Fungicide for relieving phosphorus starvation of rice as well as preparation method and application of fungicide

Abstract

The invention belongs to the technical field of symbiotic colonization of microorganisms and plants, and particularly relates to a fungicide for relieving phosphorus starvation of rice as well as a preparation method and application of the fungicide, including fungus mortierella which can effectively adapt to a soil flooding environment and is slightly influenced by anaerobic environments such as water logging when symbiotic colonization occurs between the mortierella and rice; on the basis, a bacterial mixed group of rhodococcus erythropolis and pseudomonas aeruginosa is further introduced, acid-aluminum stress of rice rhizosphere can be effectively reduced, stiffness of root tip cell walls is relieved, symbiotic colonization of mortierella to rice is further promoted, abundance of mortierella in rice rhizosphere soil is improved, and rice yield is increased. Therefore, the utilization capability of the rice on phosphorus nutrients is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of symbiotic colonization of microorganisms and plants, and particularly relates to a microbial agent for relieving phosphorus hunger of rice, and a preparation method and application thereof. BACKGROUND

[0002] Phosphorus is an essential nutrient for rice growth, and rice can only absorb and utilize orthophosphate in soil solution, but due to the adsorption of soil minerals, the precipitation and fixation of metal cations in soil solution, and the occlusion of phosphorus by metal oxides, the availability of phosphorus in soil is low; in addition, due to the poor mobility of phosphorus and the highly uneven spatial distribution pattern of soil solid phase, the phosphorus patch phenomenon is caused, which further reduces the biological availability of phosphorus. Therefore, the increase of rice yield in agricultural production has to rely on the large input of chemical fertilizers, and at the same time, considering the fixation of phosphorus in soil, the actual input amount of chemical fertilizers is required to be much higher than the crop requirement, which causes a series of resource and environmental problems, especially in the vast tropical and subtropical rice-producing countries, the dependence on imported phosphorus fertilizer further increases the national financial and social burden.

[0003] Rhizosphere microorganisms are equivalent to the second genome of plants, and more than 70% of terrestrial plants choose to establish symbiotic relationship with endophytic fungi such as mycorrhizal fungi in the face of phosphorus barren, and improve the utilization ability of phosphorus through the interception of fungal hyphae or the phosphorus-solubilizing enzyme in the interhyphal. However, in the flooded environment of paddy field, the strong anaerobic reduction condition inhibits the symbiotic colonization of mycorrhizal fungi, so that most species of mycorrhizal fungi are difficult to survive in the paddy field. In addition, the cell wall rigidity caused by acid aluminum stress may further limit the colonization of fungi. Therefore, it is of great significance to screen or culture fungal species that can adapt to the environment of paddy field, produce symbiotic colonization with rice, have hyphal structure, and at the same time alleviate the cell wall rigidity and improve the colonization rate of fungi. SUMMARY

[0004] In order to solve the above technical problems, in order to improve the symbiotic colonization ability of fungi in acid aluminum soil, especially the symbiotic colonization between fungi and rice in the flooded environment, and thus improve the utilization ability of phosphorus nutrient in the growth process of rice, the present application provides a microbial agent for relieving phosphorus hunger of rice, which comprises fungal Mortierella.

[0005] As a preferred, the microbial agent for relieving phosphorus hunger of rice further comprises bacteria Rhodococcus ruber and bacteria Pseudomonas aeruginosa.

[0006] The present application further provides a preparation method of the above-mentioned microbial agent for relieving phosphorus hunger of rice,

[0007] (1) Preparation of bacterial mixed community

[0008] Rhodococcus erythropolis (China General Microbiological Culture Collection Center, preservation number: CGMCC No. 9611) is inoculated into the culture medium in the form of a single colony, cultured for a period of time, the cell colonies obtained after culture are collected by centrifugation and dispersed in sterile water to obtain a Rhodococcus erythropolis suspension;

[0009] Pseudomonas aeruginosa (preservation number: CGMCC No. 34768) preserved in the China General Microbiological Culture Collection Center on June 5, 2025, is inoculated into the culture medium in the form of a single colony, cultured for a period of time, the cell colonies obtained after culture are collected by centrifugation and dispersed in sterile water to obtain a Pseudomonas aeruginosa suspension;

[0010] The Rhodococcus erythropolis suspension and the Pseudomonas aeruginosa suspension obtained above are mixed to obtain a bacterial mixed community suspension (SynCom);

[0011] (2) Preparation of Mortierella capitata F2 suspension

[0012] Mortierella capitata F2 (preservation number: CGMCC No. 18560) recovered is inoculated into the culture medium, cultured for a period of time, the mycelium and spores obtained after culture are collected by centrifugation and dispersed in sterile water to obtain a Mortierella capitata F2 suspension.

