WO2021217894A1

WO2021217894A1 - Serratia marcescens mb21 and use thereof

Info

Publication number: WO2021217894A1
Application number: PCT/CN2020/102196
Authority: WO
Inventors: Yitao GUO; Xinglong GUO; Guangli Wu; Guoming MENG; Ying Yuan; Yaowei Kang
Original assignee: Hebei Monband Water Soluble Fertilizer Co., Ltd
Priority date: 2020-04-28
Filing date: 2020-07-15
Publication date: 2021-11-04
Also published as: CN111394284B; CN111394284A

Abstract

Provided is Serratia marcescens MB21 and use thereof, relating to the technical field of microbiology. The Serratia marcescens MB21 was preserved in China General Microbiological Culture Collection Center, with a preservation number: CGMCC No. 19490. The strain MB21 has the ability to hydrolyze silicon and resist Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani.

Description

Serratia Marcescens MB21 and Use thereof

Cross-reference to Related Application

The present disclosure claims priority of Chinese patent application with the filing number 2020103501341 filed on April 28, 2020 with the Chinese Patent Office, and entitled “Serratia Marcescens MB21 and Use thereof” , the contents of which are incorporated herein by reference in entirety.

Technical Field

The present disclosure relates to the technical field of microbiology, in particular to Serratia marcescens MB21 and use thereof.

Background Art

The silicon element has been confirmed by the international soil science community to be a fourth plant nutrient element after nitrogen, phosphorus and potassium, which is an essential nutrient for the grass family and root crops. The application of silicon can improve the effectiveness of phosphorus in soil and the phosphorus content of plants, so that the absolute amount of potassium in plants is greatly increased or slightly increased; meanwhile, the application of silicon fertilizer has obvious improvement effect on the growth, yield and quality of crops such as rice, oat, wheat, sugarcane, and sorghum. Besides the function in the aspect of plant growth, the silicon element can also improve the disease resistance and insect resistance of plants, relieve the poisoning of metal ions, relieve salt stress, and enhance drought resistance. The predatory planting mode greatly reduces the content of effective silicon in soil, and the silicon-deficient farmland accounts for more than 50%of the area of the national farmland, so the importance of using silicon fertilizer can be seen. Since silicon is distributed very widely in nature, next to oxygen, the content of silicon in the earth’s crust occupies the second position, mainly existing in forms of silica and silicates. The silicon content in soil is about 70%, but it is in a very stable crystalline state and an amorphous state, with very low solubility, which is hard to be absorbed by plants. Therefore, screening microorganisms that can decompose insoluble silicon-containing minerals in soil, and developing biological silicon fertilizers become important ways for solving the problem of silicon fertilizers in China.

Botrytiscinerea caused by Deuteromycotina Botrytis cinerea is a worldwide plant disease, and by infecting roots, stems, leaves, flowers and fruits of fruits and vegetables, germs cause diseases such as soft rot, lodging, flower rot and fruit rot, thereby leading to the loss of nearly one billion of Euro every year. In recent years, with the increasing growth of vegetable planting areas, Botrytiscinerea has developed as one of the most harmful plant diseases.

Bipolaris Sorokiniana is one of the important diseases in wheat production, with a quite wide range of occurrence, and is popular and harmful in many areas and countries such as Asia, South America, North America, Oceania and Europe. This disease also occurs in wheat production areas of China such as North China, Northwest China, Northeast China and Huanghuai plain. By forming a large number of black embryo grains, this disease seriously deteriorates the quality of wheat, and brings great loss to the yield of wheat, wherein the yield of a diseased field is generally reduced by 20%～30%, and the yield of a seriously diseased field is reduced by 30%～70%.

Rhizoctonia solani, also called as wheat sharp eyespot, almost occurs in wheat planting areas of all temperate zones in the world, and has a quite wide damage range. After the wheat is invaded by Rhizoctonia solani, symptoms such as rotten buds, withered diseased seedlings, damaged stalks and rotten stems, and withered white ears may be caused at different growth stages, pathogenic bacteria invade the wheat and then destroy transportation tissues of the stalks and leaf sheaths, so that transportation of nutrition and the like is interrupted, further causing dead seedlings or no scion or withered whole plants and appearance of withered booting. The infection rate of a general diseased field is 20%～50%, and the infection rate of a seriously diseased field may be 80%～90%. According to statistics, the yield loss caused by Rhizoctonia solani is generally about 10%, and may reach 30%～40%in serious cases.

In summary, to screen a strain having a function of hydrolyzing (activating) silicon and meanwhile being capable of resisting diseases is of great significance in solving the above problems.

Summary

Objects of the present disclosure include, for example, providing a strain of Serratia marcescens MB21 (also known as ky21) , which strain has the ability to hydrolyze silicon and resist Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani.

Objects of the present disclosure also include, for example, providing a product containing the Serratia marcescens MB21 or use thereof.

In order to solve the above technical problems, the present disclosure specifically adopts following technical solutions:

According to one aspect of the present disclosure, the present disclosure provides a strain of Serratia marcescens MB21, which was preserved in China General Microbiological Culture Collection Center on March 19, 2020, with a preservation number: CGMCC No. 19490.

