country. Its fruit is rich in volatile components such as citral and linalool, and has high application value in the food, pharmaceutical, daily chemical, and natural fragrance industries. With the expansion of artificial cultivation of Litsea cubeba, problems such as leaf yellowing, decreased growth vigor, and premature aging during cultivation have gradually attracted attention. Among these, insufficient iron availability is one of the important factors inducing physiological premature aging in Litsea cubeba.

[0003] Iron is an essential micronutrient for chlorophyll synthesis, electron transport in photosynthesis, and the maintenance of various redox enzyme activities in plants. Iron deficiency senescence in Litsea cubeba typically manifests as yellowing of new leaves, decreased chlorophyll content, slow growth, and premature leaf drop. In severe cases, it affects plant growth, fruit yield, and essential oil quality. While Litsea cubeba cultivation areas in southern China are mostly acidic red soils, and the total amount of iron in the soil may not necessarily be deficient, the iron exists primarily in the form of insoluble ferric iron (Fe3+) or mineral-bound iron. The content of ferrous iron (Fe2+) and available iron, which can be directly absorbed and utilized by plants, is low, leading to the possibility of iron deficiency chlorosis and premature senescence in plants during their growth.

[0004] Currently, common methods for iron supplementation in production include applying inorganic iron fertilizers to the soil, foliar spraying of chelated iron, and the application of organic amendments. These methods can alleviate iron deficiency symptoms in plants to some extent, but they still have problems such as short-lasting effects, frequent application, high costs, and decreased utilization rates after soil fixation. Especially in the cultivation of perennial woody plants such as Litsea cubeba, it is difficult to maintain the supply of available iron in the rhizosphere in the long term by relying solely on exogenous iron fertilizers, and it is also difficult to improve the efficiency of iron form conversion in the rhizosphere microenvironment.

[0005] Rhizosphere microorganisms can influence soil iron activation and plant iron uptake through mechanisms such as reducing ferric iron (Fe3+), secreting siderophores, producing organic acids, and regulating the rhizosphere microenvironment. Specifically, iron reduction converts poorly soluble or less available ferric iron into more readily available ferrous iron (Fe2+), while siderophores form stable complexes with ferric iron in the environment, enhancing iron mobility and bioavailability in the rhizosphere. For perennial woody plants such as Litsea cubeba, screening rhizosphere functional strains with both iron reduction and siderophore production capabilities can help improve rhizosphere iron supply at both the iron speciation and iron activation levels.

[0006] Therefore, it is necessary to screen indigenous functional strains from the acidic rhizosphere red soil of Litsea cubeba that can adapt to this type of rhizosphere environment and have the ability to reduce ferric iron and produce iron carriers. The effects of these strains on improving leaf yellowing, plant iron content, and rhizosphere active iron supply in Litsea cubeba can be verified by pot inoculation. This will lead to the development of a microbial application method suitable for regulating iron deficiency premature aging of Litsea cubeba in acidic red soil areas in southern China. Summary of the Invention

[0007] In view of this, the present invention provides a rhizosphere iron-promoting strain that alleviates iron deficiency-induced premature aging in Litsea cubeba and its application. Specifically, it relates to a rhizosphere iron-reducing bacterium derived from acidic red soil rhizosphere of Litsea cubeba, possessing both ferric iron reduction capacity and iron-producing capacity. Klebsiella variicola LcB6, this strain can improve the level of active iron in the rhizosphere of Litsea cubeba by reducing ferric iron and secreting siderophores, thereby improving the iron absorption of plants and alleviating leaf yellowing and premature aging caused by insufficient iron availability, providing a microbial regulatory pathway for the healthy cultivation of Litsea cubeba.

