A microbial agent for alleviating phosphorus starvation in rice, its preparation method and application

Abstract

This invention belongs to the field of symbiotic colonization technology of microorganisms and plants, and specifically relates to a microbial agent for alleviating phosphorus hunger in rice, its preparation method, and its application. The agent includes the fungus *Morchella spp.*, which is effectively adapted to waterlogged soil environments and is less affected by anaerobic environments such as waterlogging when colonizing rice. Furthermore, by introducing a mixed bacterial community of *Rhodococcus rubrum* and *Pseudomonas aeruginosa*, the invention effectively reduces aluminum stress in the rice rhizosphere, alleviates the stiffness of root tip cell walls, further promotes the symbiotic colonization of *Morchella spp.* with rice, and increases the abundance of *Morchella spp.* in the rice rhizosphere soil, thereby improving the rice's ability to utilize phosphorus nutrients.

Description

Technical Field

[0001] This invention belongs to the field of symbiotic colonization technology of microorganisms and plants, and specifically relates to a microbial agent for alleviating phosphorus starvation in rice, its preparation method and application. Background Technology

[0002] Phosphorus is an essential nutrient for rice growth. Rice can only absorb and utilize phosphorus in the form of orthophosphate from the soil solution. However, due to adsorption by soil minerals, precipitation and fixation of metal cations in the soil solution, and the occlusion of phosphorus by metal oxides, the bioavailability of phosphorus in the soil is low. Furthermore, the poor mobility of phosphorus and the highly uneven spatial distribution of the soil solid phase, resulting in phosphorus spots, further reduce phosphorus bioavailability. Therefore, increasing rice yields in agricultural production inevitably relies on the large-scale input of chemical fertilizers. Considering the fixation of phosphorus in the soil, the actual amount of fertilizer required is far higher than the crop's needs, causing a series of resource and environmental problems. This is particularly true in many tropical and subtropical rice-producing countries, where dependence on imported phosphate fertilizers further exacerbates the national financial and social burden.

[0003] Rhizosphere microorganisms are essentially a plant's second genome. Faced with phosphorus deficiency, over 70% of terrestrial plants choose to establish symbiotic relationships with endophytic fungi such as mycorrhizal fungi, enhancing their phosphorus utilization through fungal hyphal interception or phosphatase in the hyphae. However, in the flooded environment of paddy fields, strong anaerobic and reducing conditions inhibit the symbiotic colonization of mycorrhizal fungi, making it difficult for most species to survive in paddy fields. Furthermore, cell wall stiffening caused by aluminum stress may further limit fungal colonization. Therefore, screening or culturing fungal species that can adapt to paddy field environments, establish symbiotic colonization with rice, and possess hyphal structures, while simultaneously alleviating cell wall stiffness and improving fungal colonization rates, is of great significance. Summary of the Invention

[0004] To address the aforementioned technical issues and enhance the ability of fungi to coexist and colonize in aluminate soils, particularly the symbiotic colonization of fungi with rice in flooded environments, thereby improving the utilization of phosphorus nutrients during rice growth, this solution provides a fungal agent for alleviating phosphorus hunger in rice, including the fungus *Morchella spp.*

[0005] Preferred microbial agents for alleviating phosphorus starvation in rice also include Rhodococcus rubrum and Pseudomonas aeruginosa.

[0006] The present invention also provides a method for preparing the above-mentioned microbial agent for alleviating phosphorus starvation in rice.

[0007] (1) Preparation of mixed bacterial communities

[0008] Rhodococcus erythropolis (China General Microbiological Culture Collection Center, accession number: CGMCC No. 9611) was inoculated into a culture medium as a single colony and cultured for a period of time. The resulting cell communities were collected by centrifugation and dispersed in sterile water to form a Rhodococcus erythropolis suspension.

[0009] Pseudomonas aeruginosa (CGMCC No. 34768), deposited at the China General Microbiological Culture Collection Center on June 5, 2025, was inoculated into a culture medium as a single colony and cultured for a period of time. The resulting cell communities were collected by centrifugation and dispersed in sterile water to form a Pseudomonas aeruginosa suspension.

[0010] The above-obtained Rhodococcus erythropoietin and Pseudomonas aeruginosa suspensions were mixed to obtain a bacterial mixed community suspension (SynCom).

[0011] (2) Preparation of fungal suspension of *Morchella spp.*

[0012] After the revived Mortierella capitata F2 (CGMCC No. 18560) was inoculated into a culture medium and cultured for a period of time, the mycelium and spores obtained were collected by centrifugation and dispersed in sterile water to form a suspension of the fungus Mortierella capitata.

