Stenotrophomonas sepilia DB3 with disease resistance and iron-dissolving promoting functions and application thereof

Abstract

The application belongs to the technical field of microorganisms and particularly relates to a strain of DB3 with disease-resistant iron-dissolving and growth-promoting functions Stenotrophomonas sepilia The strain DB3 is isolated from the rhizosphere of pear trees in calcareous soil, has good abilities of IAA production, rapid growth, dissolution of insoluble iron, iron carrier production and antagonism to various pathogenic bacteria. Through potting tests, it is shown that the DB3 bacterial agent can significantly promote the growth of peanut and pear seedlings, improve the root development and iron nutrient content, enhance the SPAD value and iron absorption of leaves, and relieve the iron deficiency chlorosis, and can be used for preparing a biological bacterial fertilizer, reducing the input of chemical iron fertilizer, and has a good development and application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain with disease-resistant, iron-dissolving, and growth-promoting functions. And its applications. Background Technology

[0002] Iron is an essential micronutrient for plant growth, playing a vital role in photosynthesis, respiration, nitrogen metabolism, and electron transport. However, despite the abundance of iron in nature, many plants still suffer from iron deficiency chlorosis due to the low bioavailability of iron. This is particularly severe in calcareous soils (with a high pH), where iron exists primarily as insoluble forms such as ferric oxide and ferric hydroxide, making it difficult for plants to directly absorb and utilize, thus exacerbating the iron deficiency problem.

[0003] Typical symptoms of iron deficiency chlorosis include leaf chlorosis, stunted plant growth, and reduced yield, which can even lead to plant death in severe cases. Statistics show that approximately 30%–40% of arable land worldwide suffers from varying degrees of iron deficiency, particularly in arid and semi-arid regions and alkaline soils. To alleviate iron deficiency in plants, researchers have employed various strategies, such as applying chemical iron fertilizers (e.g., EDDHA-Fe), localized soil acidification, and mycorrhizal fungal inoculation. However, these methods suffer from high costs, poor stability, and significant environmental impacts, making them difficult to implement on a large scale in agricultural production. Therefore, there is an urgent need to find efficient, ecological, and safe iron nutrient improvement technologies.

[0004] Plant rhizosphere growth-promoting bacteria (PGPB) play a crucial role in improving soil nutrient supply and promoting plant growth. Some rhizosphere bacteria can enhance iron absorption and utilization by plants and promote root growth by secreting siderophores, dissolving insoluble iron minerals, and secreting organic acids. To address current problems, this paper proposes using strains with highly efficient iron-dissolving capabilities to prepare inoculants to alleviate iron deficiency chlorosis in plants.

[0005] However, systematic research on strains with high iron-solubilizing capabilities is still lacking, especially regarding the development of strains with good adaptability and growth-promoting effects in calcareous soils. Therefore, screening and utilizing strains with high iron-solubilizing capabilities to prepare microbial inoculants is of great significance for alleviating iron deficiency chlorosis in plants and promoting the development of green agriculture. Summary of the Invention

[0006] In view of the above, it is necessary to develop new strains to address the problem of iron deficiency chlorosis in plants, in order to improve the efficiency of iron absorption by plants, promote plant growth, enhance stress resistance, and provide new technical support for the research and application of bio-iron fertilizers.

[0007] To achieve the above objectives, this invention screened out a strain The strain, classified and named as follows: The strain is classified and named Stenotrophomonas maltophilia DB3 in Chinese, with accession number CCTCC NO: M20251606. It is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on July 15, 2025.

[0008] The present invention also includes the components described above. Inoculants for bacterial strains.

[0009] The present invention also provides as described above. Application of the strains or the above-described bacterial agents in the production of IAA, dissolved iron, and / or siderophores.

[0010] The present invention also provides as described above. The application of the strains or the above-mentioned bacterial agents in the preparation of biocontrol agents for the prevention and control of plant pathogens.

