Application of pseudomonas stutzeri in promoting rice growth, improving salt-alkali stress resistance and controlling trichoderma reesei toxin

Abstract

The application provides an application of Pseudomonas oxidans in promoting rice growth, improving salt-alkali stress resistance and TR toxin prevention, and relates to the technical field of biotechnology.The inventors have found that under normal rice culture conditions, Pseudomonas oxidans ZJ37 has the effect of promoting the growth of the above-ground part of Nipponbare rice, and therefore the strain can be used in the planting of Nipponbare rice; under salt-alkali stress conditions, Pseudomonas oxidans ZJ37 can promote the growth of the above-ground part and root hair of Jiaheyou No.5 rice, and therefore the strain has the effect of improving the salt-alkali stress resistance of Jiaheyou No.5 rice, and has a potential application prospect in relieving plant salt-alkali stress and promoting growth.In addition, Pseudomonas oxidans ZJ37 can also inhibit the production of plant Burkholderia TR toxin, and can be used in the prevention of TR toxin.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of *Pseudomonas oxidans* in promoting rice growth, enhancing resistance to salt and alkali stress, and controlling TR toxin. Background Technology

[0002] Currently, the most commonly used methods for improving saline-alkali land, such as chemical and physical methods, are costly and may cause secondary pollution. Adaptation methods for crops, such as screening salt-tolerant varieties and genetic modification, are also time-consuming. Therefore, efficient management and rational development of saline-alkali soil resources, increasing the area of ​​arable soil, will help improve the ecological environment and promote sustainable agricultural development. In addition, there are problems such as biotoxin contamination (e.g., tropolone, TR) in grain and oil crops, but currently there are no effective control strategies for biotoxin contamination in grain crops.

[0003] Because the plant-microbe-soil system is an interactive organic whole, the rhizosphere microbiome, as the plant's second genome, especially its core microorganisms, may play an important role in promoting the host's tolerance to harsh environments and controlling biotoxins. Studies have shown that plant growth-promoting rhizobacteria (PGPRs) have the ability to establish mutually beneficial relationships with plants, enhancing the plant host's resistance to stresses such as salinity and alkalinity through various strategies, such as helping plants absorb nutrients, activating their defense mechanisms, or improving soil nutrient levels. Other studies have indicated that certain microorganisms have degradative and inhibitory effects on biotoxins.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The first objective of this invention is to provide a strain of *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans ZJ37) was developed to address the aforementioned technical issues.

[0006] A second objective of this invention is to provide a microbial agent.

[0007] A third objective of this invention is to provide *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans Application of ZJ37 in promoting the aboveground growth of Nipponbare rice.

[0008] The fourth objective of this invention is to provide *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans Application of ZJ37 in improving the salt-alkali stress resistance of Jiaheyou 5 rice.

[0009] The fifth objective of this invention is to provide *Pseudomonas oxidans* ZJ37 (Pseudarthrobacter oxydans Application of ZJ37 in promoting the aboveground growth of Jiaheyou 5 rice under saline-alkali stress conditions.

[0010] The sixth objective of this invention is to provide *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans Application of ZJ37 in promoting root hair growth of Jiaheyou 5 rice under saline-alkali stress.

[0011] The seventh objective of this invention is to provide *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans Application of ZJ37 in the prevention and treatment of TR toxin.

[0012] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a strain of *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans ZJ37), the taxonomic name of this strain is: *Pseudomonas oxysporum*, and its Latin scientific name is: Pseudarthrobacter oxydans It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on May 19, 2026, with accession number CGMCC No. 38771.

[0013] Secondly, the present invention provides a bacterial agent comprising the aforementioned *Pseudomonas oxysporum* ZJ37 ( Pseudarthrobacter oxydans ZJ37).

[0014] Thirdly, the present invention provides the above-mentioned *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans Application of ZJ37 or microbial agents in promoting the aboveground growth of Nipponbare rice.

[0015] Fourthly, the present invention provides the above-mentioned *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans Application of ZJ37 or microbial agents in improving the salt and alkali stress resistance of Jiaheyou 5 rice.

[0016] Fifthly, the present invention provides the above-mentioned *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans Application of ZJ37 or microbial agents in promoting the aboveground growth of Jiaheyou 5 rice under salt-alkali stress conditions.

[0017] As a further technical solution, the salt and alkali stress conditions are 0.5 wt% NaCl and pH 10.5.