[0013] As a preferred option, the culture medium used for inoculating Rhodococcus rubrum in step (1) is Luria-Bertani (LB) medium, in which the active ingredients include 5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride.

[0014] As a preferred method: In step (1), after Rhodococcus rubrum is inoculated into 50 mL of culture medium in the form of a single colony, it is cultured in a shaking incubator at 28°C at 180 rpm for 24 hours to reach approximately 2.3 × 10⁻⁶ colonies. 8 Density of CFU / mL.

[0015] As a preferred option, the culture medium used for inoculating Pseudomonas aeruginosa in step (1) is Luria-Bertani (LB) medium, in which the active ingredients include 5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride.

[0016] Preferably, in step (1), after Pseudomonas aeruginosa is inoculated into 50 mL of culture medium as a single colony, it is cultured in a shaking incubator at 28°C at 180 rpm for 24 hours to reach approximately 2.3 × 10⁻⁶ colonies.8 Density of CFU / mL.

[0017] As a preferred option: in step (1), the centrifugal collection is performed by centrifuging at 10000×g for 5 minutes using a disc centrifuge.

[0018] As a preferred option: in step (1), the Red City Rhodococcus suspension and the Pseudomonas aeruginosa suspension are mixed in equal volumes.

[0019] As a preferred option: the culture medium used for inoculating *Morchella* in step (2) is potato agar (PDA), the initial pH of which is 6.5. After inoculation with *Morchella*, it is cultured in a shaking incubator at 29°C at a speed of 200 rpm for 7–10 days.

[0020] As a preferred option: in step (2), the centrifugal collection is performed by centrifuging at 8000×g for 5 minutes using a disc centrifuge.

[0021] The present invention also provides an application of the above-mentioned microbial agent for alleviating phosphorus hunger in rice cultivation: first, a bacterial mixed community suspension (SynCom) is inoculated onto rice seedlings, and then a fungal suspension of *Morchella spp.* is inoculated onto the rice seedlings.

[0022] As a preferred method, the microbial agent for alleviating phosphorus hunger in rice is applied to rice cultivation in paddy fields: first, a bacterial mixed community suspension (SynCom) is inoculated onto rice seedlings located in the paddy field, and then a fungal suspension of *Morchella esculenta* is inoculated onto the rice seedlings.

[0023] Furthermore: 3 days after transplanting rice seedlings, a bacterial mixed community suspension (SynCom) was inoculated into the rhizosphere of the rice seedling at a rate of 5 mL / seedling, followed by an inoculation of a fungal suspension of *Morchella esculenta* at a rate of 5 mL / seedling into the rhizosphere.

[0024] The beneficial effects of this invention are as follows: In this solution, it was discovered that the fungus *Morchella spp.* can effectively adapt to waterlogged soil environments. When it coexists and colonizes with rice, it is less affected by anaerobic environments such as waterlogging. Thus, even in waterlogged anaerobic environments, a considerable abundance of *Morchella spp.* can still be maintained in the rhizosphere soil of rice. Furthermore, through the interception effect of *Morchella spp.* hyphae on phosphorus nutrients and the phosphorus-dissolving ability of the phospholysin in the hyphae, the utilization of phosphorus in the soil by rice is improved, thereby promoting the growth of rice.

[0025] Furthermore, the applicant discovered that under soil aluminum stress, rice root cell walls typically accumulate excessive amounts of polysaccharides due to disordered metabolism, leading to cell wall stiffness, which may limit fungal symbiotic colonization. In this proposed solution, the mixed bacterial community of *Rhodococcus rubrum* and *Pseudomonas aeruginosa* (SynCom) can reduce cell wall stiffness by increasing XET enzyme activity in rice root tips, thereby promoting the symbiotic colonization of *Morchella* on rice and increasing the abundance of native *Morchella* species in the rice rhizosphere soil.

[0026] In addition, the mixed bacterial community of Rhodococcus rubrum and Pseudomonas aeruginosa inoculated can reduce the aluminum content in the rhizosphere soil, increase the available phosphorus content, and decompose the cell wall to provide sugar sources, which will also help improve the yield-promoting effect on rice to some extent.