[0013] As preferred: the culture medium used for inoculating Rhodococcus erythropolis in step (1) is Luria-Bertani (LB) medium, and the effective components thereof include 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride.

[0014] As preferred: in step (1), after Rhodococcus erythropolis is inoculated into 50 mL of the culture medium in the form of a single colony, it is cultured in a shaking incubator at 28°C at a speed of 180 rpm for 24 hours, so as to achieve a density of about 2.3×10 8 CFU / mL.

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

[0016] As preferred: in step (1), after Pseudomonas aeruginosa is inoculated into 50 mL of the culture medium in the form of a single colony, it is cultured in a shaking incubator at 28°C at a speed of 180 rpm for 24 hours, so as to achieve a density of about 2.3×108 density of CFU / mL.

[0017] As preferred: in step (1), the centrifugal collection is by a disc centrifuge at 10000xg for 5 minutes.

[0018] As preferred: in step (1), the Rhodococcus ruber suspension and the Pseudomonas aeruginosa suspension are mixed in equal volume.

[0019] As preferred: in step (2), the medium for inoculating the Mortierella fungus is potato agar medium (PDA), the initial pH of which is 6.5, and after inoculating the Mortierella fungus, the medium is cultured in a shaking incubator at 29℃ at a speed of 200rpm for 7-10d.

[0020] As preferred: in step (2), the centrifugal collection is by a disc centrifuge at 8000xg for 5 minutes.

[0021] The application also provides an application of the above-mentioned bacterial agent for relieving phosphorus hunger of rice in rice planting: first inoculating the bacterial mixed community suspension (SynCom) on rice seedlings, and then inoculating the Mortierella fungus suspension on the rice seedlings.

[0022] As preferred: the application of the bacterial agent for relieving phosphorus hunger of rice in rice planting in a paddy field: first inoculating the bacterial mixed community suspension (SynCom) on rice seedlings in the paddy field, and then inoculating the Mortierella fungus suspension on the rice seedlings.

[0023] Further: 3 days after transplanting the rice seedlings, the bacterial mixed community suspension (SynCom) is inoculated on the rhizosphere of the rice seedlings at a dosage of 5mL / plant, and then the Mortierella fungus suspension is inoculated on the rhizosphere at a dosage of 5mL / plant.

[0024] The application has the beneficial effect that: in the present scheme, it is found that the Mortierella fungus can effectively adapt to the soil flooding environment, and has less influence on the anaerobic environment such as waterlogging when symbiotic colonization with rice, so that the Mortierella fungus still maintains considerable abundance in the rhizosphere soil of rice in the waterlogged anaerobic environment, and further improves the utilization of phosphorus in the soil by the interception effect of the Mortierella fungus hypha on phosphorus nutrients and the solubilization capacity of the hypha on phosphorus, thereby promoting the growth of rice.

[0025] Moreover, the applicant found that under the stress of soil acid aluminum, the cell wall of rice roots would usually cause excessive accumulation due to polysaccharide metabolism disorder, and then cause cell wall stiffness, which might limit the symbiotic colonization of fungi. In the present scheme, the bacterial mixed community (SynCom) of Rhodococcus hongchengii and Pseudomonas aeruginosa could cut the excess sugar chains of the cell wall by increasing the XET enzyme activity of the rice root tip, reduce the degree of cell wall stiffness, and ultimately promote the symbiotic colonization of Mortierella fungi on rice, thereby increasing the abundance of indigenous Mortierella fungi in the rhizosphere soil of rice.