[0018] Sixthly, the present invention provides the above-mentioned *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans Application of ZJ37 or microbial agents in promoting root hair growth of Jiaheyou 5 rice under saline-alkali stress conditions.

[0019] As a further technical solution, the salt and alkali stress conditions are 0.5 wt% NaCl and pH 10.5.

[0020] In a seventh aspect, the present invention provides *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans Application of ZJ37 in the prevention and treatment of TR toxin.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The inventors discovered that under normal rice cultivation conditions, *Pseudomonas oxysporum* ZJ37 promotes the aboveground growth of Nipponbare rice, thus making this strain suitable for Nipponbare rice cultivation. Under saline-alkali stress conditions, *Pseudomonas oxysporum* ZJ37 promotes the growth of aboveground parts and root hairs in Jiaheyou 5 rice, thus enhancing the salt-alkali stress resistance of Jiaheyou 5 rice and showing potential application in alleviating plant salt-alkali stress and promoting growth. Furthermore, *Pseudomonas oxysporum* ZJ37 can also inhibit *Burkholderia phytoendigiae*. Burkholderia plantarii The production of TR toxin can be used for the prevention and treatment of TR toxin. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 For LB medium Pseudarthrobacter oxydans The phenotype of ZJ37; Figure 2 To construct a phylogenetic tree; Figure 3 Comparison of seedling length differences of Nipponbare rice before and after inoculation under normal conditions; Figure 4 Comparison of the difference in seedling length of Jiaheyou No. 5 rice before and after inoculation under normal conditions; Figure 5 This represents the CK phenotype of Nipponbare under normal conditions. Figure 6 The phenotype of Nipponbare treated with ZJ37 under normal conditions; Figure 7 This is the CK phenotype of Jiaheyou No. 5 under normal conditions; Figure 8 The phenotype of Jiaheyou 5 treated with ZJ37 under normal conditions; Figure 9 Comparison of differences in rice seedling length under saline-alkali conditions; Figure 10 for Pseudarthrobacter oxydans Phenotypic characteristics of rice under saline-alkali conditions after inoculation treatment; Figure 11 Phenotypes of uninoculated rice (left: saline-alkali conditions; right: normal conditions); Figure 12 For comparison of stem thickness data; Figure 13 Bacterial antagonism experiment (left side: ZJ7, ZJ12, or ZJ19; right side: ZJ37); Figure 14 This is the result of the TR toxin test. Detailed Implementation

[0024] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0025] In a first aspect, the present invention provides a strain of *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans ZJ37), the taxonomic name of this strain is: *Pseudomonas oxysporum*, and its Latin scientific name is: Pseudarthrobacter oxydans It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on May 19, 2026, with accession number CGMCC No. 38771.

[0026] The inventors have discovered that, under normal rice cultivation conditions, the strain of this invention promotes the growth of the aboveground parts of Nipponbare rice; under saline-alkali stress conditions, the strain of this invention can promote the growth of the aboveground parts and root hairs of Jiaheyou 5 rice. Therefore, the strain of this invention has potential application prospects in alleviating plant saline-alkali stress and promoting growth.

[0027] Secondly, the present invention provides a bacterial agent comprising the aforementioned *Pseudomonas oxysporum* ZJ37 ( Pseudarthrobacter oxydans ZJ37).

[0028] The bacterial agent of this invention includes *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans ZJ37), therefore possessing *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans All the beneficial effects of ZJ37.

[0029] Thirdly, the present invention provides the above-mentioned *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans Application of ZJ37 or microbial agents in promoting the aboveground growth of Nipponbare rice.

[0030] The inventors discovered that *Pseudomonas oxysporum* ZJ37 promotes the growth of the aboveground parts of Nipponbare rice and can be used to prepare products that promote the growth of the aboveground parts of Nipponbare rice.

[0031] Fourthly, the present invention provides the above-mentioned *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans Application of ZJ37 or microbial agents in improving the salt and alkali stress resistance of Jiaheyou 5 rice.

[0032] The inventors discovered that under saline-alkali stress conditions, *Pseudomonas oxysporum* ZJ37 can promote the growth of the aboveground parts and root hairs of Jiaheyou 5 rice. Therefore, this strain has the effect of improving the salt-alkali stress resistance of Jiaheyou 5 rice and has potential application prospects in alleviating plant salt-alkali stress and promoting growth.