[0027] In summary, this solution primarily provides phosphorus activation for rice grown in phosphorus-deficient soil. Furthermore, the cultivation process of the bacterial-fungal cross-species synthetic inoculant is simple to operate. The fungi, Pseudomonas aeruginosa, and Rhodococcus rubrum in the solution are all derived from phosphorus-deficient farmland soil that has been cultivated for a long time, making it inexpensive. It can also ensure the quality and safety of food, and can be promoted and applied in the agricultural field, which is conducive to the realization of green and ecological agriculture. Attached Figure Description

[0028] Figure 1 This is a comparative diagram showing the abundance trends of arbuscular mycorrhizal fungi in the rhizosphere soil of rice seedlings after inoculation with the rhizosphere of rice seedlings in dryland and paddy fields, respectively, in the existing technology.

[0029] Figure 2 This is a comparison chart showing the results of 16S determination analysis of rhizosphere soil of rice at harvest time in Application Example 2 (RP) and Blank Control Example 1 (CK) of this application;

[0030] Figure 3 This is a comparison chart of the rice yield (g / per rice) harvested in Application Example 1, Application Example 2, and Blank Control Example 1 of this application;

[0031] Figure 4 This is a comparison chart showing the analysis results of phosphorus components in the rhizosphere soil of rice at harvest time in Application Example 1, Application Example 2, and Blank Control Example 1 of this application;

[0032] Figure 5 This is a comparison chart of the total phosphorus content in the rice harvested in Application Example 1, Application Example 2, and Blank Control Example 1 of this application;

[0033] Figure 6 This is a comparison diagram of the plasticity of the root tip cell walls of rice harvested in Application Example 2 and Blank Control Example 1 of this application. Detailed Implementation

[0034] Example 1

[0035] A method for preparing the above-mentioned microbial agent for alleviating phosphorus starvation in rice:

[0036] (1) Preparation of mixed bacterial communities

[0037] Rhodococcus erythropolis was inoculated as a single colony into 50 mL of Luria-Bertani (LB) medium (the active ingredients of which included 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride; the medium was sterilized at 121°C for 20 min after preparation, and the same applies below). The medium was then incubated in a shaking incubator at 28°C at 180 rpm for 24 hours (until approximately 2.3 × 10⁻⁶ colonies were reached). 8 The cell population (density CFU / mL) was collected by centrifuging at 10000×g for 5 minutes using a disc centrifuge. After removing residual LB medium, the cells were dispersed in 5 mL of sterile water to form approximately 2.3×10⁻⁶ cells / mL. 9 The density of CFU / mL was used as the suspension of Rhodococcus rubella.

[0038] Pseudomonas aeruginosa was inoculated as a single colony into 50 mL of Luria-Bertani (LB) medium and incubated at 28°C with shaking at 180 rpm for 24 hours (until approximately 2.3 × 10⁻⁶ colonies were reached). 8 The cell population (density CFU / mL) was collected by centrifuging at 10000×g for 5 minutes using a disc centrifuge. After removing residual LB medium, the cells were dispersed in 5 mL of sterile water to form approximately 2.3×10⁻⁶ cells / mL. 9 The density of CFU / mL was used as a suspension of Pseudomonas aeruginosa.

[0039] The above-obtained Rhodococcus rubrum suspension and Pseudomonas aeruginosa suspension were mixed in equal volumes to obtain a bacterial mixed community suspension (SynCom).

[0040] (2) Preparation of fungal suspension of *Morchella spp.*

[0041] The revived Mortierella capitata F2 was inoculated into potato agar (PDA) medium (which consisted of 200g peeled potatoes, 20g glucose, and 1000mL sterile water, sterilized at 121℃ for 20min after preparation, with an initial pH of 6.5) and cultured in a shaking incubator at 29℃ and 200rpm for 8 days (reaching approximately 2.3×10⁻⁶ ppm). 8The mycelium and spores were collected by centrifuging at 8000×g for 5 minutes using a disc centrifuge (density of CFU / mL). After removing residual PDA medium, they were dispersed in sterile water to form approximately 2.3×10⁻⁶ cells / mL. 9 The density was determined as CFU / mL for the fungal suspension of *Morchella spp.*

[0042] Application Example 1

[0043] During the cultivation period, rice seedlings were transplanted separately into flooded paddy fields. Three days later, the bacterial mixed community suspension (SynCom) prepared in step (1) of Example 1 was inoculated into the rhizosphere of the rice seedlings at a rate of 5 mL / seedling. Then, the fungal suspension of *Morchella spp.* prepared in step (2) of Example 1 was inoculated into the rhizosphere at a rate of 5 mL / seedling. The paddy fields were managed under traditional cultivation conditions. At the harvest period, the rhizosphere soil of the rice was measured and analyzed, and the rice yield, total phosphorus content, and soil phosphorus composition were determined.