According to another aspect of the present disclosure, the present disclosure further provides a microbial agent, wherein the microbial agent includes the Serratia marcescens MB21.

In one or more embodiments, the microbial agent only contains the Serratia marcescens MB21.

In one or more embodiments, the microbial agent contains the Serratia marcescens MB21 and other species of microorganisms, wherein the other species of microorganisms are selected from the group consisting of Bacillus subtilis, Bacillus mucilaginosus, Bacillus licheniformis, azotobacters, Pseudomonas aeruginosa, phosphorus solubilizing bacteria, phosphorus solubilizing fungi and actinomycetes.

In one or more embodiments, the microbial agent further contains an acceptable auxiliary material in the art, wherein the acceptable auxiliary material in the art is selected from the group consisting of carriers, substances for providing nutrition to microbial ingredients and adjuvants.

In one or more embodiments, the carriers are selected from the group consisting of activated carbon, zeolite, maifanite, sepiolite, kaolin, diatomaceous earth, montmorillonite, rice hull, starch, polyvinyl alcohol and polyethylene glycol.

In one or more embodiments, the substances for providing nutrition to microbial ingredients are selected from the group consisting of salts, peptone, hydrolyzed proteins, yeast extract, glucose, amino acids and vitamins.

In one or more embodiments, the adjuvants are selected from the group consisting of pH regulators, dispersing agents, drying agents, solvents, disintegrants, fillers and stabilizers.

According to another aspect of the present disclosure, the present disclosure further provides use of the Serratia marcescens MB21, or the microbial agent in hydrolyzing silicon.

According to another aspect of the present disclosure, the present disclosure further provides use of the Serratia marcescens MB21, or the microbial agent in resisting diseases of plants.

In one or more embodiments, the diseases include at least one of Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani.

According to another aspect of the present disclosure, the present disclosure further provides use of the Serratia marcescens MB21, or the microbial agent in preparation of a product for hydrolyzing silicon and/or for resisting diseases of plants.

According to another aspect of the present disclosure, the present disclosure further provides a product for hydrolyzing silicon and/or for resisting diseases of plants, including the Serratia marcescens MB21, or the microbial agent. In one or more embodiments, the product includes fertilizer additives, fertilizers, plant growth modifiers or soil modifiers.

In one or more embodiments, the fertilizers include microbial fertilizers.

In one or more embodiments, the product further includes other microbial or non-microbial active ingredients, wherein the other microbial or non-microbial active ingredients are selected from the group consisting of hydrolyzed proteins, amino acids, microorganisms, inorganic salts or small molecule compound drugs.

In one or more embodiments, dosage forms of the product are selected from the group consisting of granule, emulsion, powder, suspension and liquid formulation.

According to another aspect of the present disclosure, the present disclosure further provides a method for improving ability of plants to resist diseases, which method includes applying the above strain MB21, or the microbial agent including the strain MB21 to plants, wherein plant diseases are at least one selected from the group consisting of Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani.

Compared with the prior art, the present disclosure has following beneficial effects:

The Serratia marcescens MB21 provided in the present disclosure has the ability to hydrolyze silicon and resist Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani. The ability of the strain MB21 to hydrolyze silicon can increase the content of plant-absorbable silicon elements in soil, thereby improving the effectiveness of phosphorus in soil and the phosphorus content of plants, and increasing the content of potassium in plants; the increase of content of plant-absorbable silicon in the soil also can improve the disease resistance and insect resistance of plants, relieve the poisoning of metal ions, relieve salt stress and enhance the drought resistance of plants; the ability of the strain MB21 to resist Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani can improve the stress resistance of plants. Based on the above-mentioned properties of the strain MB21, the microbial agent or product including the strain MB21, and use of the strain MB21 provided in the present disclosure also have the above-mentioned beneficial effects.

Brief Description of Drawings

In order to more clearly illustrate the technical solutions in specific embodiments of the present disclosure or in the prior art, drawings which need to be used for description of the specific embodiments or the prior art will be introduced briefly below, and apparently, the drawings in the description below merely show some embodiments of the present disclosure, and a person ordinarily skilled in the art still could obtain other drawings in light of these drawings without inventive efforts.

FIG. 1 shows comparison of an evolution relationship between 16s rDNA partial sequence (1407bp) of a strain MB21 and several Serratia marcescens strains (Fast Minimum Evolution Tree Method) ;

FIG. 2 shows morphology of the strain MB21 under a microscope (10×100) ;

FIG. 3 shows determination of the ability of the strain MB21 to hydrolyze silicon on a medium for hydrolyzing silicon;

FIG. 4 shows determination of the ability of the strain MB21 to inhibit Botrytiscinerea;

FIG. 5 shows determination of the ability of the strain MB21 to inhibit Bipolaris Sorokiniana; and

FIG. 6 shows determination of the ability of the strain MB21 to inhibit Rhizoctonia solani.

Detailed Description of Embodiments

Technical solutions of the present disclosure will be described below clearly and completely in combination with examples, and apparently, the examples described are only some, but not all examples of the present disclosure. Based on the examples in the present disclosure, all of other examples obtained by a person ordinarily skilled in the art without inventive efforts shall fall within the scope of protection of the present disclosure.