[0008] Preservation Information: Iron-reducing bacteria in the rhizosphere of Litsea cubeba ( Klebsiella variicola LcB6, deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China; deposit date: May 15, 2025; accession number: CCTCC NO: M 20251072.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A rhizosphere iron-promoting bacterial strain that alleviates iron deficiency-induced premature aging in Litsea cubeba, the strain being: Litsea cubeba rhizosphere iron-reducing bacterium (… Klebsiella variicola The LcB6 accession number is CCTCC NO: M 20251072.

[0011] The present invention also provides a biological agent comprising the above-mentioned strains.

[0012] The present invention also provides a bio-fertilizer comprising the above-mentioned strains.

[0013] The present invention also provides the application of the above-mentioned strains, or the above-mentioned microbial agents, or the above-mentioned bio-fertilizers in agricultural production.

[0014] Preferred method: The bacterial strain is inoculated into a culture medium, centrifuged to collect the bacterial cells, and then resuspended to prepare a bacterial suspension; the bacterial suspension is then applied to the rhizosphere of Litsea cubeba.

[0015] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a rhizosphere iron-promoting strain that alleviates iron deficiency-induced premature aging of Litsea cubeba and its application, achieving the following technical effects: This invention yielded a functional strain derived from the rhizosphere red soil acidic soil of Litsea cubeba. Klebsiella variicola LcB6 possesses a source base adapted to the acidic rhizosphere environment of Litsea cubeba. This strain combines the ability to reduce ferric iron (Fe3+) and produce siderophores, thereby promoting the conversion of ferric iron to ferrous iron (Fe2+) and enhancing the supply of active iron in the rhizosphere of Litsea cubeba by chelating and activating iron in the rhizosphere environment through siderophores.

[0016] Inoculating the rhizosphere iron-reducing bacterium LcB6 of Litsea cubeba into the rhizosphere of Litsea cubeba can increase the chlorophyll content of Litsea cubeba leaves and the soluble iron content of the plant, increase the content of ferrous iron and available iron in the rhizosphere soil, and improve iron deficiency premature aging phenotypes such as leaf yellowing and decreased plant growth vigor.

[0017] The strain provided by this invention has a clear origin and possesses dual iron-promoting functions of iron reduction and iron-producing carrier. It is easy to apply and can be used as a functional strain for the biological regulation of iron deficiency premature aging in Litsea cubeba, providing new strain resources and application pathways for the regulation of rhizosphere iron nutrition in Litsea cubeba. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 The attached figure shows the colony morphology of LcB6 provided by this invention.

[0020] Figure 2 The attached figure shows the phenotype of the LcB6 siderogenic carrier provided by the present invention on CAS medium.

[0021] Figure 3 The attached figure is a phylogenetic analysis diagram of LcB6 based on the whole genome sequence provided by the present invention.

[0022] Figure 4 The attached figure shows the phenotypic changes of Litsea cubeba seedlings after LcB6 inoculation provided by this invention. In the figure, A represents the whole plant phenotype under different treatments, B represents the leaf phenotype under different treatments, C represents the root phenotype under different treatments, D represents the plant height change under different treatments, E represents the ground diameter change under different treatments, and F represents the rhizosphere soil pH change under different treatments. In the figure, LcB6 represents the inoculation treatment, EDTA-Fe represents the iron supplementation treatment, and Control and CK are blank control treatments.

[0023] Figure 5The attached figure shows the changes in chlorophyll content of Litsea cubeba under different treatments provided by the present invention. In this figure, LcB6 represents the inoculation treatment, EDTA-Fe represents the iron supplementation treatment, and CK represents the blank control treatment.

[0024] Figure 6 The attached figure shows the changes in Fe(II) content in the rhizosphere soil of Litsea cubeba under different treatments provided by this invention.

[0025] Figure 7 The attached figure shows the changes in iron-related indicators in the rhizosphere soil of Litsea cubeba under different treatments provided by this invention.

[0026] Figure 8 The attached figure shows the changes in iron content in different tissues of Litsea cubeba under different treatments provided by the present invention.