[0044] Application Example 2

[0045] Rice seedlings were not inoculated with a suspension of the fungus *Morchella syringae*; all other procedures were the same as in Application Example 1.

[0046] During the cultivation period, rice seedlings were transplanted in a dispersed manner to a flooded paddy field. Three days later, the bacterial mixed community suspension (SynCom) prepared in step (1) of Example 1 was inoculated at a rate of 5 mL / seedling into the rhizosphere of the rice seedlings. The paddy field was managed under traditional cultivation conditions. At the harvest period, the rhizosphere soil of the rice was measured and analyzed to determine the rice yield, total phosphorus content, and soil phosphorus composition.

[0047] Blank control example 1

[0048] The rice seedlings were neither inoculated with a bacterial mixed community suspension (SynCom) nor with a fungal suspension of *Morchella spp.*, and all other procedures were the same as in Application Example 1:

[0049] During the cultivation period, rice seedlings were transplanted in a dispersed manner to paddy fields under flooded conditions, and field management was carried out in the paddy fields according to the traditional cultivation conditions described in Application Example 1 (Application Example 2). At the harvest time, the rhizosphere soil of the rice was measured and analyzed to determine the rice yield, total phosphorus content, and soil phosphorus composition.

[0050] Appendix Figure 1 This is a comparative chart showing the changes in the absolute abundance of arbuscular mycorrhizal fungi in the rhizosphere soil of rice seedlings after inoculation with the fungi described in existing technologies in dryland and paddy fields. (See attached chart.) Figure 1It can be seen that, compared to dry fields, arbuscular mycorrhizal fungi are significantly less able to establish themselves and co-proliferate with rice roots in flooded environments, leading to difficulties in their survival.

[0051] Appendix Figure 2 This is a comparison chart showing the results of fungal abundance measurements in the rhizosphere soil of rice at harvest time in Application Example 2 (RP) and the blank control Example 1 (CK):

[0052] Appendix Figure 2 In the "CK" section, rice takes at least 100 days from seedling to mature harvest. The applicant found that after such a long period of time (without other intervention), the abundance of *Morchella spp.* in the rhizosphere soil of rice in paddy fields still remains at no less than 20%. This at least shows that *Morchella spp.* can still form an effective colonization with rice roots in a flooded environment and survive for a long time. Therefore, *Morchella spp.* is selected as the fungus that colonizes with rice in paddy fields in this application.

[0053] Then, through research and comparison, Figure 2 Regarding the "CK" and "RP" items, the applicant further discovered that: after inoculating the bacterial mixed community suspension (SynCom) of this protocol in Example 2, the abundance of the fungus *Morchella* in the rice rhizosphere soil was significantly increased (by more than 75%), and... Figure 2 The study listed various fungi, but only *Morchella* showed a significant increase in abundance under the intervention of the SynCom mixed community suspension, while the abundance of other species almost all decreased, with some even disappearing. The increased abundance of *Morchella* in the rhizosphere soil reflects a more pronounced degree of symbiotic colonization between *Morchella* and rice roots. This indicates that, in addition to its own contribution to rice growth, the SynCom mixed community suspension also helps promote the symbiotic colonization between *Morchella* and rice roots, and this promoting effect is only observed in *Morchella*. Therefore, this study further inoculated rice with both *Morchella* and SynCom simultaneously to obtain this unexpected technical effect.

[0054] Appendix Figure 3 This graph compares the rice yield (g / per rice) harvested in Application Example 1, Application Example 2, and Blank Control Example 1. "CK" represents Blank Control Example 1; "RP" represents Application Example 2; and "RP+Y61" represents Application Example 1. Additionally, the vertical axis corresponding to each point in the graph represents the sampled plot (area 1m²). 2 The specific rice yield is shown in the diagram; the vertical axis corresponding to the entire rectangular frame represents the main range of rice yield variation in this application example; the vertical axis corresponding to the horizontal line within the rectangular frame represents the average rice yield in this application example. It is evident that this scheme improves rhizosphere soil fertility levels through cross-species microbial communities to increase crop yield.

[0055] Appendix Figure 4 This section compares the analysis results of phosphorus components in the rhizosphere soil of rice at harvest time in Application Example 1, Application Example 2, and Blank Control Example 1. "CK" represents Blank Control Example 1; "RP" represents Application Example 2; and "RP+Y61" represents Application Example 1. Additionally, the regions on the vertical bar corresponding to each application example, from top to bottom, represent the following based on their color intensity:

[0056] Available phosphorus (P, which can be absorbed by crops relatively easily, is an important indicator of soil fertility).