If no specific conditions are specified in the examples, they are carried out under normal conditions or conditions recommended by manufacturers. If manufacturers of reagents or apparatuses used are not specified, they are conventional products commercially available.

Unless otherwise defined herein, scientific and technical terms used in the present disclosure should have meanings that are commonly understood by those ordinarily skilled in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the present disclosure.

According to one aspect of the present disclosure, the present disclosure provides Serratia marcescens MB21. The strain in the present disclosure was preserved on March 19, 2020, with the preservation number of CGMCC No. 19490, and the classification name of Serratia marcescens, and the name of preservation unit is China General Microbiological Culture Collection Center (CGMCC for short) , with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; and postcode of 100101.

In the present disclosure, by screening approximately 300,000 strains from 36 soil samples, a strain having the function of hydrolyzing silicon while having the function of resisting diseases (including Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani) -Serratia marcescens-is obtained. Serratia marcescens is generally present in soil, water, plants, animals and intestinal tract and respiratory tract of human beings, and is a kind of Gram-negative bacillus that is widely present in natural world and is capable of producing water-insoluble yellow, violet or red pigments. The Serratia marcescens has low nutritional requirements, and some strains may produce pigments, facilitating the labeling, therefore, from the year 1906 to 1960s, the Serratia marcescens was considered by physicians as harmless saprophytic organism, and used as a biological standard for studying microbial propagation. Currently, some non-toxic strains remain as model bacteria for air microbiological studies. Serratia marcescens has been reported in many fields, in which research in the pharmaceutical field is mostly concentrated on prodigiosin, and prodigiosin has a variety of biological activities: it can be used for inhibiting delayed type hypersensitivity and rejection reaction after organ transplantation, and has antibacterial, antimalarial, antifungal and antiprotozoal activities and so on; in the field of environmental restoration: some strains in Serratia marcescens have the capability of restoring the environment, some can hydrolyze harmful substances, and some can enrich heavy metals; in the field of chemical engineering, some ingredients of Serratia marcescens are catalysts for producing biodiesel, which has great application value, and lays a foundation for the production of glycerin and free fatty acid; in the field of biological control: the Serratia marcescens has an antagonistic effect on Aeromonas hydrophila, Fusarium oxysporum vasinfectum and methicillin-resistant Staphylococcus aureus; the live bacterial preparation and metabolites thereof have certain pathogenicity to Lepidopteran pests common to vegetables such as tobacco budworm, cotton bollworm, cabbage caterpillar and diamondback moth. The effect of Serratia marcescens is in a plurality of aspects, and along with the increase of people’s demand on resources and the requirement of human sustainable development, the utilization of microorganisms is also increasing, especially such resources as Serratia marcescens having a wide range of functions.

The Serratia marcescens MB21 (also named as ky21, hereinafter abbreviated as strain MB21, both MB21 and ky21 appearing herein and in the drawings refer to Serratia marcescens MB21 with the preservation number CGMCC No. 19490) provided in the present disclosure has the ability to hydrolyze silicon and resist diseases, including Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani. The 16s DNA partial sequence of the strain MB21 provided in the present disclosure is as represented by SEQ ID NO: 1, and analysis of the partial sequence (SEQ ID NO: 1) indicates that the strain MB21 has 99.97%or more homology to Serratia marcescens, but is relatively far from the strain JCM13046 belonging to a different species Serratia ureilytia in the evolutionary genetic relationship. The colony characteristics of the strain MB21 are as follows: after being cultured and growing on R2A medium for 2d, the colony is in a round shape, red and opaque, smooth and relatively wet in surface, with a regular edge, halo, and central bulge, and has a diameter of about 3.14 μm as measured by a microscope, the colony is round, shiny, viscous, and slightly raised, and has a smooth and orderly edge. Tests indicate that the ability of the strain MB21 to hydrolyze silicon is significantly superior to control Bacillus amyloliquefaciens strain DSM7; meanwhile, the inhibitory effect of the strain MB21 on pathogenic bacteria of Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani is also superior to that of Bacillus amyloliquefaciens strain DSM7.

According to another aspect of the present disclosure, the present disclosure further provides a microbial agent containing the above strain MB21, and as the microbial agent contains the above strain MB21, the microbial agent has all of the beneficial effects of the strain MB21, which will not be repeated herein. It can be understood that the microbial agent may only contain the strain MB21, so that the microbial agent has the function of the strain MB21; the microbial agent may also contain other kinds of microorganisms, and the strain MB21 may act as a main active ingredient or an auxiliary active ingredient in the microbial agent, wherein the other kinds of microorganisms in the microbial agent include, but are not limited to, Bacillus subtilis, Bacillus mucilaginosus, Bacillus licheniformis, azotobacters, Pseudomonas aeruginosa, phosphorus solubilizing bacteria, phosphorus solubilizing fungi, actinomycetes, or the like.

It should be noted that the types of strains contained in the above microbial agent may be selected according to actual needs and applications, and the present disclosure does not limit the specific types of strains contained in the microbial agent. All microbial agents containing the strain MB21 should fall within the scope claimed in the present disclosure.

