Methylobacterium strain and application thereof

Abstract

The invention discloses a methylobacterium strain and application thereof, the methylobacterium strain is methylobacterium asperum DSD, and the preservation number of the methylobacterium strain in the China Center for Type Culture Collection (CCTCC) is CCTCC NO: M2023445. The methylobacterium strain disclosed by the invention can be applied to rice planting, the biomass of rice can be improved in a foliage spraying or root application mode, and meanwhile, the cadmium content of the overground part and root system of the rice can be reduced. The operation method is simple and easy to implement, the selected methylobacterium strain has the advantages of being low in cost, easy to obtain, environmentally friendly and the like, and the technical problem that the effect of microorganisms on the aspects of cadmium reduction and growth promotion of rice is single is solved to a certain extent.

Description

Technical Field

[0001] The invention belongs to the technical field of safe production of microorganisms and agricultural products, and specifically relates to a methylbacterium strain and application thereof. Background Art

[0002] Heavy metal pollution is one of the current problems in the field of agriculture and environment. Cadmium has attracted much attention due to its high toxicity, difficulty in degradation and easy enrichment. In the process of agricultural production, atmospheric deposition, fertilizers, pesticides and irrigation water are important input pathways of Cd into the farmland system. Researchers have proposed corresponding agronomic measures for reducing cadmium in rice, including physical, chemical and biological methods, such as foliar spraying of fulvic acid, application of melatonin, and application of quicklime to the soil. These methods have a certain effect on reducing the absorption of cadmium by crops, but may have a certain negative impact on the ecological environment of farmland. In addition, the cadmium content in the edible part of the plant can be reduced by breeding low cadmium accumulation varieties, but due to the particularity of plant varieties, their growth conditions may have certain defects, such as low yield and low stress resistance. In contrast, microbial methods have many potential advantages and have been widely used in the removal and remediation of harmful heavy metals.

[0003] Some bacteria such as Bacillus, Pseudomonas, Azotobacter, Escherichia coli, Mycobacterium and Halomonas have been reported to be used for bioremediation of heavy metals. However, when these strains are put into practical application for reducing cadmium in crops, their activity and quality in large-scale cultivation are still difficult to guarantee, and they are prone to microbial loss during application, low cadmium reduction efficiency and single cadmium reduction effect. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a methylbacterium strain and its application that can simultaneously promote plant growth and reduce cadmium content. The present invention aims to reduce the cadmium content in rice tissue, increase rice biomass, and improve rice field productivity and rice safety.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0006] A methylobacterium strain is a methylobacterium currus DSD strain, and its collection number in China Center for Type Culture Collection is CCTCC NO: M 2023445.

[0007] As a general technical concept, the present invention also provides an application of the above-mentioned Methylobacterium strain in rice cultivation.

[0008] In the above application, preferably, the Methylobacterium strain is applied to the seedling stage of rice planting.

[0009] In the above application, preferably, the Methylobacterium strain is applied to the leaves or rhizosphere of rice.

[0010] In the above application, preferably, the Methylobacterium strain is added to ultrapure water at a volume fraction of 2% and sprayed evenly on the rice leaves.

[0011] In the above application, preferably, the Methylobacterium strain is added to a hydroponic box at a volume fraction of 2% and applied to the rhizosphere of rice.

[0012] In the above application, preferably, the Methylobacterium strain is applied once.

[0013] In the above application, preferably, the rice seeds are hydroponically transplanted after they have 3 leaves and 1 heart, and the Methylobacterium strain is applied 6 to 9 days after transplanting.

[0014] Compared with the prior art, the advantages of the present invention are:

[0015] The present invention provides a strain of Methylobacterium currus DSD, with a deposit number of CCTCCNo: M 2023445. The Methylobacterium currus strain provided by the present invention improves the growth of rice, promotes the growth of rice, reduces the absorption of cadmium by rice, and thus reduces the cadmium content in rice tissues. Taking the control group to which the Methylobacterium currus strain is not applied as the control group, the application of the Methylobacterium currus strain can significantly reduce the cadmium content in rice tissues. Compared with the control group, after the Methylobacterium currus strain is applied to the rice leaves, the cadmium content in the aboveground part of the rice seedlings is reduced by 15.78%-31.89%, and the cadmium content in the root system (underground part) is reduced by 30.57%-35.92%. Compared with the control group, after the Methylobacterium currus strain is applied to the rice rhizosphere, the cadmium content in the aboveground part of the rice seedlings is reduced by 23.04%-43.50%, and the cadmium content in the root system is reduced by 8.31%-31.42%.

[0016] At the same time, the Methylobacterium vesicularis strain provided by the present invention can further increase the biomass of rice seedlings. Compared with the control group, after the Methylobacterium vesicularis strain was applied to the rice leaves, the aboveground dry weight of the rice seedlings increased by 21.93%-50.86%, and the dry weight of the rice root system increased by 22.56%-46.38%. Compared with the control group, after the Methylobacterium vesicularis strain was applied to the rice rhizosphere, the aboveground dry weight of the rice seedlings increased by 11.73%-27.80%, and the dry weight of the rice root system increased by 20.43%-31.79%.