[0011] The present invention further explains that the plant pathogens are Alternaria, Bakanae diseased rice, and / or Fusarium oxysporum.

[0012] The present invention also provides as described above. Application of the strains or the above-mentioned microbial agents in the preparation of bio-organic fertilizer.

[0013] The present invention also provides as described above. The application of the strains or the above-mentioned microbial agents in promoting plant growth.

[0014] The present invention also provides as described above. Application of strains or the above-mentioned microbial agents in improving iron nutrition in plants.

[0015] The present invention further explains that the plant is peanut and / or pear.

[0016] This invention also provides applications as described above. A method for promoting plant growth using strains or the aforementioned microbial agents, the method comprising: […]. The bacterial suspension was applied to the root zone of the plant seedlings.

[0017] To further clarify, the final concentration of the bacterial suspension is... earth.

[0018] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0019] The results obtained by the screening of this invention This strain possesses disease-resistant, iron-dissolving, and growth-promoting functions. The strain DB3 was isolated from the rhizosphere of pear trees in calcareous soil in Xinxiang, Henan Province. It exhibits excellent IAA production, rapid growth, ability to dissolve insoluble iron oxide, and iron carrier production. When prepared as an inoculant and applied to the rhizosphere, this strain significantly promotes the aboveground and root growth of peanut and wild pear seedlings, increases plant biomass, improves root morphology, and effectively alleviates symptoms of iron deficiency chlorosis. Specifically, after inoculation with DB3, the total dry weight of peanut seedlings increased by 47.05%, and the total dry weight of wild pear seedlings increased by 59.96%. The SPAD value of wild pear leaves, active iron content, and total iron content in roots significantly increased by 33.42%, 22.45%, and 74.41%, respectively. Furthermore, this strain exhibits certain antagonistic effects against pathogens such as Alternaria alternata, Bakanae diseased rice, and Fusarium oxysporum. The strain DB3 of this invention can be used as a biological agent to prepare efficient and environmentally friendly iron nutrient improvement products, replacing some chemical iron fertilizers. It is suitable for agricultural environments with low iron availability, such as calcareous soils, and has broad application prospects. Attached Figure Description

[0020] Figure 1 This is a colony morphology diagram of strain DB3.

[0021] Figure 2 This is a phylogenetic tree diagram of strain DB3.

[0022] Figure 3 This is a graph showing the antagonistic effect of strain DB3 against multiple pathogens.

[0023] Figure 4 This is a phenotypic result of strain DB3 on peanut seedling growth.

[0024] Figure 5 The figure shows the effect of strain DB3 on the aboveground dry weight of peanut seedlings.

[0025] Figure 6 The figure shows the effect of strain DB3 on the dry weight of the underground part of peanut seedlings.

[0026] Figure 7 The figure shows the effect of strain DB3 on the total dry weight of peanut seedlings.

[0027] Figure 8 The figure shows the effect of strain DB3 on the root morphology of peanut seedlings.

[0028] Figure 9 The figure shows the effect of strain DB3 on the root length of peanut seedlings.

[0029] Figure 10 The figure shows the effect of strain DB3 on the root surface area of ​​peanut seedlings.

[0030] Figure 11The figure shows the effect of strain DB3 on the aboveground dry weight of pear seedlings.

[0031] Figure 12 The figure shows the effect of strain DB3 on the dry weight of the underground part of pear seedlings.

[0032] Figure 13 The figure shows the effect of strain DB3 on the total dry weight of the roots of pear seedlings.

[0033] Figure 14 The figure shows the effect of strain DB3 on the SPAD value of pear leaves.

[0034] Figure 15 The figure shows the effect of strain DB3 on the active iron content in pear leaves.

[0035] Figure 16 The figure shows the effect of strain DB3 on the total iron content in the roots of Pyrus pyrifolia. Detailed Implementation

[0036] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0037] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0038] The culture media, reagents, and instruments involved in this experiment are all commercially available.