It can be understood that apart from the microbial ingredients, the microbial agent further may include an acceptable auxiliary material in the art, wherein the acceptable auxiliary material refers to an ingredient which does not affect the physiological function of the active ingredient in the microbial agent, and meanwhile assists the microbial agent, and examples of the auxiliary material include, but are not limited to, carriers, substances for providing nutrition to microbial ingredients, adjuvants and so on. Examples of the carriers include, but are not limited to, one or more of activated carbon, zeolite, maifanite, sepiolite, kaolin, diatomaceous earth, montmorillonite, rice hull, starch, polyvinyl alcohol and polyethylene glycol. The substances for providing nutrition to microbial ingredients include, but are not limited to, one or more of salts, peptone, hydrolyzed proteins, yeast extract, glucose, amino acids and vitamins; and the adjuvants include, but are not limited to, one or more of pH regulators, dispersing agents, drying agents, solvents, disintegrants, fillers and stabilizers.

It should be noted that types and ingredients of the auxiliary material contained in the above microbial agent may be selected according to actual needs and applications, and the present disclosure does not limit the specific types and ingredients of the auxiliary material contained in the microbial agent, as long as the auxiliary material is the above acceptable auxiliary material in the art. All microbial agents containing the strain MB21 should fall within the scope claimed in the present disclosure.

Based on the ability of the strain MB21 to hydrolyze silicon provided in the present disclosure, the present disclosure further provides use of the strain MB21 or the microbial agent containing the strain MB21 in hydrolyzing silicon. Applying the strain MB21 or the microbial agent containing the strain MB21 to hydrolyze silicon may increase the content of plant-absorbable silicon elements in soil, thereby improving the effectiveness of phosphorus in soil and the phosphorus content of plants, and increasing the content of potassium in plants; the increase of content of plant-absorbable silicon in the soil also can improve the disease resistance and insect resistance of plants, relieve the poisoning of metal ions, relieve salt stress and enhance the drought resistance of plants. Silicon to be hydrolyzed by the strain MB21 is mainly water-insoluble silicon, and examples of water-insoluble silicon include, but are not limited to, magnesium silicate, potassium feldspar, talc, zeolite, muscovite, calcium silicate, aluminum silicate and so on.

Based on the above use, the present disclosure further provides a method for hydrolyzing silicon, which method includes applying the above strain MB21, or the microbial agent containing the strain MB21, to silicon to be hydrolyzed, wherein the silicon to be hydrolyzed is water-insoluble silicon, and examples of the water-insoluble silicon include, but are not limited to, magnesium silicate, potassium feldspar, talc, zeolite, muscovite, calcium silicate, aluminum silicate and so on.

Based on the ability of the strain MB21 to resist diseases provided in the present disclosure, the present disclosure further provides use of the strain MB21 or the microbial agent containing the strain MB21 in resisting diseases of plants. The strain MB21 or the microbial agent containing the strain MB21 can be applied to resist at least one of Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani. When the strain MB21 or the microbial agent containing the strain MB21 is applied to resist diseases, it can be used to simultaneously resist Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani, and also can be only used to resist one or two diseases thereof, for example, only to resist Botrytiscinerea, or to resist Bipolaris Sorokiniana and Rhizoctonia solani, or to resist Botrytiscinerea and Bipolaris Sorokiniana, or to resist Botrytiscinerea and Rhizoctonia solani.

Based on the above use, the present disclosure further provides a method for improving plants’ ability to resist diseases, which method includes applying the above strain MB21, or the microbial agent containing the strain MB21 to plants, wherein plant diseases is at least one selected from the group consisting of Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani.

Based on the ability of the strain MB21 to hydrolyze silicon and resist diseases provided in the present disclosure, the present disclosure further provides use of the strain MB21 or the microbial agent containing the strain MB21 in preparation of a product for hydrolyzing silicon and/or for resisting diseases of plants, wherein the diseases include, but are not limited to, at least one of Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani. Applying the strain MB21 or the microbial agent containing the strain MB21 to the preparation of a product may endow the product with the ability to hydrolyze silicon and the ability to resist diseases, and using the product to hydrolyze silicon and/or resist diseases of plants may obtain the beneficial effects when the strain MB21 or the microbial agent containing the strain MB21 is applied to hydrolyze silicon and/or resist diseases of plants, and the details will not be repeated herein.

Based on the above use, the present disclosure further provides a product for hydrolyzing silicon and/or for resisting diseases of plants, and the product includes a stain MB21 or a microbial agent containing the strain MB21. The product may be used simultaneously for hydrolyzing silicon and resisting diseases of plants, and also may be used only for hydrolyzing silicon or only for resisting diseases of plants. The present disclosure does not limit the form of the product, and examples of the product include, but are not limited to, fertilizer additives, fertilizers, plant growth regulators, or soil modifiers and the like. The product may further include other functional ingredients, including but not limited to other microbial or non-microbial active ingredients, such as proteins, hydrolyzed proteins, amino acids, microorganisms, inorganic salts or small molecule compound drugs and the like; the product further may contain an acceptable auxiliary material in the art, wherein the acceptable auxiliary material refers to an ingredient which does not affect the physiological function of the active ingredient in the product, and meanwhile assists the product, and examples of the auxiliary material include, but are not limited to, carriers, substances for providing nutrition to microbial ingredients, adjuvants and so on. Examples of the carriers include, but are not limited to, one or more of activated carbon, zeolite, maifanite, sepiolite, kaolin, diatomaceous earth, montmorillonite, rice hull, starch, polyvinyl alcohol and polyethylene glycol. The substances for providing nutrition to microbial ingredients include, but are not limited to, one or more of salts, peptone, hydrolyzed proteins, yeast extract, glucose, amino acids and vitamins; and the adjuvants include, but are not limited to, one or more of pH regulators, dispersing agents, drying agents, solvents, disintegrants, fillers and stabilizers. Dosage forms of the product, for example, may be, but not limited to, granule, emulsion, powder, suspension or liquid formulation and so on.

