CN113817625A

CN113817625A - Flavobacterium acidicum and application thereof in improvement of saline-alkali soil

Info

Publication number: CN113817625A
Application number: CN202011100077.8A
Authority: CN
Inventors: 李玉义; 逄焕成; 张晓霞; 张晓丽; 常芳弟; 王国丽; 冀宏杰
Original assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Current assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Priority date: 2020-10-13
Filing date: 2020-10-13
Publication date: 2021-12-21
Anticipated expiration: 2040-10-13
Also published as: CN113817625B

Abstract

The invention discloses a flavobacterium acidicum and application thereof in improving saline-alkali soil. The Flavobacterium acidicum is Flavobacterium acidicum 237T2, and the registration number of the Flavobacterium acidicum in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 19869. The acid flavobacterium acidiflavus disclosed by the invention has the advantages that the pH value of a microenvironment of rhizosphere soil is reduced, the acid flavobacterium acidiflavus can be symbiotic with plants, the growth of the plants is promoted, and the acid flavobacterium acidiflavus is suitable for unfavorable growth environment of saline-alkali soil.

Description

Flavobacterium acidicum and application thereof in improvement of saline-alkali soil

Technical Field

The invention relates to flavobacterium acidicum and application thereof in improving saline-alkali soil in the field of microorganisms.

Background

Soil salinization is one of the most serious environmental problems in the world at present, and is also a direct influence factor for limiting high-efficiency utilization of farmlands and causing low agricultural productivity level. The saline soil is widely distributed in more than 100 countries and regions of the world, and the area reaches 10 hundred million hm². In addition, 2.3X 10⁹hm²In irrigated land of 4.5X 10⁷hm²Is damaged by salinization and occupies 19.5 percent of the irrigation area. The salinized soil in China has large area, and the total area of various salinized soils is about 9900 kilohm²Mainly concentrated in northwest, north China, northeast and coastal areas. Excessive salinity in soil can cause changes in the physical and chemical properties of soil, leading to deterioration of the growing environment of most crops, and soil salinization is also a major cause of reduced agricultural yield in many arid and semi-arid regions of the world.

Saline-alkali soil is an important farmland reserve resource in China, and how to develop and utilize saline-alkali soil resources is one of the major problems in agricultural production in China. For a long time, the general outline of the measures for improving saline-alkali soil by people at home and abroad includes four types: water conservancy improvement measures including irrigation, drainage, silt discharge, rice planting, seepage prevention and the like; secondly, agricultural improvement measures: comprises land leveling, deep ploughing and upturned soil sunning, improved farming, soil dressing, fertilization, seeding, crop rotation, interplanting and the like; ③ chemical improvement measures: including the application of modifying substances such as gypsum, phosphogypsum, calcium sulfite, calcium superphosphate, etc.; biological improvement measures: comprises planting salt-tolerant plants and pasture, green manure, afforestation and the like. Because each measure has a certain application range and condition. Therefore, it must be treated comprehensively according to local conditions. At present, the microbial inoculum for improving the saline-alkali soil becomes a research hotspot, and the action of the microbial inoculum is mainly reflected in that microorganisms in the microbial inoculum can propagate in the soil, the soil nutrient condition is improved, the soil enzyme activity is improved, the microbial diversity is increased, and the microbial flora is changed, so that the effect of regulating the growth of crops is achieved.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve saline-alkali soil and/or how to improve the photosynthetic performance of plants and/or how to promote the growth of plants and/or how to improve the biomass of plants and/or how to improve the yield of crops and/or how to improve the saline-alkali tolerance of plants.

In order to solve the technical problems, the invention firstly provides a flavobacterium acidicum.

The Flavobacterium acidificans provided by the invention is Flavobacterium acidificum 237T2, and the registration number of the Flavobacterium acidificum in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 19869. The strain has been preserved in China general microbiological culture Collection center (CGMCC for short) in 25.5.2020. Hereinafter abbreviated to Flavobacterium acidicum 237T 2.

The flavobacterium acidificum 237T2 has the shape of a rod, is gram-negative, has round bacterial colony, irregular edge, smooth surface, moist, yellow, slightly convex and opaque.

Flavobacterium acidicum 237T2 has 16S rDNA shown in sequence 1 in the sequence table.

The following applications of the culture of flavobacterium acidi 237T2 or/and the metabolite of flavobacterium acidi 237T2 or/and flavobacterium acidi 237T2 also belong to the protection scope of the present invention:

u1, application in improving saline-alkali soil or alkaline soil or application in preparing products for improving saline-alkali soil or alkaline soil,

u2, in the application of reducing the pH value of saline-alkali soil or alkaline soil or in the preparation of products for reducing the pH value of saline-alkali soil or alkaline soil,

u3, in improving the saline-alkali tolerance or alkali tolerance of plants or in preparing products for improving the saline-alkali tolerance or alkali tolerance of plants,

u4 for reducing Na content in saline-alkali soil or alkaline soil⁺Application in absorption or preparation of reducing Na content in saline-alkali soil or alkaline soil of plant pair⁺The use in an absorbent product for the manufacture of,

u5 for increasing K in saline-alkali soil or alkaline soil⁺Application in absorption or preparation of K improving saline-alkali soil or alkaline soil of plant pair⁺The use in an absorbent product for the manufacture of,

u6 for increasing Ca content in saline-alkali soil or alkaline soil²⁺Application in absorption or preparation of Ca improving saline-alkali soil or alkaline soil of plant pair²⁺The application in the absorption of water is that,