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

[0027] In summary, the present scheme mainly provides a phosphorus activation function for rice growing in phosphorus-poor soil, and the culture process of the bacterial-fungal cross-border synthetic microbial agent is simple. The Mortierella fungi, Pseudomonas aeruginosa and Rhodococcus hongchengii in the present scheme are all derived from long-term cultivated phosphorus-deficient farmland soil, which is low in cost and can ensure the quality and safety of food. The present scheme can be applied in the field of agriculture and is conducive to the realization of green ecological agriculture. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 For the prior art, the comparison chart of the abundance trend of arbuscular mycorrhizal fungi in the rhizosphere soil of rice seedlings inoculated with arbuscular mycorrhizal fungi in dry land and paddy field, respectively;

[0029] Figure 2 For the present application, the comparison chart of the results of 16S determination and analysis of the rhizosphere soil of rice at harvest in application example 2 (RP) and blank control example 1 (CK);

[0030] Figure 3 For the present application, the comparison chart of the yield (g / per rice) of rice harvested in application example 1, application example 2 and blank control example 1;

[0031] Figure 4 For the present application, the comparison chart of the analysis results of phosphorus components in the rhizosphere soil of rice at harvest in application example 1, application example 2 and blank control example 1;

[0032] Figure 5 For the present application, the comparison chart of the total phosphorus content in the rice harvested in application example 1, application example 2 and blank control example 1;

[0033] Figure 6 For the present application, the comparison chart of the plasticity degree of the root tip cell wall of rice harvested in application example 2 and blank control example 1. DETAILED DESCRIPTION

[0034] Example 1

[0035] A preparation method of the above-mentioned bacterial agent for relieving phosphorus hunger of rice:

[0036] (1) Preparation of bacterial mixed community

[0037] Rhodococcus erythropolis was inoculated into 50 mL of Luria-Bertani (LB) medium (the effective components include 5 g / L of yeast extract, 10 g / L of tryptone, and 10 g / L of sodium chloride, which is sterilized at 121°C for 20 min after preparation and then used, and the same below) in the form of single colony, and then cultured in a shaking incubator at 28°C at a speed of 180 rpm for 24 hours (to reach a density of about 2.3 x 10 8 CFU / mL), and then the cell community obtained by culture was collected by disc centrifuge at 10000 x g for 5 min, and then dispersed in 5 mL of sterile water after removing the residual LB medium to form a density of about 2.3 x 10 9 CFU / mL as a Rhodococcus erythropolis suspension;

[0038] Pseudomonas aeruginosa was inoculated into 50 mL of Luria-Bertani (LB) medium in the form of single colony, and then cultured in a shaking incubator at 28°C at a speed of 180 rpm for 24 hours (to reach a density of about 2.3 x 10 8 CFU / mL), and then the cell community obtained by culture was collected by disc centrifuge at 10000 x g for 5 min, and then dispersed in 5 mL of sterile water after removing the residual LB medium to form a density of about 2.3 x 10 9 CFU / mL as a Pseudomonas aeruginosa suspension;

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

[0040] (2) Preparation of Mortierella capitata F2 suspension

[0041] Mortierella capitata F2 recovered was inoculated into potato agar medium PDA (the medium includes 200 g of peeled potato, 20 g of glucose, and 1000 mL of sterile water, which is sterilized at 121°C for 20 min after preparation and then used, and the initial pH is 6.5), and then cultured in a shaking incubator at 29°C at a speed of 200 rpm for 8 days (to reach a density of about 2.3 x 10 8The mycelium and spores were collected by centrifugation at 8000×g for 5 minutes, and the residual PDA medium was removed and dispersed in sterile water to form a 2.3×10 9 The density of CFU / mL was used as the suspension of the fungus Mortierella sp.

[0042] Application Example 1

[0043] During the cultivation period, rice seedlings were transplanted to a flooded paddy field. 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 / plant. The fungal suspension of Mortierella fungi prepared in step (2) of Example 1 was then inoculated into the rhizosphere at a rate of 5 mL / plant. The paddy field was then managed according to conventional cultivation conditions. At the harvest period, the rhizosphere soil of the rice seedlings was analyzed, and rice yield, total phosphorus content, and soil phosphorus components were measured.

[0044] Application Example 2

[0045] The rice seedlings were not inoculated with the fungus Mortierella suspension, and the remaining operations were the same as those in Application Example 1:

[0046] During the cultivation period, rice seedlings were transplanted into a flooded paddy field. 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 / plant. The paddy field was then managed according to conventional cultivation conditions. At the harvest period, the rhizosphere soil of the rice was analyzed to determine rice yield, total phosphorus content, and soil phosphorus components.