In some optional embodiments, the dosage forms of the product, for example, may be, but not limited to, liquid, emulsion, microemulsion, suspension, powder, granule, wettable powder or water dispersible granule and so on.

It should be noted that specific forms of the above product may be selected according to actual needs and applications, and the present disclosure does not limit the specific forms (including dosage forms and specific ingredients) of the product. All products containing the strain MB21 should fall within the scope claimed in the present disclosure.

In some optional embodiments, the product is a fertilizer, and after application of the fertilizer added with the strain MB21 or the microbial agent containing the strain MB21, the plants’ ability to resist Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani may be improved, and silicon in soil is promoted to be hydrolyzed into a plant-absorbable form, thus further improving the plants’ disease resistance, insect resistance, salt stress resistance, drought resistance and other properties. In some preferred embodiments, the fertilizer is preferably a microbial fertilizer, which not only can effectively utilize the insoluble silicon-containing minerals in the soil, but also can resist diseases and improve the soil, thereby achieving multiple purposes. The present disclosure does not limit the conventional component in the fertilizer, including but not limited to, urea, ammonium sulfate, ammonium chloride, calcium phosphate, potassium phosphate, potassium nitrate, monopotassium phosphate or other kinds of minerals and trace elements, and the like, which acts as inorganic nutrient; human and animal excreta, straw, bean pulp, tea seed, sludge, fallen leaf, dry grass, cassava residue, sugar residue, and the like, which acts as organic ingredient; and pH regulators, sustained-release agents, sustained-release carriers, synergists, and the like, which acts as adjuvant.

It should be noted that the specific components of the above fertilizers or the microbial fertilizers may be selected according to actual needs and applications, and the present disclosure does not limit the specific ingredients of the fertilizers or the microbial fertilizers. All products containing the strain MB21 should fall within the scope claimed in the present disclosure.

In some optional embodiments, the product is a fertilizer additive, and separately preparing the strain MB21 or the microbial agent containing strain MB21 into the fertilizer additive facilitates cooperative use of the fertilizer additive with a fertilizer without the strain MB21, so as to endow the fertilizer without the strain MB21 with the abilities to hydrolyze silicon and resist diseases. Apart from the strain MB21 or the microbial agent containing the strain MB21, the fertilizer additive further may contain other functional ingredients, for example, being but not limited to, nutrient ingredients for providing nutrition to specific plants, or other disease-resistant ingredients; and the fertilizer additives may also contain a conventional auxiliary material, for example, being but not limited to, pH regulator, drying agent, dispersing agent and corrosion inhibitor.

It should be noted that the specific components of the above fertilizer additive may be selected according to actual needs and applications, and the present disclosure does not limit the specific components of the fertilizer additive. All products containing the strain MB21 should fall within the scope claimed in the present disclosure.

In some optional embodiments, the product is a plant growth modifier, and the strain MB21 or the microbial agent containing the strain MB21 in the plant growth modifier, after being applied to the soil, may promote the silicon in the soil to be hydrolyzed into a plant-absorbable form, thereby further improving disease resistance, insect resistance, salt stress resistance and drought resistance and other properties of plants; and the strain MB21 or the microbial agent containing the strain MB21 in the plant growth modifier, after being applied to the soil, may increase the content of available silicon that can be absorbed by plants in the soil, increase the silicon element intake amount of plants, promote the plant growth and improve the yield, enhance the plants’ defensive ability to resist diseases and pests, and meanwhile, also may reduce damage to plants caused by abiotic stresses such as salt stress, drought stress, heavy metal poisoning, radiation, nutrient imbalance, and freezing damage; meanwhile, the disease resistance of the plants to Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani also can be improved, such that the stress resistance of plants can be improved comprehensively. The plant growth modifier may further contain other substances for regulating the growth of plants, optionally including active ingredients for improving the stress resistance of plants, including but not limited to anti-drought, cold resistant, anti-insect and anti-heavy metal ingredients and so on; and optionally including ingredients that regulate plant growth, including but not limited to auxin, gibberellin, cytokinin, abscisic acid and so on.

It should be noted that the specific components of the above plant growth modifier may be selected according to actual needs and applications, and the present disclosure does not limit the specific components of the plant growth modifier. All products containing the strain MB21 should fall within the scope claimed in the present disclosure.

In some optional embodiments, the product is a soil modifier, and the strain MB21 in the soil modifier may hydrolyze silicon in the soil into a plant-absorbable form, thereby improving the soil structure, and meanwhile inhibiting the growth of pathogenic bacteria of Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani in the soil. The soil modifier further may contain an acceptable auxiliary material in the art, including but not limited to hydrolytic acrylonitrile, polyacrylamide, humic acid, sludge, straw, wood chip, peat, limestone and silicates.