[0033] Fifthly, the present invention provides the above-mentioned *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans Application of ZJ37 or microbial agents in promoting the aboveground growth of Jiaheyou 5 rice under salt-alkali stress conditions.

[0034] The inventors discovered that *Pseudomonas oxidans* ZJ37 promotes the growth of the aboveground parts of Jiaheyou 5 rice under saline-alkali stress conditions, and can be used to prepare products that promote the growth of the aboveground parts of Jiaheyou 5 rice under saline-alkali stress conditions.

[0035] As a further technical solution, the salt and alkali stress conditions are 0.5 wt% NaCl and pH 10.5.

[0036] Sixthly, the present invention provides the above-mentioned *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans Application of ZJ37 or microbial agents in promoting root hair growth of Jiaheyou 5 rice under saline-alkali stress conditions.

[0037] The inventors discovered that *Pseudomonas oxidans* ZJ37 promotes the growth of root hairs in Jiaheyou 5 rice under saline-alkali stress conditions, and can be used to prepare products that promote the growth of root hairs in Jiaheyou 5 rice under saline-alkali stress conditions.

[0038] As a further technical solution, the salt and alkali stress conditions are 0.5 wt% NaCl and pH 10.5.

[0039] In a seventh aspect, the present invention provides *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans Application of ZJ37 in the prevention and treatment of TR toxin.

[0040] Oxidative pseudoarbacterium ZJ37 can also inhibit Burkholderia phytoendcirrhosa. Burkholderia plantarii The production of TR toxin can be used for the prevention and treatment of TR toxin.

[0041] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0042] Example 1 1. Preparation of soil suspension and isolation and purification of bacterial strains Rhizosphere soils of salt-tolerant plants native to the Aral Sea region of Uzbekistan were collected. 10.0 g of rhizosphere soil was taken and 40 mL of sterile water was added. The mixture was shaken by a vortex mixer for 10 min, then treated with an ultrasonic cleaner for 60 s. After standing for 15 min until the layers separated, 1 mL of the supernatant was taken and serially diluted to 10-fold and 100-fold. 80 μL of the diluted solution was plated on LB medium.

[0043] Incubate the bacteria upside down in a 28°C incubator. Pick a single colony and inoculate it into LB liquid medium. Incubate at 28°C and 180 rpm for 24 hours to obtain a pure culture. Add an equal volume of 50% glycerol and store. 80℃.

[0044] The strain was streaked in LB solid medium (9cm diameter petri dish) and incubated at 28°C. Colonies were round, milky white, opaque, smooth, with regular edges, and approximately 1-2 mm in diameter. Figure 1 As shown.

[0045] 2. Identification of strains Species identification was performed using the 16S rRNA gene sequence. The primers used for PCR amplification were 27F / 1492R (27F: 5'). AGAGTTTGATCCTGGCTCAG 3' (SEQ ID NO.1) and 1492R: 5' ACGGCTACCTTGTTACGACTT 3' (SEQ ID NO. 2) yielded the gene sequence of strain ZJ37, the nucleotide sequence of which is as follows:

[0046] Based on standard strain sequences from the NCBI database, homology was analyzed through sequence alignment, and a phylogenetic tree was constructed using MEGA12 software to further determine the species affiliation of the isolated strains. Phylogenetic analysis was performed using neighbor-joining (1000 bootstrap), and the constructed phylogenetic tree is shown below. Figure 2 As shown. Preliminary identification indicates this strain is *Pseudomonas oxidans* (…). Pseudarthrobacter oxydans ), named *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans ZJ37).

[0047] In addition, other strains were screened from soil in Uzbekistan and identified and named... Bacillus subtilis ZJ7 Variovorax paradoxus ZJ12, Bacillus paralicheniformis ZJ19.

[0048] 3. Rice inoculation experiment Enrichment culture was performed at 28°C using standard liquid LB medium. Pseudarthrobacter oxydans ZJ37, its OD value was measured.

[0049] After the seeds of Japanese Haru and Jiaheyou 5 rice germinated and developed young roots, they were soaked in a bacterial solution diluted to 0.1 OD for 6 hours. The control group was soaked in sterile water. The inoculated rice seeds were placed in 96-well hydroponic boxes, with 9 replicates per treatment, and prepared Yoshida rice nutrient solution (pH 5.8) was added. The boxes were then incubated in an incubator under a cycle of 28℃ for 8 hours, 25℃ for 20 hours, and 12 hours of light and 12 hours of darkness for 7 days.