u7 for reducing Na content in saline-alkali soil or alkaline soil⁺Absorbing and improving K in saline-alkali soil or alkaline soil by plants⁺Application in absorption or preparation of reducing Na content in saline-alkali soil or alkaline soil of plant pair⁺Absorbing and improving K in saline-alkali soil or alkaline soil by plants⁺The use in an absorbent product for the manufacture of,

u8 for reducing Na content in saline-alkali soil or alkaline soil⁺Absorbing and improving Ca in saline-alkali soil or alkaline soil by plants²⁺Application in absorption or preparation of reducing Na content in saline-alkali soil or alkaline soil of plant pair⁺Absorbing and improving Ca in saline-alkali soil by plants²⁺The use in an absorbent product for the manufacture of,

u9, in improving the photosynthetic performance of plants or in preparing products for improving the photosynthetic performance of plants,

u10 for reducing intercellular CO of plant₂Application in concentration or preparation of plant intercellular CO reduction₂The application of the water-soluble polymer in a concentration product,

u11, in improving the net photosynthetic rate of plants or in preparing products for improving the net photosynthetic rate of plants,

u12, application in improving the conductivity of plant stomata or application in preparing products for improving the conductivity of plant stomata,

u13, application in improving plant transpiration rate or application in preparing a product for improving plant transpiration rate,

u14, application in improving the water utilization efficiency of plant leaves or application in preparing products for improving the water utilization efficiency of plant leaves,

u15, in increasing the chlorophyll content of plants or in preparing products for increasing the chlorophyll content of plants,

u16, use in the production of auxin (IAA) or use in the preparation of a product for the production of auxin (IAA),

u17, application in promoting plant growth or application in preparing plant growth promoting products,

u18, use for increasing crop yield or use for preparing a product for increasing crop yield,

u19, use for increasing plant biomass or use for preparing a product for increasing plant biomass.

In the application, the product can be a microbial inoculum, a microbial ecological agent containing the microbial inoculum or a biological fertilizer containing the microbial inoculum.

In the above-mentioned application, the culture of Flavobacterium acidicum 237T2 may be a substance obtained by culturing Flavobacterium acidicum 237T2 in a microorganism culture medium (i.e., a fermentation product, such as a fermentation broth containing Flavobacterium acidicum 237T2 and a substance secreted into a liquid medium, or a solid fermentation broth containing Flavobacterium acidicum 237T28 and a substance secreted into a solid medium).

In order to solve the technical problems, the invention also provides a microbial inoculum, a microecological preparation containing the microbial inoculum or a biological fertilizer containing the microbial inoculum.

The microbial inoculum provided by the invention contains metabolites of Flavobacterium acidocaldarium 237T2 or/and Flavobacterium acidocaldarium 237T 2.

The microbial inoculum can be any one of the following microbial inoculants:

a1, bacterial agent for improving saline-alkali soil or alkaline soil,

a2, a microbial inoculum for reducing the pH value of saline-alkali soil or alkaline soil,

a3, bacterial agent for improving saline-alkali resistance or alkali resistance of plant,

a4, reducing Na content in saline-alkali soil or alkaline soil⁺The absorbed microbial inoculum is the microbial inoculum,

a5, increasing K content in saline-alkali soil or alkaline soil⁺The absorbed microbial inoculum is the microbial inoculum,

a6, increasing Ca content in saline-alkali soil or alkaline soil²⁺The absorbed microbial inoculum is the microbial inoculum,

a7, reducing Na content in saline-alkali soil or alkaline soil⁺Absorbing and improving K in saline-alkali soil or alkaline soil by plants⁺The absorbed microbial inoculum is the microbial inoculum,

a8, reducing Na content in saline-alkali soil or alkaline soil⁺Absorbing and improving Ca in saline-alkali soil or alkaline soil by plants²⁺The absorbed microbial inoculum is the microbial inoculum,

a9, a bacterial agent for improving the photosynthetic performance of plants,

a10, reducing intercellular CO of plant₂The bacterial agent with the concentration is added into the culture medium,

a11, a bacterial agent for increasing the net photosynthetic rate of plants,

a12, a microbial inoculum for improving the stomatal conductance of plants,

a13, a microbial inoculum for improving plant transpiration rate,

a14, a microbial inoculum for improving the water utilization efficiency of plant leaves,

a15, a bacterial agent for increasing the chlorophyll content of plants,

a16, an auxin (IAA) -producing microbial inoculum,

a17, bacterial agent for promoting plant growth,

a18, microbial inoculum for improving crop yield,

a19, microbial inoculum for increasing plant biomass.

The active ingredients of the microbial inoculum can be metabolites of flavobacterium acidi 237T2 or/and flavobacterium acidi 237T2, the active ingredients of the microbial inoculum can also contain other biological ingredients or non-biological ingredients, and the other active ingredients of the microbial inoculum can be determined by a person skilled in the art according to the effects of the microbial inoculum.

The microbial inoculum may also include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier is a mineral material or a biological material; the mineral material may be at least one of grass peat, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material is at least one of straws, pine shells, rice straws, peanut shells, corn flour, bean flour, starch, grass peat and animal manure of various crops; the liquid carrier can be water; in the microbial inoculum, the metabolites of Flavobacterium acidocaldarium 237T2 or/and Flavobacterium acidocaldarium 237T2 can exist in the form of cultured living cells, fermentation liquor of the living cells, filtrate of cell culture or mixture of the cells and the filtrate. The preparation formulation of the microbial inoculum can be various preparation formulations, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.