[0017] A methylobacterium strain, wherein the methylobacterium strain is the Methylobacterium currus DSD (Methylobacteriumcurrus DSD), which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2023445. The deposit address is Wuhan University, China, and the deposit date is March 31, 2023. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with specific preferred embodiments, but the protection scope of the present invention is not limited thereby. The materials and instruments used in the following embodiments are all commercially available.

[0019] Example 1

[0020] A methylobacterium strain of the present invention, the methylobacterium strain is a methylobacterium currus DSD (Methylobacteriumcurrus DSD), which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2023445, the deposit unit address is Wuhan University, China, and the deposit date is March 31, 2023.

[0021] The Methylobacterium strain is a Gram-negative bacterium and one of the main components of the plant microbial community. It was identified as Methylobacterium vesicularis through physiological and biochemical identification and molecular biological identification. Its main biological characteristics are: after 24-48 hours of slant culture, it forms pink dot-like colonies with a round and smooth surface. It can grow well under aerobic or anaerobic conditions, with the optimal growth temperature of 30°C and pH=7.

[0022] The Methylobacterium vegetative strain of the present invention has the effects of promoting rice growth and reducing cadmium. The preparation method of the Methylobacterium vegetative strain comprises the following steps:

[0023] (1) Activation: The culture medium for Methylobacterium vesicularis is beef powder peptone medium (NA), and its formula is: beef powder: 3g / L, peptone: 10g / L, NaCl: 5g / L, and the medium base fluid is deionized water. The culture medium is sterilized at high temperature and then cooled. The frozen glycerol bacteria of Methylobacterium vesicularis are inoculated into the culture medium, and the culture is shaken on a constant temperature shaker at a culture temperature of 30°C and a rotation speed of 180r / min for 48h to obtain activated Methylobacterium vesicularis.

[0024] (2) Cultivation: The activated Methylobacterium vedosum strain obtained above was expanded and inoculated into a new beef powder peptone medium at an inoculum rate of 2% to 5%, and cultured on a constant temperature shaker at 30° C. and a speed of 180 r / min for 24 to 48 h. The OD value was adjusted with ultrapure water. 600 =1, and the Methylobacterium vedosum strain was obtained.

[0025] The present invention has no special limitation on the sources of the components in the NA medium, and any conventional commercially available product in the art can be used. The components of the NA medium in the present invention were purchased from Beijing Coolbo Technology Co., Ltd.

[0026] The Methylobacterium vesiculosum strain of the invention improves the growth condition of rice under Cd stress, reduces the absorption of Cd by rice, and simultaneously promotes the growth of rice and increases the biomass of rice.

[0027] Example 2

[0028] An application of the methylbacterium strain of the present invention in rice planting adopts the methylbacterium strain of Example 1.

[0029] Test plants:

[0030] Rice (Zhongjiazao 45) was purchased from Hunan Academy of Agricultural Sciences.

[0031] Test strains:

[0032] Methylobacterium currus DSD (Deposit No.: CCTCC NO: M 2023445). The preparation method is shown in Example 1.

[0033] Culture medium:

[0034] Beef powder peptone medium (NA): beef powder: 3g / L, peptone: 10g / L, NaCl: 5g / L.

[0035] Implementation of hydroponic experiment:

[0036] The hydroponic experiment was conducted in a hydroponic box using nutrient solution to cultivate rice. The nutrient solution formula required for rice is: 4mmol / L Ca(NO3)2·4H2O, 6mmol / L KNO3, 1mmol / L NH4H2PO4, 2mmol / L MgSO4·7H2O, 10μmol / L H3BO3, 1.8μmol / L MnSO4, 0.2μmol / L NaMoO4, 0.31μmol / L CuSO4, 5μmol / L ZnSO4 and 50μmol / L Fe-EDDHA, and the pH is adjusted to 6.0. The rice seeds were transplanted to the hydroponic box after 3 leaves and 1 heart. The strain was applied on the 7th day after the rice was transplanted. The Methylobacterium velociformis strain was added to ultrapure water at 2% (v / v) and sprayed evenly on the rice leaves to ensure that uniform water mist can be observed on all leaves. The spraying was once. At the same time, the atmospheric deposition of exogenous Cd was simulated by spraying Cd solution on rice leaves. Exogenous Cd was designed to spray Cd solution on the leaves after 9 am during the entire rice seedling growth stage. The concentration of Cd solution was 2 mg / L, and it was carried out twice, on the 14th and 21st days after rice transplanting. When spraying Cd solution, ensure that uniform water mist can be observed on all leaves. In order to prevent Cd solution from entering the nutrient solution, plastic film was used to cover the surface of the hydroponic box, and the film was removed after the leaves were almost dry. After the rice seedlings were cultivated to the 28th day, the whole rice plant was harvested and the whole rice plant was divided into the aboveground part and the underground part. After rinsing with deionized water, it was sterilized at 105℃ for 30 minutes, and dried at 70℃ to constant weight, and the plant biomass was measured. After the biomass was measured, the plants were crushed according to the aboveground part and the underground part, and the plant samples were digested after crushing. After the digestion was completed, the Cd content of the plants was measured.