[0039] Example 1:

[0040] This embodiment is Screening, identification and biological characteristics of strains

[0041] 1. Isolation and purification of strain DB3: 1g of rhizosphere soil from pear orchards in calcareous soil of Xinxiang, Henan Province was collected and added to a 50ml Erlenmeyer flask containing glass beads and 9ml of SM buffer. The mixture was shaken at 170°C for 30min to obtain a soil suspension. The suspension was then serially diluted with sterile water to a final concentration of 10. -5 ~10 -7 The concentration suspension was spread on TSA medium and incubated at 30°C for 48 hours. Single bacteria were then picked using the streak plate method to purify the strain.

[0042] 2. Identification of strain DB3: The specific procedure for morphological identification is as follows: DB3 strain is inoculated onto TSB medium and incubated in a constant temperature incubator. After 24 hours of incubation, observe for the appearance of round colonies with smooth surfaces on the petri dish. Figure 1 Molecular identification (e.g.) Figure 2 This strain was classified and named Its Chinese classification name is Stenotrophomonas maltophilia.

[0043] The applicant has already submitted The strain is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M20251606; deposit address: Wuhan University, Wuhan, China; deposit date: July 15, 2025.

[0044] Example 2:

[0045] This embodiment is Functional study of strains

[0046] 1. Determination of the iron-soluble capacity of the fermentation broth of the strain: The activated strain was added at an inoculum rate of 1% to a solution containing iron. In TSB medium containing solid powder, a non-inoculated treatment (CK) was also performed, with three replicates for each treatment, at 28°C. The culture was shaken and incubated for 48 h. The supernatant of the fermentation broth was collected and treated with mixed acid. The iron content was determined by ICP-OES after digestion.

[0047] 2. Qualitative determination of IAA function of strain DB3: DB3 strain was added to TSB medium containing L-tryptophan at a 1% inoculum and incubated at 30℃ and 170 r·min. -1 After shaking for 48 hours, the supernatant was obtained by centrifugation at 10,000 rpm for 5 minutes. 100 μl of the supernatant was mixed with an equal volume of Salkowski colorimetric solution. 100 μl of uninoculated culture medium was mixed with an equal volume of Salkowski colorimetric solution as a blank control. The mixture was reacted for 30 minutes in the dark. The solution turning red indicates that the strain has the ability to produce IAA.

[0048] 3. Determination of bacterial growth capacity: After inoculating with seed culture of DB3 strain, the OD600 values ​​of the bacterial culture at 0, 4, 8, 12, 16, 20 and 24 hours were measured to determine its 24-hour growth status.

[0049] 4. Determination of siderophore production capacity: Following the method described by Schwyn and Neilands, the bacterial suspension cultured in LNM medium under iron-limited conditions for 48 h was centrifuged at 3500 rpm for 10 min through a well plate filter. The supernatant was mixed with CAS detection solution at a 1:1 ratio and allowed to stand for 2 h. The absorbance (A) at OD630 nm was then measured. The control was sterile LNM medium, which was also mixed with CAS detection solution in the same proportion, and the absorbance (Ar) was measured. The relative content of siderophores was calculated using the formula [(Ar-As) / Ar]×100%.

[0050] All of the above functional characteristic measurements were based on The results of the bacterial strain assay are shown in Table 1:

[0051] Table 1 Analysis of growth-promoting ability

[0052]

[0053] As shown in Table 1, strain DB3 has the ability to produce IAA, iron carriers, and dissolve insoluble iron oxide.

[0054] Example 3:

[0055] This embodiment is Plate confrontation test between strain and multiple pathogens

[0056] Multiple pathogens were cultured in PDA medium, followed by the activation of the tested pathogenic fungus (Alternaria). Abbreviation (Aa), rice bakanae disease fungus Abbreviation (FFJ), Fusarium oxysporum cucumber-specific type Abbreviation (Foc), Fusarium oxysporum banana-specific type The strain (abbreviated as Foc4) was used to prepare 5 mm mycelial discs, which were then transferred to the center of a new PDA plate, with spot inoculations made around the pathogen. DB3 bacterial suspension with a concentration of 0.80 was used. After the bacterial suspension dried, the plates were placed in an incubator at 28°C for incubation. Sterile water was spotted around the perimeter as a control group. Each treatment was performed in triplicate. The plate phenomena were observed and photographed after 5 days of incubation.