[0057] Primary phosphorus (Primary-P) is a "potential stockpile" of phosphorus nutrients in the soil, but it is released slowly and is not as easily absorbed by crops as available phosphorus.

[0058] Secondary phosphorus (which can be gradually converted into a form that can be absorbed by plants after weathering, with an effect similar to the "available phosphorus" mentioned above);

[0059] Residual phosphorus (P, phosphorus fixed in the soil, not easily lost, poorly available, and difficult for crops to absorb) is represented by the vertical coordinate range corresponding to the span of each colored area, which indicates the specific content.

[0060] The attached figure compares the effects of different microbial communities on rhizosphere soil fertility. In Example 1, which represents the application of this scheme, the simultaneous use of *Morchella spp.* and SynCom resulted in the highest content of phosphorus nutrients (the sum of available phosphorus and secondary phosphorus) in the rhizosphere soil, which are more easily absorbed by rice crops. This shows that this scheme improves rhizosphere soil fertility by using cross-species microbial communities to increase crop yield.

[0061] Appendix Figure 5 This graph compares the total phosphorus content in rice harvested from Application Example 1, Application Example 2, and Blank Control Example 1. "CK" represents Blank Control Example 1; "RP" represents Application Example 2; and "RP+Y61" represents Application Example 1. Furthermore, the vertical axis corresponding to each point in the graph represents the specific value of total phosphorus content in each sampled rice; the vertical axis corresponding to the entire rectangular frame represents the main range of variation in total phosphorus content in the rice of that application example; and the vertical axis corresponding to the horizontal line within the rectangular frame represents the average total phosphorus content in the rice of that application example.

[0062] Appendix Figure 6This figure shows a comparison of the plasticity of the root tip cell walls of rice harvested in Application Example 2 and Blank Control Example 1, where "CK" represents Blank Control Example 1 and "RP" represents Application Example 2. This figure illustrates that the intervention through inoculation with a mixed bacterial community of Rhodococcus rubrum and Pseudomonas aeruginosa (SynCom) reduced the stiffness of the rice root cell walls in this protocol.

Claims

1. A microbial agent for alleviating phosphorus starvation in rice, characterized in that: The microbial agent comprises a mixed community of fungi (Morchella spp.) and bacteria; the mixed bacterial community is obtained by mixing equal volumes of Rhodococcus rubrum suspension and Pseudomonas aeruginosa suspension. The Rhodococcus hongchengensis was deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 9611. The Rhodococcus hongchengensis was inoculated into a culture medium in the form of single colonies, and the cell communities obtained by centrifugation were collected and dispersed in sterile water to form a Rhodococcus hongchengensis suspension. The *Pseudomonas aeruginosa* strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34768. The *Pseudomonas aeruginosa* strain is inoculated into a culture medium as a single colony and cultured. The cell communities obtained by centrifugation are collected and dispersed in sterile water to form a *Pseudomonas aeruginosa* suspension. The aforementioned *Morchella* species is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 18560.

2. A method for preparing a microbial agent for alleviating phosphorus starvation in rice as described in claim 1, characterized in that: (1) Preparation of fungal suspension of *Morchella spp.* After the revived *Morchella* was inoculated into a culture medium and cultured for a period of time, the mycelium and spores obtained were collected by centrifugation and dispersed in sterile water to form a suspension of the fungus *Morchella*.

3. The microbial agent for alleviating phosphorus starvation in rice as described in claim 1, characterized in that: The culture medium is Luria-Bertani medium, whose active ingredients include 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride.

4. The microbial agent for alleviating phosphorus starvation in rice as described in claim 1, characterized in that: The Rhodococcus rubrum and Pseudomonas aeruginosa were separately inoculated into 50 mL of the culture medium as single colonies, and then cultured in a shaking incubator at 28°C and 180 rpm for 24 hours, and then centrifuged separately.

5. The method for preparing the microbial agent for alleviating phosphorus starvation in rice as described in claim 2, characterized in that: The culture medium mentioned in step (1) is potato agar medium with an initial pH of 6.

5. After inoculation with the spore-forming fungus, it is cultured in a shaking incubator at 29°C at a speed of 200 rpm for 7 to 10 days.

6. The application of the microbial agent for alleviating phosphorus starvation in rice as described in claim 1 in rice cultivation.

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