According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.

Hereinbefore, the metabolite of F.acidocaldarius 237T2 may be a fermentation broth of F.acidocaldarius 237T 2. The fermentation broth of Flavobacterium acidocaldarium 237T2 can be prepared as follows: culturing the flavobacterium acidificans 237T2 in a liquid fermentation culture medium, and collecting a fermentation liquid (containing the flavobacterium acidificans 237T2 and substances secreted into the liquid culture medium), wherein the fermentation liquid is a metabolite of the flavobacterium acidificans 237T 2.

The culture of the above-mentioned Flavobacterium acidocaldarium 237T2 also falls within the scope of the present invention. The culture of the above-mentioned Flavobacterium acidicum 237T2 can have at least one of the following functions:

b1, improving saline-alkali soil or alkaline soil,

b2, reducing the pH value of saline-alkali soil or alkaline soil,

b3, a bacterial agent for improving the saline-alkali resistance or alkali resistance of plants,

b4, reducing Na content in saline-alkali soil or alkaline soil⁺The absorption is carried out by the absorption device,

b5, increasing K content in saline-alkali soil or alkaline soil⁺The absorption is carried out by the absorption device,

b6, increasing Ca content in saline-alkali soil or alkaline soil²⁺The absorption is carried out by the absorption device,

b7, reducing saline-alkali soil of plant pairOr Na in alkaline soil⁺Absorbing and improving K in saline-alkali soil by plants⁺The absorption is carried out by the absorption device,

b8, reducing Na content in saline-alkali soil or alkaline soil⁺Absorbing and improving Ca in saline-alkali soil by plants²⁺The absorption is carried out by the absorption device,

b9, improving the photosynthetic performance of the plant,

b10, reducing intercellular CO of plant₂The concentration of the active ingredients in the mixture is,

b11, improving the net photosynthetic rate of plants,

b12, improving the conductivity of the plant stomata,

b13, improving the plant transpiration rate,

b14, improving the water utilization efficiency of the plant leaves,

b15, increasing the chlorophyll content of the plant,

b16, producing auxin (IAA),

b17, promoting the growth of plants,

b18, improving the yield of the crops,

and B19, increasing the biomass of the plant.

Any one of the following applications of the microbial inoculum also belongs to the protection scope of the invention:

c1, application in improving saline-alkali soil or alkaline soil or application in preparing saline-alkali soil or alkaline soil improving products,

c2, in the application of reducing the pH value of saline-alkali soil or alkaline soil or in the preparation of products for reducing the pH value of saline-alkali soil or alkaline soil,

c3, the application in improving the saline-alkali resistance of plants or the application in preparing products for improving the saline-alkali resistance of plants,

c4, reducing Na content in saline-alkali soil or alkaline soil⁺Application in absorption or preparation of reducing Na content in saline-alkali soil or alkaline soil of plant pair⁺The use in an absorbent product for the manufacture of,

c5 improving K in saline-alkali soil or alkaline soil⁺Application in absorption or preparation of K improving saline-alkali soil or alkaline soil of plant pair⁺The use in an absorbent product for the manufacture of,

c6 Ca in saline-alkali soil or alkaline soil for increasing plant pair²⁺Application in absorption or preparation of Ca improving saline-alkali soil or alkaline soil of plant pair²⁺The application in the absorption of water is that,

c7, reducing Na content in saline-alkali soil or alkaline soil⁺Absorbing and improving K in saline-alkali soil or alkaline soil by plants⁺Application in absorption or preparation of reducing Na content in saline-alkali soil or alkaline soil of plant pair⁺Absorbing and improving K in saline-alkali soil or alkaline soil by plants⁺The use in an absorbent product for the manufacture of,

c8, reducing Na content in saline-alkali soil or alkaline soil⁺Absorbing and improving Ca in saline-alkali soil or alkaline soil by plants²⁺Application in absorption or preparation of reducing Na content in saline-alkali soil or alkaline soil of plant pair⁺Absorbing and improving Ca in saline-alkali soil or alkaline soil by plants²⁺The use in an absorbent product for the manufacture of,

c9, application in improving plant photosynthetic performance or application in preparing products for improving plant photosynthetic performance,

c10 reducing intercellular CO of plant₂Application in concentration or preparation of plant intercellular CO reduction₂The application of the water-soluble polymer in a concentration product,

c11, application in improving net photosynthetic rate of plants or application in preparing products for improving net photosynthetic rate of plants,

c12, application in improving the conductivity of plant stomata or application in preparing products for improving the conductivity of plant stomata,

c13, application in improving plant transpiration rate or application in preparing products for improving plant transpiration rate,

c14, the application of the plant leaf water utilization efficiency improving agent or the application of the plant leaf water utilization efficiency improving agent in the preparation of products,

c15, application in increasing the chlorophyll content of plants or application in preparing products for increasing the chlorophyll content of plants,

c16, use in the production of auxin (IAA) or use in the preparation of a product for the production of auxin (IAA),

c17, application in promoting plant growth or application in preparing products for promoting plant growth,

c18, use for increasing crop yield or for preparing a product for increasing crop yield,

c19, use for increasing plant biomass or use for preparing a product for increasing plant biomass.

In the application, the product can be a microbial ecological preparation containing the microbial inoculum or a biological fertilizer containing the microbial inoculum.

In the present application, the plant or crop may be a monocot or a dicot. The dicotyledonous plant may be a plant of the order platycodonales. The tubular florales plant may be a solanaceae plant. The Compositae plant can be Helianthus plant. The plant of the genus Helianthus may be Helianthus annuus.