[0050] The seedling lengths of the two rice seedling groups (inoculated and control groups) were measured, as shown in Tables 1 and 2 below. Statistical analysis was performed using GraphPad Prism 10 software. Considering homogeneity of variance testing, and using the growth phenotype of uninoculated rice under normal conditions as a control, an unpaired t-test was conducted on the seedling length measurement data. A p-value < 0.05 was considered statistically significant.

[0051] Table 1. Comparison of seedling length differences in Japanese white rice under normal conditions (cm)

[0052] Table 2 Comparison of seedling length differences of Jiaheyou 5 rice under normal conditions (cm)

[0053] Statistical analysis showed that, under normal conditions, compared to uninoculated rice seeds, Pseudarthrobacter oxydans Strain ZJ37 significantly promoted the growth of Nipponbare rice seedlings, with a significantly increased seedling length compared to the control (CK). Figure 3 , Figure 5 and Figure 6 As shown.

[0054] However, under normal conditions, inoculation treatment had no significant effect on the seedling growth of Jiaheyou 5 rice. Figure 4 , Figure 7 and Figure 8 As shown.

[0055] Example 2 1. Rice inoculation experiment Bacteria screened from Uzbek soil were enriched and cultured in conventional liquid LB medium at 28°C, including... Bacillus subtilis ZJ7 Variovorax paradoxus ZJ12 and Bacillus paralicheniformis ZJ19 Pseudarthrobacter oxydans ZJ37 (all identified by the inventors through sequencing) and the above mixed bacteria were used to determine their OD values.

[0056] After the rice seeds (Jiaheyou 5) sprouted and developed roots, they were soaked in a bacterial solution diluted to 0.1 (obtained by dilution with LB broth) for 6 hours. The control group was soaked in LB broth. The inoculated rice seeds were placed in 96-well hydroponic boxes with 6 replicates per treatment, and prepared Yoshida rice nutrient solution was added. The salt-alkali stress conditions for rice were 0.5% NaCl and pH 10.5. The rice was cultured in an incubator under a cycle of 28℃ for 8 h, 25℃ for 20 h, and 12 h light and 12 h dark for 7 days. Two control groups were set up: uninoculated rice was cultured under salt-alkali conditions (CK salt-alkali) and normal conditions (CK normal) (without NaCl added and the nutrient solution pH adjusted to 5.8).

[0057] The seedling length and stem diameter of rice seedlings in each treatment group and the control group were measured, as shown in Tables 3 and 4 below. Figure 9 , Figure 10 , Figure 11 and Figure 12 As shown. Statistical analysis of the data was performed using GraphPad Prism 10 software. Considering the homogeneity of variance test, the growth phenotype of uninoculated rice under saline-alkali conditions was used as a control. Dunnett's multiple comparisons test was performed on the stem diameter and seedling length measurements. If P < 0.05, it indicates a significant difference.

[0058] Table 3. Length of rice seedlings (cm) under saline-alkali conditions

[0059] Table 4. Stem thickness data (cm) after inoculation treatment

[0060] Statistical analysis showed that, compared to uninoculated rice seeds, Pseudorhrobacter oxydans Strain ZJ37 significantly promoted the growth of rice seedlings under saline-alkali conditions, with seedling length significantly increased compared to the control (CK) saline-alkali treatment, as shown in the following example. Figure 9 As shown in the figure. Therefore, we speculate that ZJ37 can help rice alleviate the effects of salinity and promote its growth.

[0061] After treatment with ZJ7, ZJ12, ZJ19, and ZJ37 inoculants, under saline-alkali conditions, the number of root hairs in rice inoculated with ZJ37 increased, such as... Figure 10 , Figure 11 As shown in the figure. Therefore, it is speculated that ZJ37 can help rice promote root hair growth under salt and alkali stress, that is, the strain induces the formation of "high-density root hair type root structure" and improves the absorption efficiency of unit root.

[0062] Based on the above experimental results, we hypothesize that ZJ37 may exert different effects under different environmental conditions and in different rice varieties through different mechanisms and pathways. Under saline-alkali conditions, it may alleviate saline-alkali stress by adjusting root structure (e.g., increasing root hairs); while under normal conditions, the fungus may optimize resource allocation by increasing seedling length, and this effect is also closely related to the characteristics of the host variety and environmental factors.