[0047] Blank control example 1

[0048] The rice seedlings were neither inoculated with the bacterial mixed community suspension (SynCom) nor with the fungal Mortierella suspension. The remaining operations were the same as those in Application Example 1:

[0049] During the cultivation period, rice seedlings were transplanted into flooded paddy fields in a dispersed manner. Field management was then carried out according to the conventional cultivation conditions described in Application Example 1 (Application Example 2). At harvest, the rhizosphere soil of the rice plants was analyzed to determine rice yield, total phosphorus content, and soil phosphorus components.

[0050] Attachment Figure 1 The following is a comparison of the abundance (absolute abundance) of arbuscular mycorrhizal fungi in the rhizosphere of rice seedlings after they were inoculated into the rhizosphere of rice seedlings in dry fields and paddy fields. Figure 1It can be seen that compared with the dry field, the arbuscular mycorrhizal fungi cannot effectively colonize the rice roots in the flooded environment, leading to the difficulty of the survival of the fungi.

[0051] The Figure 2 For the above application example 2 (RP) and blank control example 1 (CK), the comparison chart of the fungal abundance determination results of the rhizosphere soil of the harvested rice is as follows:

[0052] The Figure 2 In the "CK" item, the rice will experience more than 100 days from seedling to mature harvest, and the applicant found that after such a long time, the Mortierella fungus still maintains an abundance of not less than 20% in the rhizosphere soil of the rice in the paddy field (without other intervention), which at least indicates that the Mortierella fungus can still form effective colonization with the rice roots in the flooded environment, thereby surviving for a long time, so the Mortierella fungus is selected as the fungus for colonization with the rice in the paddy field in the present application;

[0053] Further research and comparison of the "CK" item and the "RP" item in the Figure 2 The applicant further found that after inoculating the bacterial mixed community suspension (SynCom) of the present application in application example 2, the abundance of the fungal Mortierella in the rhizosphere soil of the rice can be obviously improved (the increase is as high as more than 75%), and Figure 2 In the above, a plurality of fungi are listed, but only the Mortierella fungus has an obvious increase in abundance under the intervention of the mixed community suspension (SynCom), and the abundance of the rest of the fungi almost decreases, and some even disappear at last, and the increase in the abundance of the Mortierella fungus in the rhizosphere soil also reflects that the colonization of the Mortierella fungus with the rice roots is more obvious. This indicates that in addition to the contribution of the mixed community suspension (SynCom) to the growth of the rice, it also helps to promote the colonization degree of the Mortierella fungus with the rice roots, and only the Mortierella fungus has this promoting effect. Therefore, the Mortierella fungus and the SynCom are inoculated on the rice at the same time in the present application to obtain the unexpected technical effect.

[0054] The Figure 3 For the above application example 1, application example 2, and blank control example 1, the comparison of the yield (g / per rice) of the harvested rice is as follows, wherein "CK" represents blank control example 1; "RP" represents application example 2; "RP+Y61" represents application example 1. In addition, the vertical coordinates corresponding to each point in the figure represent the specific rice yield of each sampled sample field (area 1m 2 It can be seen that the present application improves the fertility level of the rhizosphere soil by using the cross-border microbial community to improve the yield of crops.

[0055] Figure 2 Figure 4 For the comparison of the analysis results of phosphorus components in the rhizosphere soil of rice harvested in the above application example 1, application example 2, and blank control example 1, wherein “CK” represents blank control example 1; “RP” represents application example 2; “RP+Y61” represents application example 1. In addition, according to the different color depths of each area from top to bottom on the vertical columnar bar corresponding to each application example, the following are represented in turn:

[0056] Available-P (available-P, which can be more easily absorbed by crops, is an important indicator of soil fertility);

[0057] Primary-P (Primary-P, which belongs to the “potential stock” of phosphorus nutrients in the soil, but has a slow release speed and is not as easy to be absorbed by crops as available-P);

[0058] Second-P (Second-P, which can be gradually converted into a form that can be absorbed by plants after weathering, similar to the above-mentioned “available-P”);

[0059] Residual-P (Residual-P, which is fixed in the soil, is not easy to lose, has poor availability, and is difficult to be absorbed by crops), the range of the ordinate corresponding to the span of each color area in the vertical direction represents the specific content size.