The method for culturing the flavobacterium acidificum 237T2 also belongs to the protection scope of the invention.

The method for culturing the flavobacterium acidificans 237T2 provided by the invention comprises the step of culturing the flavobacterium acidificans 237T2 in a culture medium for culturing microorganisms.

The method for preparing the microbial inoculum also belongs to the protection scope of the invention.

The method for preparing the microbial inoculum provided by the invention comprises the step of taking metabolites of the flavobacterium acidi 237T2 and/or the flavobacterium acidi 237T2 as components of the microbial inoculum to obtain the microbial inoculum.

In the above method, the microbial inoculum may be a liquid microbial inoculum. In the above method, the flavobacterium acidifier 237T2 can be cultured in a fermentation medium to obtain a fermentation broth, and the fermentation broth is mixed with a carrier to obtain the liquid microbial inoculum. The carrier may be peatmoss.

The invention utilizes the flavobacterium acidifier 237T2 to prepare the saline-alkali soil improvement microbial inoculum, because the microbial inoculum contains a large amount of beneficial viable bacteria substances and a plurality of natural fermentation active substances, the microbial inoculum can be propagated in the soil of a root zone to form a microbial dominant flora beneficial to the growth of crops, thereby regulating the nutrition environment of the rhizosphere, manufacturing and assisting the crops to absorb nutrition, improving and restoring the microecological balance of the soil, and being capable of improving the physicochemical property and the soil structure of the saline-alkali soil, thereby improving permeability, accelerating salt leaching, cutting off capillary tubes, preventing the saline-alkali from returning upwards and reducing the soil salinity. The flavobacterium acidificans 237T2 disclosed by the invention has the advantages of reducing the pH value of the microenvironment of rhizosphere soil, being capable of symbiotically growing with plants, promoting the growth of the plants and adapting to the unfavorable growth environment of saline-alkali soil. The mechanism for improving the saline-alkali soil is as follows: the activity of the flavobacterium acidicum 237T2 can generate a large amount of metabolic derivatives, and insoluble elements solidified by saline-alkali soil can be effectively replaced for plants to absorb and utilize; secondly, the turf contains a large amount of organic matters, has high organic matter content and rich humic acid, and also contains cellulose, hemicellulose, nitrogen, phosphorus, potassium and other substances; ③ the flavobacterium acidicum 237T2 can reduce the salinity and alkalinity of the soil. The acid flavobacterium 237T2 is screened from plants, has the advantages of symbiosis with plants, and can generate IAA and promote the growth of crops.

Biological material preservation instructions.

Classification nomenclature of biological materials: flavobacterium acidicum.

Latin literature name of biomaterial: flavobacterium acidificum.

Strain number of biological material: 237T 2.

The preservation unit is called as follows: china general microbiological culture Collection center.

The preservation unit is abbreviated as: CGMCC.

Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.

The preservation date is as follows: year 2020, 5 and 25.

The preservation number is: CGMCC No. 19869.

Drawings

FIG. 1 is a phylogenetic tree constructed based on the 16S rRNA gene sequence.

FIG. 2 shows the effect of liquid microbial inoculum of Flavobacterium acidicum 237T2 on the pH value of saline-alkali soil. The difference between treatments with different lower case letters was significant (P < 0.05).

FIG. 3 shows the qualitative detection result of the secretion of auxin by Flavobacterium acidocaldarium 237T 2. The left panel shows the treatment with the fermentation broth of Flavobacterium acidocaldarium 237T2, and the right panel shows the negative Control (CK).

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The soil salinity content and pH determination methods in the following examples are as follows: taking a five-point sampling method, taking 100g of soil (hereinafter, the soil is referred to as initial soil) at each point, taking the soil back to a laboratory, naturally drying the soil in the air, grinding the soil, sieving the soil by a 2mm sieve, extracting a supernatant of a soil solution at a soil-water ratio of 1:5, and measuring the pH and the conductivity of the soil by a pH meter (FE20) and a conductivity meter (DDS-307) respectively, wherein the conductivity is converted into the content of the salt of the soil according to an empirical formula: soil salinity content (g.kg)^-1) Conductivity × 0.064 × 5 × 10/1000.

Plant K in the examples below⁺、Na⁺And Ca²⁺The content determination method comprises the following steps: weighing about 0.5g of plant leaves in a digestion tank, and adding 4ml of concentrated nitric acid (super grade pure), H₂O₂(guaranteed reagent) 2 ml. Standing for 2 hours, capping, screwing, shaking gently, placing on a microwave digestion instrument for digestion according to a set program, after digestion, uncapping, placing on an acid dispelling instrument, dispelling acid at about 160 ℃ until the acid is completely dried, transferring with 5% nitric acid to a volumetric flask with the volume of 50ml, performing dry filtration, and detecting with an atomic absorption spectrophotometer.

Na in the following examples⁺/K⁺＝Na⁺content/K⁺Content of Na⁺/Ca²⁺＝Na⁺content/Ca²⁺And (4) content.

Net photosynthetic Rate, stomatal conductance, intercellular CO in the examples below₂The concentration and transpiration rate measurements were as follows: at 8 a.m.: 00-11: 30 the measurement of the plant leaves is carried out by using an LI-6400 type portable photosynthetic apparatus (Beijing Gaotai technologies Co., Ltd.), and the light intensity is uniformly set to 1900 umol/(m)²·s)。

The method of measuring Water Use Efficiency (WUE) in the following examples is as follows: WUE is GY/(P +. DELTA.W).