[0063] 2. Antagonistic effect of mixed bacterial strains In the rice inoculation culture experiment, the mixed bacterial strains did not significantly improve the host's stress tolerance. This is presumably because the growth of different bacteria has a mutual inhibitory effect, weakening the potential growth-promoting bacteria's effect on rice seedlings. Therefore, a confrontation experiment was conducted on the selected bacteria for inoculation, as follows: Figure 13 As shown.

[0064] The results of the confrontation experiment show that ZJ37 is easily inhibited by other colonies, which provides a possible reason why mixed strains cannot play a significant role in promoting growth.

[0065] 3. Experiment on inhibiting pathogenic toxins Burkholderia plantarum Burkholderia plantarii It is one of the important pathogens causing bacterial damping-off in rice seedlings. It has strong infectivity, and the toxin it produces, tropolone (TR), is cytotoxic to a variety of plants and animals.

[0066] Inoculation and toxin testing treatment: Three groups were set up: CK: control group consisting of seeds soaked in sterile water and roots drenched; BP group: seeds soaked in sterile water and pathogen B. PLANTING Root irrigation treatment group, BP+ZJ37 group: ZJ37 seed soaking, B. PLANTING Root irrigation treatment group.

[0067] Enrichment culture was performed at 28°C using standard liquid LB medium. Pseudorhrobacter oxydans ZJ37 and Burkholderia plantarii , and measure its OD value.

[0068] After the seeds showed signs of sprouting, the BP+ZJ37 group seeds were soaked in ZJ37 bacterial solution diluted to 0.1 with an OD600 value for 6 hours. The control group and BP group seeds were soaked in sterile water. The inoculated rice seeds were then cultivated in soil culture medium and placed in an incubator under cycling conditions of 28℃ for 8 h, 25℃ for 20 h, and 12 h light, 12 h dark. The BP group and BP+ZJ37 group used diluted B... PLANTING Root irrigation with bacterial solution.

[0069] After 10 days of cultivation, rice seedling tissues were collected, and the TR toxin content was detected by GC-MS / MS. Statistical analysis was performed, and the results are shown in Table 5. Figure 14 As shown.

[0070] TR toxin content after surface treatment (mg / kg)

[0071] The experimental results show that the ZJ37 strain of this invention has a good inhibitory effect on the TR toxin of Burkholderia plantarum and can be used for the prevention and control of TR toxin.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans ZJ37), characterized in that, The *Pseudomonas oxidans* ZJ37 ( Pseudarthrobacter oxydans ZJ37 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38771.

2. A microbial agent, characterized in that, Including the *Pseudomonas oxidans* ZJ37 as described in claim 1 ( Pseudarthrobacter oxydans ZJ37).

3. The *Pseudomonas oxidans* ZJ37 as described in claim 1 ( Pseudarthrobacter oxydans Application of the microbial agent (ZJ37) or the microbial agent according to claim 2 in promoting the aboveground growth of Nipponbare rice.

4. The *Pseudomonas oxidans* ZJ37 as described in claim 1 ( Pseudarthrobacter oxydans Application of the microbial agent (ZJ37) or the microbial agent according to claim 2 in improving the salt and alkali stress resistance of Jiaheyou 5 rice.

5. The *Pseudomonas oxidans* ZJ37 as described in claim 1 ( Pseudarthrobacter oxydans Application of the microbial agent (ZJ37) or the microbial agent according to claim 2 in promoting the aboveground growth of Jiaheyou 5 rice under salt-alkali stress.

6. The application according to claim 5, characterized in that, The salt and alkali stress conditions are 0.5 wt% NaCl and pH 10.

5.

7. The *Pseudomonas oxidans* ZJ37 as described in claim 1 ( Pseudarthrobacter oxydans Application of the microbial agent (ZJ37) or the microbial agent according to claim 2 in promoting root hair growth of Jiaheyou 5 rice under salt-alkali stress.

8. The application according to claim 7, characterized in that, The salt and alkali stress conditions are 0.5 wt% NaCl and pH 10.

5.

9. The *Pseudomonas oxidans* ZJ37 as described in claim 1 ( Pseudarthrobacter oxydans The application of the microbial agent (ZJ37) or the microbial agent according to claim 2 in the prevention and control of TR toxin.

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