It should be noted that the specific components of the above soil modifier may be selected according to actual needs and applications, and the present disclosure does not limit the specific components of the soil modifier. All products containing the strain MB21 should fall within the scope claimed in the present disclosure.

Based on the above-mentioned use, the present disclosure further provides a method for increasing the content of available silicon in soil, which method includes applying a strain MB21, or a product containing the strain MB21 to soil, wherein the available silicon can be absorbed by plants, and examples of the product include, but are not limited to, fertilizer additives, fertilizers, plant growth modifiers, soil modifiers and so on.

The technical solutions and beneficial effects of the present disclosure are further described below in combination with limited examples.

Example 1

1. Screening a microbial strain having a function of hydrolyzing silicon:

1.1 Acquisition of soil samples

Representative soil such as sand, clay and black soil was selected. The samples were derived from different areas such as agricultural fields, grasslands, and forest soil, especially rice fields, wheat fields and soil using silicon fertilizers for many years were sampled, 15-20 grams of sample was acquired from each point, meanwhile the origin (province, county) , acquisition year and month, the source of soil (plants, sandy soil, or others) were labeled, and the acquired samples were stored in 80%glycerol tubes and preserved in -80 ℃ refrigerator.

1.2 Screening of microbial strains having the function of hydrolyzing silicon

First step enrichment: 1 g of soil sample was taken and shaken up in 10 mL of purified water, 100 μL of mixed liquid thereof was taken and underwent shake culture in nutrient-deficient R2A liquid medium (this nutrient-deficient R2A liquid medium contained yeast powder 0.5 g, tryptone 0.5 g, glucose 0.5 g, dipotassium hydrogen phosphate 0.3 g, sodium pyruvate 0.3 g, magnesium silicate 5.0 g and water 1 L) at 30 ℃ for 3 days, the bacterial solution was diluted 10 ^-6, 10 ^-7 and 10 ^-8 times and then applied to R2A solid medium (this R2A solid medium contained yeast powder 0.5 g, tryptone 0.5 g, peptone 0.75 g, glucose 0.5 g, soluble starch 0.5 g, dipotassium hydrogen phosphate 0.3 g, sodium pyruvate 0.3 g, magnesium silicate 2.5 g, agar 15 g and water 1 L) , and functional strains were picked.

Second step enrichment: 100 μL of the bacterial solution enriched in the previous step was taken and underwent shake culture in 1/2N nutrient-deficient R2A medium (this 1/2N nutrient-deficient R2A medium contained yeast powder 0.25 g, tryptone 0.25 g, glucose 0.5 g, dipotassium hydrogen phosphate 0.3 g, sodium pyruvate 0.3 g, magnesium silicate 5.0 g and water 1 L) at 30 ℃ for 3 days, the bacterial solution was diluted 10 ^-6, 10 ^-7 and 10 ^-8 times and then coated a vessel, and strains having the function of hydrolyzing silicon were picked.

Third step enrichment: 100 μL of the bacterial solution enriched in the second step was taken and underwent shake culture in 1/2N nutrient-deficient R2A medium at 30 ℃ for 3 days, the bacterial solution was diluted 10 ^-6, 10 ^-7 and 10 ^-8 times and then coated a vessel, and strains having the function of hydrolyzing silicon were picked.

1.3 Repeated verification

In order to ensure the sustainability of the strains’ ability to hydrolyze silicon, the strains picked from an enrichment plate were inoculated in R2A solid medium containing 0.25%magnesium silicate, and cultured at 30 ℃, experimental results were observed every day, and it was verified whether the picked colonies had the function of hydrolyzing silicon, such that false positive colonies were excluded.

1.4 Result

Approximately 200,000-300,000 microbial strains from 129 soil samples of different provinces and cities acquired by the above method were screened for microbial strains for hydrolyzing silicon, as a result, 100-200 target strains having the function of hydrolyzing silicon were screened therefrom, and after multiple times of repeated verification, a strain capable of hydrolyzing silicon was obtained, named MB21 (also named as ky21) . In addition, this strain also has multiple functions of resisting diseases (Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani) , and the like, therefore, this strain was taken as a subject of study, and researches of subsequent tests were developed therefor.

2. Determination of strain 16sDNA:

2.1 Extracting bacterial DNA by a CTAB method

A single colony was inoculated in 5 mL of R2A, and cultured overnight under a condition of a temperature of 30 ℃, to serve as a seed culture solution, then 1 mL of the seed culture solution was taken into 100 mL of R2A liquid medium, and cultured under a condition of 37 ℃ and 220 r/min, and after 16 h of culturing, the resultant was centrifuged at 5000 r/min for 10 minutes, and the supernatant was discarded. After adding 10 mL of TE to the precipitation for centrifugal washing, the thalli were dissolved with 10 mL of TE, and the resultant was fully mixed and then stored at -20 ℃ for later use. 3.5 mL of the bacterial suspension was taken, 10%SDS 184 μL was added and fully mixed, then 10 mg/mL protease K 37 μL was added and fully mixed, after 1 h of warm bath under the condition of 37 ℃, 5 mol/L NaCl 740 μL was added, and then CTAB/NaCl 512 μL was added and fully mixed, followed by incubation under the condition of 65 ℃ for 10 min. An equal volume of chloroform/isoamyl alcohol was added and fully mixed, followed by centrifugation at 10000 r/min for 5 min, the supernatant was retained, the supernatant and an equal volume of phenol/chloroform/isoamyl alcohol (25: 24: 1) are fully mixed and then centrifuged at 10000 r/min for 5 min, the supernatant was retained, and then fully mixed with 0.6 times of isopropanol, followed by centrifugation at 10000 r/min for 5 min, DNA precipitation was collected, then the DNA precipitation was centrifuged and washed with 70%ethanol; and the DNA was dissolved with 1 mL of TE, and RNase A with a final concentration of 20 μg/mL was added, and stored at 4 ℃.