[0057] Result: As Figure 3 As shown, this indicates that strain DB3 can significantly antagonize Alternaria. Rice seedling blight pathogen Fusarium oxysporum cucumber-specific strain and Fusarium oxysporum banana-specific .

[0058] Example 4:

[0059] This embodiment is Study on the growth-promoting effect of microbial agents on peanut seedlings

[0060] (1) Peanut seedling cultivation: After soaking peanut seeds in sterile water for 12 hours, add 1% hydrogen peroxide for 20 minutes for disinfection, and finally rinse with sterile water. Spread the disinfected seeds evenly on a culture medium with sterile water-soaked filter paper, and place them in a 28℃ constant temperature incubator for 3-5 days to germinate. Select peanut seeds with white sprouts and sow them in sterilized substrate for seedling cultivation. When the peanut seedlings have 2-3 true leaves, select healthy and uniform seedlings and transplant them into a sterilized mixed soil with a calcareous soil:quartz sand ratio of 3:2.

[0061] (2) Preparation of DB3 bacterial suspension: The DB3 strain, preserved in glycerol tubes at -80℃, was purified by streaking on TSA medium. After incubation at 28℃ for 12 h, a single colony was picked and transferred to TSB liquid medium, and then incubated at 28℃. The strain was cultured overnight under specific conditions to activate it. Then, the seed culture was inoculated into fresh TSB liquid medium at a 1% (volume fraction) inoculation rate and incubated at 28°C and 170 rpm. -1 DB3 fermentation broth was obtained by shaking culture for 24 hours. The OD was adjusted to 1.0, centrifuged, and the bacterial cells were collected, resuspended three times, and diluted with sterile water to a viable cell concentration of 10⁻⁶. 7 The bacterial suspension at CFU / mL is ready for use.

[0062] (3) Inoculation: Inoculate the prepared DB3 bacterial suspension (final concentration: The soil was inoculated into the rhizosphere of peanut seedlings, with the uninoculated solution serving as a control (CK). Each treatment was replicated 12 times, and samples were collected after 30 days of culture to determine peanut phenotypic traits.

[0063] Experimental results:

[0064] 1. For example Figure 3-7 As shown, DB3 inoculant significantly promoted peanut seedling growth. Compared with the control (CK), the aboveground dry weight, root dry weight, and total dry weight of peanut seedlings treated with DB3 inoculant were significantly increased by 43.5%, 80.0%, and 38.1%, respectively.

[0065] 2. For example Figure 8-10 As shown, DB3 inoculant can improve the root morphology of peanut seedlings, increasing root length and root surface area by 55.3% and 55.2%, respectively.

[0066] Example 5:

[0067] This embodiment is Study on the effect of microbial agents on improving iron nutrition in pear

[0068] (1) Pear seedling cultivation:

[0069] Select healthy pear seeds and soak them in sterile water for 24 hours. Then soak them in 75% alcohol for 30 seconds, rinse 3-5 times with sterile water, add 2% sodium hypochlorite and soak for 20 minutes, finally rinsing thoroughly with sterile water. Spread the sterilized seeds evenly on a culture medium lined with sterile water-moistened filter paper, and place them in a 28℃ constant temperature incubator to germinate for 3-5 days. Select germinated seeds and sow them in sterilized substrate for seedling cultivation. When the pear seedlings in the substrate have 2-3 true leaves, select healthy and uniform seedlings and transplant them into sterilized substrate pots for pot cultivation.