[0037] Example 3

[0038] An application of the methylbacterium strain of the present invention in rice planting adopts the methylbacterium strain of Example 1.

[0039] The test plants, test strains and culture medium are the same as those in Example 2.

[0040] Implementation of hydroponic experiment:

[0041] The hydroponic experiment was conducted in a hydroponic box using nutrient solution to cultivate rice. The nutrient solution formula required for rice: 4mmol / L Ca(NO3)2·4H2O, 6mmol / L KNO3, 1mmol / L NH4H2PO4, 2mmol / L MgSO4·7H2O, 10μmol / L H3BO3, 1.8μmol / L MnSO4, 0.2μmol / L NaMoO4, 0.31μmol / L CuSO4, 5μmol / L ZnSO4 and 50μmol / L Fe-EDDHA, and the pH was adjusted to 6.0. The rice seeds were transplanted to the hydroponic box after 3 leaves and 1 heart. The strain was applied on the 7th day after the rice was transplanted. The Methylobacterium velociformis strain was directly added to the hydroponic box at 2% (v / v) and applied to the rhizosphere of the rice, and applied once in total. At the same time, Cd solution (CdCl2·2.5H2O) was added to the hydroponic box at a final concentration of 2 mg / L. The rice seedlings were harvested after culturing for 28 days, and the rice plants were divided into the aboveground and underground parts. After rinsing with deionized water, the plants were sterilized at 105℃ for 30 minutes, and dried at 70℃ to constant weight, and the plant biomass was measured. After the biomass was measured, the plants were crushed according to the aboveground and underground parts, and the plant samples were digested after crushing. After the digestion was completed, the Cd content of the plants was measured.

[0042] Comparative Example 1

[0043] The test plants were the same as those in Example 2. The hydroponic experiment was carried out in the same manner as in Example 2 except that the Methylobacterium vedosum strain was not applied. Other operating steps were exactly the same as those in Example 2.

[0044] Comparative Example 2

[0045] The test plants were the same as those in Example 3. The hydroponic experiment was carried out in the same manner as in Example 3 except that the Methylobacterium vedosum strain was not applied. Other operating steps were exactly the same as those in Example 3.

[0046] Table 1 Dry weight of aboveground and underground parts of rice under different treatments

[0047] Example 2 Example 3 Comparative Example 1 Comparative Example 2 Aboveground dry weight (g / plant) 0.377±0.033 0.078±0.004 0.278±0.017 0.065±0.031 Underground dry weight (g / plant) 0.083±0.007 0.031±0.003 0.063±0.003 0.025±0.002

[0048] As can be seen from Table 1, the Methylobacterium vedosum strain provided by the present invention can significantly increase the aboveground and underground dry weights whether sprayed on the rice leaves or applied to the rice rhizosphere. Compared with Comparative Example 1, the aboveground and underground dry weights in Example 2 increased by 35.59% and 31.58%, respectively. Compared with Comparative Example 2, the aboveground and underground dry weights in Example 3 increased by 20.41% and 25.63%, respectively.

[0049] Table 2 Cd content in aboveground and underground parts of rice under different treatments

[0050]

[0051] As can be seen from Table 2, the Methylobacterium vehicularis strain provided by the present invention can significantly reduce the cadmium content in rice tissues whether it is sprayed on the rice leaves or applied to the rice rhizosphere. Among them, the Methylobacterium vehicularis strain has a better effect on controlling the absorption of atmospheric deposition cadmium by rice through foliar spraying. Compared with Comparative Example 1, the cadmium content in the aboveground and underground parts of Example 2 was reduced by 23.95% and 32.47%, respectively. Compared with Comparative Example 2, the cadmium content in the aboveground and underground parts of Example 3 was reduced by 34.60% and 16.62%, respectively.

[0052] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as above in the preferred embodiment, it is not used to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A Methylobacterium strain, characterized in that The methylobacterium strain is the methylobacterium currus DSD strain, and its collection number in the China Center for Type Culture Collection is CCTCC NO: M2023445.

2. Use of the Methylbacterium strain as claimed in claim 1 in rice cultivation.

3. The use according to claim 2, characterized in that: The methylbacterium strain is applied to the seedling stage of rice planting.

4. The use according to claim 3, characterized in that: The Methylobacterium strain is applied to the leaves of rice or to the rhizosphere of rice.

5. The use according to claim 4, characterized in that: The methylbacterium strain is added into ultrapure water at a volume fraction of 2%, and is evenly sprayed on the rice leaves.

6. The use according to claim 4, characterized in that: The methylbacterium strain was added into a hydroponic box at a volume fraction of 2% and applied to the rhizosphere of rice.

7. The use according to any one of claims 4 to 6, characterized in that: The Methylobacterium strain was applied once.

8. The use according to any one of claims 4 to 6, characterized in that: The rice seeds were hydroponically transplanted after they had 3 leaves and 1 heart, and the Methylobacterium strain was applied 6 to 9 days after transplanting.

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