[0060] The figure reflects the influence of different microbial communities on the fertility level of the rhizosphere soil, wherein the content of phosphorus nutrients (the sum of available-P and secondary-P) that can be more easily absorbed by rice crops in the rhizosphere soil is the highest after the use of Mortierella and SynCom in application example 1 of the present scheme, which shows that the present scheme improves the fertility level of the rhizosphere soil through cross-border microbial communities to improve crop yield.

[0061] Figure 3 Figure 5 For the comparison of the total phosphorus content in the rice harvested in the above application example 1, application example 2, and blank control example 1, wherein “CK” represents blank control example 1; “RP” represents application example 2; “RP+Y61” represents application example 1. In addition, the ordinate corresponding to each point in the figure represents the specific value of the total phosphorus content in the sampled rice; the ordinate corresponding to the entire rectangular frame represents the main change range of the total phosphorus content in the rice in the application example; the ordinate corresponding to the horizontal line in the rectangular frame represents the average of the total phosphorus content in the rice in the application example.

[0062] Figure 4 Figure 6A comparison chart of the plasticity of the root tip cell walls of the rice harvested in the above-described application example 2 and the blank control example 1, in which "CK" represents the blank control example 1; "RP" represents the application example 2. The chart illustrates that the rigidity of the rice root system cell walls is reduced after the intervention by the bacterial mixed community (SynCom) of Rhodococcus rhodnophilus and Pseudomonas aeruginosa.

Claims

1. A bacterial agent for alleviating phosphorus starvation in rice, characterized by: The bacterial agent includes the fungus Mortierella sp.

2. The bacterial agent for alleviating phosphorus starvation in rice according to claim 1, characterized in that: The bacterial agent also includes bacteria Rhodococcus erythroxysporum and bacteria Pseudomonas aeruginosa.

3. A method for preparing a bacterial agent for alleviating phosphorus starvation in rice according to claim 2, characterized in that: (1) Preparation of mixed bacterial communities The Rhodococcus ruberii bacteria is inoculated into a culture medium in the form of a single colony and cultured for a period of time, and then the cultured cell colonies are collected by centrifugation and dispersed in sterile water to form a Rhodococcus ruberii bacteria suspension; The Pseudomonas aeruginosa is inoculated into a culture medium in the form of a single colony and cultured for a period of time, and then the cultured cell colonies are collected by centrifugation and dispersed in sterile water to form a Pseudomonas aeruginosa suspension; Mixing the Rhodococcus rubervillei suspension and the Pseudomonas aeruginosa suspension obtained above to obtain a mixed bacterial community suspension; (2) Preparation of Mortierella fungus suspension The revived Mortierella fungi are inoculated into a culture medium and cultured for a period of time. Mycelia and spores obtained by culture are collected by centrifugation and dispersed in sterile water to prepare a Mortierella fungi suspension.

4. The method for preparing the bacterial agent for alleviating phosphorus starvation of rice according to claim 3, wherein: The culture medium described in step (1) is Luria-Bertani culture medium, wherein the effective ingredients include 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride.

5. The method for preparing the bacterial agent for alleviating phosphorus starvation of rice according to claim 3, wherein: In step (1), the Rhodococcus erythroxysporum and the Pseudomonas aeruginosa are inoculated into 50 mL of the culture medium in the form of single colonies, and then cultured in a shaking incubator at 28°C at a speed of 180 rpm for 24 hours, and then centrifuged respectively.

6. The method for preparing the bacterial agent for alleviating phosphorus starvation of rice according to claim 3, wherein: In step (1), the Rhodococcus erythroxysporum suspension and the Pseudomonas aeruginosa suspension are mixed in equal volumes to obtain the bacterial mixed community suspension.

7. The method for preparing the bacterial agent for alleviating phosphorus starvation of rice according to claim 3, characterized in that: The culture medium in step (2) is a potato agar culture medium, the initial pH of the potato agar culture medium is 6.5, and after inoculating the Mortierella sp., the culture medium is cultured at 200 rpm in a shaking incubator at 29° C. for 7 to 10 days.

8. Use of the bacterial agent for alleviating phosphorus starvation of rice as claimed in claim 1 or 2 in rice cultivation in paddy fields.

9. Use of the bacterial agent for alleviating phosphorus starvation of rice as claimed in claim 2 in rice cultivation.

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