[0027] Figure 9 The attached figure is a process flow diagram provided by the present invention. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] This invention discloses a rhizosphere iron-promoting bacterial strain that alleviates iron deficiency-induced premature aging in Litsea cubeba and its application. See the process flow diagram. Figure 9 .

[0030] Example 1 Screening of iron-reducing bacteria in the rhizosphere of Litsea cubeba Rhizosphere soil from *Litsea cubeba* was collected from the experimental forest farm of the Hunan Academy of Forestry Sciences. The soil type was red soil with an acidic pH of 5.2. Rhizosphere soil closely adhering to the roots of *Litsea cubeba* was collected as screening material for bacterial strains. 1 g of rhizosphere soil sample was weighed and added to a 250 mL Erlenmeyer flask containing 50 mL of LB liquid medium. The sample was then incubated at 37℃ and 200 r / min for 24 h with shaking.

[0031] After enrichment culture, the enriched bacterial solution was taken and serially diluted 10-fold with 0.15 mol / L NaCl solution to prepare 10... -5 10 -6 10 -7 and 10 -8 Diluent. Spread 200 μL of the diluent onto a primary screening plate containing ferric citrate and incubate at 37°C for 24–48 h.

[0032] Based on the colony growth on the plates, select single colonies with clear morphology and good growth, and transfer them to new screening plates for streak purification. Repeat the streak purification process three times to obtain pure culture strains. Number the purified strains and inoculate them into slant agar plates. Incubate at 37°C for 24 h, then store at 4°C for later use.

[0033] After enrichment culture, initial plate screening, and streak purification, a total of 14 candidate strains were obtained, numbered B1–B14. After multiple streak tests, the candidate strains formed morphologically consistent single colonies. The colony morphology of strain B6 is shown in […]. Figure 1 .Depend on Figure 1 It can be seen that strain B6 forms relatively stable single colonies on LB solid medium, indicating that after purification, this strain can be used for subsequent iron reduction capacity determination, siderophore detection and pot re-inoculation experiments.

[0034] Example 2 Determination of the iron-reducing ability of iron-reducing bacteria Candidate strains B1–B14 were inoculated into LB broth and cultured at 37°C with shaking for 36 h. After culture, the bacterial suspension was collected, centrifuged, and the supernatant was discarded, retaining the bacterial cell pellet. The bacterial cells were resuspended in sterile water, and the suspension concentration was adjusted to OD0.05. 600 =1.0, which is used as the bacterial suspension to be tested.

[0035] A certain volume of the bacterial suspension to be tested was inoculated into a reaction system containing ferric iron (Fe²⁺) substrate. A blank control (no inoculation) was used. The mixture was incubated at 37°C with shaking. Samples were taken at set times to determine the concentration of ferrous iron (Fe²⁺) in the reaction system. + Content, and based on the initial trivalent iron Fe³⁺ + Calculation of Fe³ addition amount + Reduction rate. Fe³ + The reduction rate is calculated using the following formula: Fe³ + Reduction rate (%) = Fe² generated in the reaction system + Content / Initial Fe³ + Content × 100%.

[0036] The test results showed that Fe²⁺ in the reaction system after B6 treatment + The content increased significantly, Fe³ + The reduction rate was higher than that of the control treatment, indicating that this strain can reduce ferric iron to ferrous iron and has a strong iron reducing ability. Multiple candidate strains obtained from screening were compared under the same conditions, and the results of the ferric iron reducing ability determination of different candidate strains are shown in Table 1.

[0037] Table 1 shows that different candidate strains have different effects on Fe³⁺. + The reducing abilities of the strains varied significantly. Specifically, strain B6 showed the highest Fe²⁺ reduction at 36 hours. + The amount generated reached 55 mM, Fe³ + The reduction rate reached 87%, which was higher than that of other candidate strains, indicating that B6 has a strong ability to reduce ferric iron among the candidate strains. Therefore, B6 was identified as the target strain for subsequent siderophore detection, strain identification, and pot inoculation verification, and was named LcB6.