Wherein GY is yield (kg); p is precipitation (mm); the delta W is the difference (mm) of the water storage capacity of the soil at the beginning and the end of the observation period.

The chlorophyll content was measured as follows in the following examples: the plant leaf parts were measured using a portable chlorophyll meter (SPAD-502, KONICAMINOLTA SENSING, INC.) at 8 a.m.: 00-8: 30. the SPAD value is a parameter that measures the relative content of chlorophyll in a plant.

The fragrance concentration index, simpson index, ACE index and Chao1 index were determined as follows in the following examples: collecting sunflower leaves, storing in a refrigerator at 4 deg.C, and determining by Mergiz biological medicine science and technology Limited.

All data in the following examples were significantly analyzed using IBM SPSS Statistics 23.

Example 1 isolation and characterization of Flavobacterium acidicum (Flavobacterium aceticum) 237T2

Isolation of Flavobacterium acidicum (Flavobacterium acidificum)

Collecting rice seeds in a greenhouse of an agricultural resource and agricultural division institute of China academy of agricultural sciences in Haisheng area of Beijing, husking, and sterilizing the surface of the rice seeds, wherein the method comprises the following steps: washing 1g of hulled rice seed with sterile water to remove surface dust, etc., sequentially soaking in 70% ethanol for 3min, 5% sodium hypochlorite for 5min, 70% ethanol for 30s, and finally washing with sterile water for 5-7 times, simultaneously smearing the sterilized rice seed on a TSA (Shanghai ai research Biotechnology Co., Ltd.) plate under light pressure once, and culturing at 28 deg.C for 72h to check the surface sterilization effect. Grinding the surface sterilized seeds in a sterile mortar to powder, transferring the powder to 45ml of sterile water containing glass beads, shaking at 28 deg.C for 30min, and curingColumns, respectively from 10^-3、10^-4And 10^-5Spreading 0.1ml of the diluted solution on a plate, repeating for 3 times at each dilution, culturing at 28 deg.C for 3-5 days, picking up single colony, and separating to obtain endophytic bacterium 237T 2.

Identification of Flavobacterium acidicum (Flavobacterium acidificum)

1. Morphological identification

The endophytic bacteria 237T2 in the logarithmic growth phase were observed for colony thallus status by plate streaking, and mainly included the size, color, transparency, wettability of the colony, colony surface status (whether flat, protruding, wrinkled, recessed, etc.), and colony edge status (whether regular, irregular, radial, etc.). On the other hand, endophytic bacterium 237T2 in the logarithmic growth phase was gram-stained, and then the morphology of the cells was observed by an optical microscope. Biochemical identification was performed using the API20NE system.

The results show that the endogenous bacterium 237T2 is rod-shaped, gram-negative, round in colony, irregular in edge, smooth in surface, wet, yellow, slightly protruding and opaque. The results of the physiological and biochemical characteristics of Flavobacterium acidicum 237T2 are shown in Table 1, and the results are negative in the nitrate reduction utilization test, can decompose tryptophan to generate indole, can hydrolyze glucose to generate acid, is positive in the hydrolysis of esculin, is positive in the hydrolysis of gelatin, is negative in the hydrolysis of arginine, is negative in the hydrolysis of urea, and is positive in the PNPG test. The assimilation test of glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, gluconate, malic acid and citric acid is positive, and the assimilation test of capric acid, adipic acid and phenylacetic acid is negative.

TABLE 1. Flavobacterium acidicum 237T2 physiological and biochemical indexes

Test of	Substrate	Reaction of	Results
				NO₃	Potassium nitrate	Reduction and utilization of nitrate	-
TRP	L-tryptophane	Indole production	+
				GLU	D-glucose	Acidification of glucose	+
ADH	L-arginine	Arginine double hydrolase	-
				URE	Urea	Urea hydrolysis	-
ESC	Esculin	Hydrolysis of esculin	+
				GEL	Gelatin	Hydrolysis of gelatin	+
PNPG	4-nitroso-βD-methyl galactose	Galactosidase enzyme	+
				GLU	D-glucose	Glucose assimilation reaction	+
ARA	L-arabinose	Arabinose assimilation reaction	+
				MNE	D-mannose	Mannose assimilation reaction	+
MAN	D-mannitol	Assimilation of mannitol	+
				NAG	N-acetyl glucosamine	N-acetyl-glucosamine assimilation	+
MAL	D-maltose	Assimilation of maltoseReaction of	+
				GNT	Gluconate	Assimilation of gluconate	+
CAP	Decanoic acid	Assimilation of capric acid	-
				ADI	Adipic acid	Adipic acid assimilation reaction	-
MLT	Malic acid	Assimilation of malic acid	+
				CIT	Citric acid	Assimilation of citric acid	+
PAC	Phenylacetic acid	Phenylacetic acid assimilation	-

2. 16S rRNA gene and genome sequence homology analysis

Extraction of total genomic DNA of endophytic bacterium 237T2 adopts a rhizobacteria genome extraction kit. The 16S rRNA gene was amplified using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The reaction system is 50 μ L: comprises 10 XBuffer (5 μ L), 4 μ L dNTP (2.5m mol/L), 2 μ L primer 27F (10m mol/L), 0.25 μ L primer 1492R (10m mol/L)2 μ L, Taq enzyme (5U/L), 3 μ L DNA as template, ddH₂O make up to 50. mu.L. PCR reaction procedure: 5min at 94 ℃; 1min at 94 ℃, 1min at 52 ℃, 1min at 72 ℃ and 30 cycles; 10min at 72 ℃. The PCR products were sent to Shanghai Producer for bidirectional sequencing and the sequence was analyzed by http:// eztaxon-e.ezbiocloud. net/ezt _ identity.