2.2 Amplification and sequencing:

PCR amplified 16S rDNA, and PCR reaction procedure is as shown in Table 1:

Table 1

The primers for PCR amplification were 16S rDNA universal primers:

27f: 5’-AGAGTTTGATCCTGGCTCAG-3’, as represented by SEQ ID NO: 2;

1492r: 5’-GGTTACCTTGTTACGACTT-3’, as represented by SEQ ID NO: 3.

The PCR product was subjected to 1.5%agarose gel electrophoresis, the electrophoresis product was recovered, purified and sequenced, and homologous sequences were searched by Blast in GenBank according to the obtained 16S rDNA sequence, and the homologous sequences were analyzed and compared, to establish a phylogenetic tree, and results are as shown in FIG. 1.

2.3 16S sequencing result for MB21 strain

Upon determination of the 16sDNA sequence (1407 bp) of MB21 strain, a partial sequence having 1407 bp was obtained, and result is as represented by SEQ ID NO: 1:

Analysis of the sequence of SEQ ID NO: 1 indicates that this strain has a high degree of homology to Serratia marcescens of 99.97%or more. Through further comparison of the homology with several strains of Serratia marcescens strain (Fast Minimum Evolution Tree Method) (see FIG. 1 for the result) , it was found that this strain was closest to evolution of all strains of Serratia marcescens, but was relatively far from the strain JCM13046 belonging to a different species Serratia ureilytia in the evolutionary genetic relationship (as shown in FIG. 1, showing evolutionary genetic relationship between the strain MB21 provided in the present disclosure to Serratia marcescens) .

3. Strain morphology observation:

The screened strain was inoculated onto an R2A plate and cultured at 30 ℃ for 2d, to observe colony’s size, shape, color, glossiness, viscosity, raised shape, transparency, edge characteristics and presence and absence of spore, etc.

3.1 Results of strain morphology observation:

Upon observation, after the strain MB21 (Serratia marcescens, Serratia) was cultured and grew on the R2A medium for 2d, the colony was in a round shape, red and opaque, smooth and relatively wet in surface, with a regular edge, halo, and central bulge, and has a diameter of about 3.14 μm as measured by a microscope, the colony was round, shiny, viscous, and slightly raised, and had a smooth and orderly edge. Morphology of the strain MB21 under a microscope (10×100) is as shown in FIG. 2.

Example 2

Determination of the ability of the strain MB21 to hydrolyze silicon

The strain MB21 cultured and growing on R2A medium for 2d was inoculated into a medium for hydrolyzing silicon (i.e. silicon-mineral-containing medium) , with a Bacillus amyloliquefaciens (strain DSM7) being set as a positive control, and cultured at room temperature for 2d, and after rinsing the colonies with water, the diameter of circles formed by hydrolysis of silicon was measured in unit of mm.

The ability to hydrolyze silicon can be determined and evaluated by the following formula:

Ability to Hydrolyze Silicon = Diameter (mm) of Circle formed by Hydrolysis of Silicon + X;

X is a weighting coefficient, and is correspondingly -1, 0, 1 or 2 according to the degree of transparency of the circle formed by hydrolysis of silicon by the strain.

(Note: X is a weighting coefficient, which is selected from -1, 0, 1, or 2 according to the transparency of the circle formed by hydrolysis of silicon by the strain. The number “2” represents a completely transparent hydrolysis circle; the number “1” represents a translucent hydrolysis circle; the number “0” represents that the hydrolysis circle is opaque, but a sign of hydrolysis is visible on the surface of the medium, that is to say, the hydrolysis circle is substantially unobservable by human eyes, but a weak sign of hydrolysis is visible at the position inoculated with the bacterium after the colony is washed with water; “-1” represents no hydrolysis activity. )

Results of determination of the ability of the strain MB21 to hydrolyze silicon are as shown in Table 2 and FIG. 3, and the determination results indicate that the ability of the strain MB21 to hydrolyze silicon is significantly stronger than that of the control Bacillus amyloliquefaciens strain DSM7.

Table 2. Ability of the Strain to Hydrolyze Silicon on Medium for Hydrolyzing Silicon

Example 3

Determination of fungicidal activity of the strain MB21 to plant fungi (Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani) :

The strain MB21 cultured and growing on the R2A medium for 2d was applied on a plate by a crossing method, and a small fungal agar block with mycelia (Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani) was drawn and inoculated in the center of PDA medium, with the medium only inoculated with pathogenic fungi being taken as a control, and cultured at 30 ℃ for 7～10 d. When the plate was filled with the control pathogenic fungi, the width of an antagonism belt between the strain and each indicator fungus was measured in unit of mm.