[0038]

[0039] Example 3 LcB6 Ferrocarrier Production Capacity Testing The siderophore-producing ability of LcB6 was evaluated using CAS assay medium. Purified LcB6 was inoculated onto CAS solid assay medium and incubated at 37℃ for 24–48 h. Color changes around the colonies were observed. Results showed a distinct discoloration zone around the LcB6 colonies, indicating that this strain can produce siderophores and has the ability to chelate ferric iron in the environment.

[0040] The siderogenic phenotype of LcB6 on CAS medium is shown in [reference needed]. Figure 2 .

[0041] Depend on Figure 2 It can be seen that a distinct discoloration zone appeared around the culture medium after LcB6 inoculation; LcB6 can change the CAS medium from blue to yellow or orange-yellow, indicating that this strain has the ability to produce siderophores. Combined with the Fe(III) reduction rate measurement results in Example 2, it shows that LcB6 has both ferric iron reduction and siderophore production functions.

[0042] Example 4 Strain identification LcB6 was inoculated onto LB solid medium and incubated at 37°C for 24 h. Colony morphology was then observed. The results showed that the colonies of this strain were round, smooth, with regular edges, and the colony morphology was relatively uniform.

[0043] Single colonies of LcB6 were picked and inoculated into LB broth and cultured at 37°C with shaking for 24 h. 50 μL of the bacterial culture was transferred to a centrifuge tube and lysed at 100°C for 15 min to serve as a PCR template. PCR amplification was performed using universal primers 27F and 1492R for bacterial 16S rDNA. After agarose gel electrophoresis, samples with clear bands were selected for sequencing.

[0044] The obtained 16S rDNA sequence was submitted to the NCBI database for BLAST homology alignment, and a phylogenetic tree was constructed using the neighbor-joining method with MEGA 11 software. Phylogenetic analysis showed that LcB6 clustered with Klebsiella strains in the same branch. Genomic DNA was extracted from the strain, and whole-genome sequencing and assembly were performed. The obtained whole-genome assembly sequence was submitted to the Type (Strain) Genome Server (TYGS) for genome-wide classification and identification analysis.

[0045] TYGS species identification results show that LcB6 belongs to a known species. Klebsiella variicola Pairwise comparisons showed that LcB6 and Klebsiella variicola The dDDH d4 value of DSM 15968 was 93.2%, with a confidence interval of 91.3%–94.7%, and the difference in G+C content was 0.03%. A phylogenetic tree constructed based on the whole genome sequence showed that LcB6 and... Klebsiella variicola DSM 15968 clustered into closely related branches. Based on TYGS species identification results, dDDH values, differences in G+C content, and phylogenetic analysis results, this strain was identified as... Klebsiella variicola LcB6.

[0046] Phylogenetic analysis results of LcB6 based on whole genome sequence are shown below. Figure 3 The identification and preservation information of the strains are shown in Table 2. Figure 3 It can be seen that LcB6 and Klebsiella variicola DSM 15968 clustered into a closely related branch. Table 2 shows that the TYGS genome-wide classification results of LcB6 support its classification as belonging to... Klebsiella variicola , and Klebsiella variicola The dDDH d4 value of DSM 15968 was 93.2%, and the difference in G+C content was 0.03%. Based on the results of colony morphology, 16S rDNA sequence analysis, whole-genome TYGS classification, and phylogenetic analysis, this strain was identified as *Litsea cubeba* rhizosphere iron-reducing bacterium. Klebsiella variicola LcB6. This strain has been preserved in the culture, with the preservation number CCTCC NO: M 20251072.

[0047]

[0048] Example 5 Potted plant re-grafting experiment LcB6 was inoculated into LB liquid medium and cultured at 37°C with shaking for 24 h to obtain the fermentation broth. After the culture was completed, the cells were collected by centrifugation, resuspended in sterile water, and the concentration of the suspension was adjusted to OD200. 600 =1.0, used as a bacterial suspension for inoculation treatment.