The obtained 16S rRNA fragment has the length of 1286bp, and the sequence of the fragment is detailed in a sequence 1 in a sequence table. The similarity between the endophytic bacterium 237T2 and Flavobacterium acidophilum model strain is the highest in the result of the Eztaxon website comparison, and is 99.45%, and a phylogenetic tree (figure 1) constructed according to the 16S rRNA gene sequence shows that 237T2 and Flavobacterium acidophilum are gathered into one type, and the result shows that the endophytic bacterium 237T2 is Flavobacterium acidicum (Flavobacterium acidophilum).

According to the above characteristics, endophytic bacterium 237T2 was identified as Flavobacterium acidificum. Flavobacterium acidicum 237T2 has been deposited in China general microbiological culture Collection center (CGMCC) at 25.5.2020, and the accession number of the CGMCC is CGMCC No. 19869. Hereinafter abbreviated to Flavobacterium acidicum 237T 2.

Example 2 Flavobacterium acidicum (Flavobacterium acidificum)237T2 secretes auxin

According to the reference (Eric G lickm ann, Yves Dessauux. A detailed education of the specificity of the salkowski reagent for induced compounds produced by bacteriophoretic bacteria J]Applied and environmental Microbiology,1995(2): 793-. Inoculating Flavobacterium acidicum 237T2 to R₂A liquid culture medium (Shanghai limited in research and development industries)Company), shaking at 28 ℃ for 4d with shaking at 180rpm to obtain 237T2 fermentation broth.

The qualitative detection was performed as follows: 50 μ L of 237T2 fermentation broth was mixed with 50 μ L of Salkowski colorimetric solution (Salkowski colorimetric solution prepared from 50mL of 35% HClO)₄Solution and 1mL of 0.5M FeCl₃Liquid formed by mixing the solutions) is dripped on a white ceramic plate and is placed for 0.5h at room temperature in a dark place, and then the IAA can be secreted by the red color. mu.L of 237T2 fermentation broth was treated with 50. mu.L of 50mg/L IAA colorimetric solution (50mg/L IAA colorimetric solution is obtained by adding IAA to Salkowski colorimetric solution to give 50mg/L IAA content) as a positive control, and 50. mu. L R of the total amount of the colorimetric solution was added₂A liquid medium was treated with 50. mu.L of 50mg/L IAA solution as a negative Control (CK). The experiment was performed in triplicate.

The qualitative detection result shows that the color of the bacterial liquid turns red after the Salkowski colorimetric solution is added into the 237T2 fermentation liquid dropwise, which indicates that 237T2 can secrete plant growth hormone (IAA) (FIG. 3).

And (3) quantitative detection: centrifuging the 237T2 fermentation liquid at 12000rpm for 1min, collecting supernatant 400ul, adding equivalent Salkowski colorimetric solution, standing in dark place for 30min, and measuring OD₅₃₀The value is obtained. Standard curves were prepared using analytically pure IAA gradient dilutions. Simultaneously, absorbing 237T2 fermentation liquor 800ul to 48-hole enzyme label plate, and measuring OD on an enzyme label instrument₆₀₀. The experiment was performed in triplicate.

Adopting IAA solutions with different concentrations and Salkowski colorimetric solution to mix in equal volume, standing for 30min in dark at room temperature, and determining OD₅₃₀Values, IAA at different concentrations were configured as follows:

quantification of IAA concentration: 250 mg/L: 0.025g IAA +100ml absolute ethanol

200 mg/L: 8.0ml 250mg/L IAA +2.0ml absolute ethanol

150 mg/L: 6.0ml 250mg/L IAA +4.0ml absolute ethanol

100 mg/L: 4.0ml 250mg/L IAA +6.0ml absolute ethanol

50 mg/L: 2.0ml 250mg/L IAA +8.0ml absolute ethanol

35 mg/L: 1.4ml 250mg/L IAA +8.6ml absolute ethanol

30 mg/L: 1.2ml 250mg/L IAA +8.8ml absolute ethanol

25 mg/L: 1.0ml 250mg/L IAA +9.0ml absolute ethanol

0.0 mg/L: 10.0ml of absolute ethanol.

The OD of 237T2 supernatant and Salkowski colorimetric solution was measured₅₃₀Value sum 237T2 fermentation liquid OD₆₀₀The quantitative result (Table 2) was obtained by conversion using a standard curve equation, and the IAA content in the 237T2 fermentation broth was 276.3978 mg/L.

TABLE 2 quantitative determination of IAA secretion by Flavobacterium acidicum 237T2

OD530	OD600	OD530/OD600	IAA concentration (mg/L)
				0.7105	0.2618	2.7139	276.3978

Example 3 preparation of microbial inoculum for improving saline-alkali soil

Inoculating activated Flavobacterium acidicum 237T2 into 500mL triangular flask containing 100mL fermentation medium, culturing at 37 deg.C under shaking at 180rpm for 48h to obtain Flavobacterium acidicum 237T2 fermentation liquid, wherein the content of Flavobacterium acidicum 237T2 in the fermentation liquid of Flavobacterium acidicum 237T2 is 10⁹cfu/ml. Wherein, the fermentation medium is a neutral TSB medium, and the preparation method comprises the following steps: according to the commercial medium instructions, 30g of dry TSB (BD Co.) powder was added and steamedDistilled water is added to 1000ml, then the pH value is adjusted to 7.0 by NaOH, and the solution obtained by sterilizing the solution for 20 minutes at 121 ℃ is the neutral TSB culture medium.