Determination results of the ability of the strain MB21 to resist diseases (Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani) are as shown in Table 3 and FIGS. 4～6, and the determination results indicate that the antagonistic bacterium MB21 had a bacteriostatic effect on the three plant pathogenic fungi, Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani, with stronger ability than the control Bacillus amyloliquefaciens strain DSM7. The bacteriostatic belt of the strain MB21 was 2～10 mm, while the bacteriostatic belt of the control Bacillus amyloliquefaciens strain DSM7 was only 0～3 mm.

Table 3. Bacteriostatic Ability of Antagonistic Bacterium MB21 Strain

Finally, it should be explained that the various examples above are merely used for illustrating the technical solutions of the present disclosure, rather than limiting the present disclosure; although the detailed description is made to the present disclosure with reference to various preceding examples, those ordinarily skilled in the art should understand that they still could modify the technical solutions recited in various preceding examples, or make equivalent substitutions to some or all of the technical features therein; and these modifications or substitutions do not make the corresponding technical solutions essentially depart from the scope of the technical solutions of various examples of the present disclosure.

Industrial Applicability

The Serratia marcescens MB21 provided in the present disclosure has the ability to hydrolyze silicon and resist Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani. The ability of the strain MB21 to hydrolyze silicon can increase the content of plant-absorbable silicon elements in soil, thereby improving the effectiveness of phosphorus in soil and the phosphorus content of plants, and increasing the content of potassium in plants; the increase of content of plant-absorbable silicon in the soil also can improve the disease resistance and insect resistance of plants, relieve the poisoning of metal ions, relieve salt stress and enhance the drought resistance of plants; the ability of the strain MB21 to resist Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani can improve the stress resistance of plants.

Claims

A strain of Serratia marcescens MB21, preserved in China General Microbiological Culture Collection Center on March 19, 2020, with a preservation number: CGMCC No. 19490.
A microbial agent, wherein the microbial agent comprises the Serratia marcescens MB21 according to claim 1.
The microbial agent according to claim 2, wherein the microbial agent only contains the Serratia marcescens MB21.
The microbial agent according to claim 2, wherein the microbial agent contains the Serratia marcescens MB21 and other species of microorganisms, wherein the other species of microorganisms are selected from the group consisting of Bacillus subtilis, Bacillus mucilaginosus, Bacillus licheniformis, azotobacters, Pseudomonas aeruginosa, phosphorus solubilizing bacteria, phosphorus solubilizing fungi and actinomycetes.
The microbial agent according to claim 2, wherein the microbial agent further contains an acceptable auxiliary material in the art, and the acceptable auxiliary material in the art is selected from the group consisting of carrier, substance for providing nutrition to microbial ingredients and adjuvant.
The microbial agent according to claim 5, wherein the carrier is selected from the group consisting of activated carbon, zeolite, maifanite, sepiolite, kaolin, diatomaceous earth, montmorillonite, rice hull, starch, polyvinyl alcohol and polyethylene glycol.
The microbial agent according to claim 5 or 6, wherein the substance for providing nutrition to microbial ingredients is selected from the group consisting of salts, peptone, hydrolyzed proteins, yeast extract, glucose, amino acids and vitamins.
The microbial agent according to any one of claims 5-7, wherein the adjuvant is selected from the group consisting of pH regulators, dispersing agents, drying agents, solvents, disintegrants, fillers and stabilizers.
Use of the Serratia marcescens MB21 according to claim 1, or the microbial agent according to any one of claims 2-8 in hydrolyzing silicon.
Use of the Serratia marcescens MB21 according to claim 1, or the microbial agent according to any one of claims 2-8 in resisting diseases of plants.
The use according to claim 10, wherein the diseases comprise at least one of Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani.
Use of the Serratia marcescens MB21 according to claim 1, or the microbial agent according to any one of claims 2-8 in preparation of a product for hydrolyzing silicon and/or for resisting diseases of plants.
The use according to claim 12, wherein the diseases comprise at least one of Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani.
A product for hydrolyzing silicon and/or for resisting diseases of plants, comprising the Serratia marcescens MB21 according to claim 1, or the microbial agent according to any one of claims 2-8.
The product according to claim 14, wherein the product comprises fertilizer additives, fertilizers, plant growth modifiers or soil modifiers.
The product according to claim 15, wherein the fertilizers comprise microbial fertilizers.
The product according to any one of claims 14-16, wherein the product further comprises other microbial or non-microbial active ingredients, wherein the other microbial or non-microbial active ingredients are selected from the group consisting of hydrolyzed proteins, amino acids, microorganisms, inorganic salts and small molecule compound drugs.
The product according to any one of claims 14-17, wherein a dosage form of the product is selected from the group consisting of granule, emulsion, powder, suspension and liquid formulation.
A method for improving ability of plants to resist a disease, wherein the method comprises applying to the plants the Serratia marcescens MB21 according to claim 1, or the microbial agent according to any one of claims 2-8, wherein the disease of the plants is at least one selected from the group consisting of Botrytiscinerea, Bipolaris Sorokiniana and Rhizoctonia solani.