[0070] (2) Inoculation treatment:

[0071] Preparation of bacterial suspension: A bacterial suspension of Burkholderia metallica DB3 obtained according to the aforementioned method was prepared, and its final concentration was adjusted to... Soil was then inoculated into the rhizosphere of *Pyrus pyrifolia* seedlings, with no inoculation as the control (CK). Each treatment was replicated 12 times, and samples were collected after 30 days of culture to determine the phenotypic traits of *Pyrus pyrifolia*.

[0072] Experimental results:

[0073] 1. For example Figure 11-13 As shown, inoculation with DB3 inoculant significantly promoted the growth of *Pyrus pyrifolia* seedlings. Compared with the control (CK), the aboveground dry weight, root dry weight, and total dry weight of *Pyrus pyrifolia* seedlings treated with DB3 inoculant suspension were significantly increased by 45.0%, 113.33%, and 58.7%, respectively.

[0074] 2. For example Figure 14-16 As shown, inoculation with DB3 bacterial agent can significantly improve the root morphology of pear. Compared with the control CK, the SPAD content of pear treated with DB3 bacterial suspension increased significantly by 36.92%. Compared with the control, the active iron content in leaves and the total iron content in roots of pear treated with DB3 bacterial agent increased significantly by 25.2% and 83.7%, respectively.

[0075] In summary, the above experiments demonstrate that the *Stenotrophomonas maltophilia* DB3, screened by the applicant, possesses excellent abilities in producing IAA (inorganic acid), rapid growth, dissolving insoluble iron, and producing iron carriers. Plate tests confirm its antagonistic effects against pathogens such as *Alternaria alternata*, *Bakanaeda jasminoides*, and *Fusarium oxysporum*. Pot experiments revealed that DB3 significantly promotes the aboveground and root growth of peanut and pear seedlings, increases plant biomass, improves root morphology, and effectively enhances the SPAD value, active iron content, and total iron content in pear leaves and roots, alleviating iron deficiency chlorosis symptoms. This indicates that this strain can promote plant growth by improving iron availability and absorption efficiency, thus replacing chemical iron fertilizers to a certain extent, reducing fertilizer input, and mitigating environmental impact. Therefore, the strain DB3 of this application can serve as an important material for bio-iron fertilizer or bio-growth-promoting agents, and its application in calcareous soils and iron-deficient areas has significant ecological and economic value.

[0076] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Stenotrophomonas sepilia The DB3 strain, with accession number CCTCC NO: M20251606, is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on July 15, 2025.

2. Containing the as described in claim 1 Stenotrophomonas sepilia DB3 strain inoculant.

3. As described in claim 1 Stenotrophomonas sepilia The application of the DB3 strain or the bacterial agent of claim 2 in the production of IAA, iron dissolution and / or siderophore production.

4. As described in claim 1 Stenotrophomonas sepilia The application of DB3 strain or the bacterial agent of claim 2 in the preparation of biocontrol agents for controlling plant pathogens; wherein the plant pathogen is Alternaria (Alternaria). Alternaria alternata ), rice seedling blight pathogen ( Fusarium fujikuroi Nirenberg ), Fusarium oxysporum cucumber-specific type ( Fusarium oxysporum ) and / or Fusarium oxysporum banana-specific ( Fusarium oxysporum f.sp. Cubense 4).

5. As described in claim 1 Stenotrophomonas sepilia The application of DB3 strain or the microbial agent of claim 2 in the preparation of bio-organic fertilizer.

6. As described in claim 1 Stenotrophomonas sepilia The application of the DB3 strain or the microbial agent of claim 2 in promoting plant growth, wherein the plant is a peanut seedling.

7. As described in claim 1 Stenotrophomonas sepilia The application of the DB3 strain or the microbial agent of claim 2 in improving iron nutrition in plants, wherein the plant is *Pyrus pyrifolia*.

8. Application as described in claim 1 Stenotrophomonas sepilia A method for promoting plant growth using DB3 strain or the microbial agent of claim 2, characterized in that, The method is as follows: [The method is described in the original text, which is not translated here.] Stenotrophomonas sepilia DB3 strain suspension was applied to the root zone of plant seedlings.

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