And (3) uniformly mixing the turfy soil (carrier) and the fermentation liquor of the flavobacterium acidi 237T2 to obtain the microbial agent for improving the saline-alkali soil. The content of the flavobacterium acidifier 237T2 in the microbial inoculum for improving the saline-alkali soil is 5 multiplied by 10⁸cfu/g。

Example 4 laboratory test for lowering pH of saline-alkali soil Using Flavobacterium acidicum 237T2

Inoculating Flavobacterium acidicum 237T2 into neutral TSB culture medium (same as example 2), shaking and culturing at 30 deg.C and 180rpm (rotation radius of 20mm) for 48h to obtain Flavobacterium acidicum 237T2 fermentation liquid, wherein the content of Flavobacterium acidicum 237T2 in the Flavobacterium acidicum 237T2 fermentation liquid is 10⁹cfu/ml。

The fermentation broth of Flavobacterium acidi 237T2 was applied to medium saline-alkali soil having a pH of 8.45 (saline-alkali soil was taken from corn field in the test station of the Ministry of Long irrigation district, Enoted in the Hetao irrigation district, Pengyuan county, inner Mongolia) on day 12 month 3, the fermentation broth of Flavobacterium acidi 237T2 was applied to soil at an amount of 2kg per mu, and the pH changes were measured at day 12 month 4, day 12 month 6, day 12 month 9 and day 12 month 18, respectively, and the medium saline-alkali soil having a pH of 8.45, to which the fermentation broth of Flavobacterium acidi 237T2 was not applied, was used as a Control (CK). The flavobacterium acidi 237T2 has a significant effect on reducing the pH of soil, and the reduction effect is immediate, the pH value of the fermentation broth of flavobacterium acidi 237T2 is significantly reduced by 0.34 unit (P <0.05) compared with CK on the first day, and the reduction range of the pH value of the fermentation broth of flavobacterium acidi 237T2 on the 3 rd to 15 th days (i.e. 12 months, 6 days to 18 days) is similar to that of the first day (12 months, 4 days) (fig. 2).

Example 5 field experiment of improving saline-alkali soil by promoting crop growth with Flavobacterium acidicum 237T2

1. Test site

The field plot experiment is arranged in the field of Wuyuan county, inner Mongolia, and the field soil is silt loam. Before fertilization, a five-point sampling method is adopted, 100g of soil (hereinafter referred to as initial soil) is taken at each point and taken back to a laboratory, and salt content, pH (potential of hydrogen) and nutrient content (the nutrient content detection method is that a soil sample with 0-40cm layers is taken after the sunflower is harvested, taken back to the laboratory for natural air drying, crushed and sieved, and then soil organic matter, quick-acting nitrogen (alkaline hydrolysis method) and quick-acting potassium (ammonium acetate extraction) are measured, and the specific measurement method is that soil agriculture analysis (Baysantin, 2005), soil salt content and pH are measured by taking the sampled soil back to the laboratory for natural air drying, grinding, sieving by a 2mm sieve, extracting a soil solution supernatant with a soil-water ratio of 1:5, measuring the soil conductivity by a conductivity meter (DDS-307), and converting the soil conductivity into the soil salt content according to an empirical formula, wherein the soil salt content (g.kg-1) is as conductivity multiplied by 0.064 × 5 10/1000. The results are shown in Table 3.

TABLE 3 physicochemical Properties of the initial soil

2. Design and implementation of experiments

In 2019, field micro-district tests are carried out in Wuyuan county, inner Mongolia, 5-10 months. A random block design is adopted, 2 treatment areas are randomly arranged, and each treatment area is provided with three repetitions. The 2 treatment zones are respectively a non-microbial agent application treatment zone (CK) and a microbial agent treatment zone (JJ).

The study area was divided into 6 cells, each cell area 1.8m × 1.8 m. And the processing is distributed in a random drawing mode. The above 2 treatment zones were operated in the same manner except that they were inoculated with F.acidocaldarius 237T 2.

Edible sunflower JK601 is sowed in 2019, 5 and 26 days, and base fertilizers are applied in both treatments according to the local method when the sunflower is sowed, wherein the specific fertilizing method is as follows: 0.067kg of organic silicon water-soluble slow-release fertilizer compound fertilizer is applied per square meter to serve as base fertilizer. In 2019, 6 months and 18 days, the two treatment areas are subjected to seedling stage topdressing, and the microbial agent treatment area is 600kg/hm²The microbial agent for improving saline-alkali soil of example 3 was added in such an amount that the cell content of Flavobacterium acidocaldarium 237T2 in the soil was 3X 10¹⁰cfu/m²Without applyingTreating with bacterial agent at a rate of 600kg/hm²The amount of peatmoss (carrier in the saline-alkali soil improvement microbial agent of example 3); the application method comprises ditching and strip applying 5-10cm near the root of the plant, with the depth of 10-15cm, and covering with soil after applying. During the test period, photosynthesis of leaves of the sunflower in the bud stage (Table 4), plant height and SPAD value (Table 5), soil salinity, pH and plant ion content in the harvest stage (Table 6), and soil bacteria diversity (Table 7) were measured.