[0049] Two-year-old Litsea cubeba seedlings with uniform growth were selected as experimental materials. The cultivation substrate was prepared by mixing nutrient soil, perlite, and loess in a volume ratio of 3:3:1. The experiment included LcB6 inoculation treatment, EDTA-Fe iron supplementation treatment, and a sterile water control treatment. For the LcB6 inoculation treatment, the bacterial suspension was applied via rhizosphere irrigation, 50 mL per plant, with a concentration of OD0.05. 600 =1.0; EDTA-Fe iron supplementation treatment was administered via rhizosphere irrigation with 0.5 mmol / L EDTA-Fe solution, 50 mL per plant; the sterile water control treatment was administered with an equal volume of sterile water concurrently. All treatments were applied every 7 days for a total of 3 applications. All treatments were cultured under identical conditions, and samples were taken at 0 and 60 days post-treatment to determine plant phenotype, chlorophyll content, soluble iron content in roots, stems, and leaves, as well as Fe(II) and DTPA-Fe content in the rhizosphere soil.

[0050] Phenotypic characteristics of the plant, leaves, and roots after regrafting from pots are shown below. Figure 4 Changes in chlorophyll content are shown in Figure 5 Changes in Fe(II) content in rhizosphere soil are shown in [reference needed]. Figure 6 Changes in rhizosphere soil iron-related indicators are shown in [the table below]. Figure 7 The changes in soluble iron content in different tissues are shown in the figure. Figure 8 The improvement of LcB6 treatment relative to CK at 60 d is shown in Table 3, and the measured data at 0 d and 60 d are shown in Table 4.

[0051]

[0052]

[0053] Depend on Figure 4 It can be seen that, compared with CK, the LcB6-treated plants had greener leaves and better root development, indicating that LcB6 re-inoculation can improve the growth status of Litsea cubeba seedlings. Figure 5 It can be seen that the chlorophyll content of LcB6 treatment was higher than that of CK after 60 days; Figure 6 and Figure 7 It can be seen that LcB6 treatment can increase active iron-related indicators such as Fe(II) and DTPA-Fe in rhizosphere soil; Figure 8It can be seen that LcB6 treatment can increase the soluble iron content in the roots, stems and leaves of Litsea cubeba.

[0054] As shown in Tables 3 and 4, after 60 days of treatment, the chlorophyll content in the LcB6 treatment increased from 3.6 mg / g FW in the control group (CK) to 5.7 mg / g FW, an increase of 58.3%; the Fe(II) content in the rhizosphere soil increased from 3.6 mg / kg to 5.5 mg / kg, an increase of 52.8%; and the DTPA-Fe content increased from 7.4 mg / kg to 9.0 mg / kg, an increase of 21.6%. These results indicate that LcB6 can improve the supply of active iron in the rhizosphere of *Litsea cubeba*, improve the iron nutrition status of the plants, and thus alleviate the iron deficiency-induced premature aging phenotype of *Litsea cubeba*.

[0055] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0056] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A rhizosphere iron-promoting bacterial strain that alleviates iron deficiency-induced premature aging in Litsea cubeba, characterized in that, The strain mentioned is: *Litsea cubeba* rhizosphere iron-reducing bacteria (… Klebsiella variicola The LcB6 accession number is CCTCC NO: M 20251072.

2. A biological agent, characterized in that, It includes the strain described in claim 1.

3. A bio-fertilizer, characterized in that, It includes the strain described in claim 1.

4. The application of the strain of claim 1, the microbial agent of claim 2, or the bio-fertilizer of claim 3 in agricultural production.

5. The application as described in claim 4, characterized in that, The bacterial strain was inoculated into a culture medium, centrifuged to collect the bacterial cells, and then resuspended to prepare a bacterial suspension; the bacterial suspension was then applied to the rhizosphere of Litsea cubeba.

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