(1) Effect of Flavobacterium acidicum 237T2 on photosynthetic property of saline-alkali soil

TABLE 4 influence of microbial agents for improving saline-alkali soil on photosynthetic property of saline-alkali soil

Note: the different lower case letters following the same column of data indicate significant differences (P < 0.05).

As can be seen from Table 4, the net photosynthetic rate, stomatal conductance, transpiration rate, leaf water utilization efficiency and SPAD of the microbial inoculum-treated area are respectively improved by 52.90%, 46.15%, 33.33%, 14.51% and 12.99% compared with the area without the microbial inoculum; and the microbial inoculum treatment zone is used for reducing intercellular CO₂The concentration has obvious effect, and is reduced by 20.29 percent compared with a region without applying the microbial inoculum. It is demonstrated that the flavobacterium acidificum 237T2 can improve the photosynthetic performance of plants in saline-alkali soil.

The planting area of the inner Mongolia sunflower accounts for about 30 percent of the total area of the national sunflowers, and the total yield accounts for about 39 percent of the total yield of the national sunflowers. In recent years, planting sunflower has become the mainstay of local agriculture and is one of the major economic sources for the local farmers to expand reproduction. Photosynthesis is a very complex biological oxidation-reduction process, can assimilate simple inorganic matters around into organic matters for self utilization, and is an important influence factor in the processes of dry matter accumulation and yield formation of crops. In the total dry matter of the plants, the organic matter formed by photosynthesis accounts for about 95%. Net photosynthetic rate, stomatal conductance and intercellular CO₂Concentration can be measuredPhotosynthetic performance of the plant. The soil salinization can affect the respiration, photosynthesis and the like of plants, so the soil salinization is one of important factors for restricting the continuous development of crops and agriculture in the river irrigation areas. The research on the photosynthetic physiological characteristics of the sunflowers in the saline-alkali soil has great significance for improving the productivity of the sunflowers.

(2) Influence of flavobacterium acidicum 237T2 on plant height, overground part biological quality and yield of saline-alkali soil plants

TABLE 5 influence of microbial inoculum for improving saline-alkali soil on plant height, overground part biological quality and yield of saline-alkali soil

Treatment of	Plant height (cm)	Dry weight of aerial biomass (g)	Yield (kg. hm)^-2)
				CK	93.05a	125.85a	2050a
JJ	107.33b	186.20b	2400b

As can be seen from Table 5, the plant height of the microbial agent treatment zone is significantly increased by 15.35% compared with CK, the overground part biomass is significantly increased by 47.95% compared with CK, and the yield is significantly increased by 17.07% compared with CK. The results show that the flavobacterium acidificum 237T2 can promote plant growth and improve plant yield in saline-alkali soil.

(3) Influence of flavobacterium acidificans 237T2 on pH of saline-alkali soil and plant ion content

TABLE 6 microbial inoculum for improving saline-alkali soil for saline-alkali soil pH and plant K⁺、Na⁺And Ca²⁺Influence of the ion content

Treatment of

pH

K⁺(g·kg^-1)

Na⁺(g·kg^-1)

Ca²⁺(g·kg^-1)

Na⁺/K⁺

Na⁺/Ca²⁺

CK

8.45a

29.55a

12.84a

3.43a

0.43a

3.74a

JJ

8.21b

36.33b

10.45b

4.76b

0.29b

2.19b

As can be seen from Table 6, the pH value of the soil in the microbial agent treatment area can be obviously reduced by 0.24 unit compared with that of CK; the application of the saline-alkali soil improvement microbial agent of example 3 can increase K of plants to various degrees⁺And Ca²⁺Content of Na is reduced⁺In which K is⁺The content is increased by 22.94 percent compared with CK; ca²⁺The content is increased by 38.78 percent compared with CK; and Na⁺The content is reduced by 22.87 percent compared with CK.

The results show that the acid flavobacterium 237T2 can obviously reduce the pH value of saline-alkali soil and can obviously increase the K of plants⁺And Ca⁺Content of Na is obviously reduced⁺And (4) content. Flavobacterium acidicum 237T2 reduces plant Na⁺The absorption of the fertilizer increases the saline-alkali resistance of crops.

(4) Influence of flavobacterium acidicum 237T2 on bacterial diversity of saline-alkali soil

TABLE 7 influence of microbial Agents for improving saline-alkali soil on the bacterial diversity of saline-alkali soil

Treatment of	Shannon index	Simpson index	chao1 index	ACE index
					CK	5.078±0.306a	0.021±0.006a	2749.026±144.236a	2745.531±134.382a
JJ	5.544±0.497a	0.070±0.046a	2888.423±186.830a	2828.206±159.916a

As can be seen from table 7, the diversity of the bacteria treated with 237T2 was improved compared to CK, but the effects thereof were not significant, but the aroma index, simpson index, chao1 index, and ACE index were respectively improved by 9.18%, 233.33%, 5.07%, and 3.01% compared to CK.

The soil microbial diversity is the most important control factor for the stability of a soil ecosystem and is also an important index for measuring the balance of the soil ecosystem. The fragrance concentration index and the simpson index are indices used to estimate the microbial diversity in a sample, wherein a greater fragrance concentration index value indicates a higher microbial community diversity in the sample, and the simpson index is the opposite; the ACE index and the Chao1 index are indices used to estimate the abundance of a bacterial population, and the larger the value, the higher the abundance.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> institute of agricultural resources and agricultural regionalism of Chinese academy of agricultural sciences

<120> Flavobacterium acidicum and application thereof in improvement of saline-alkali soil

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1286

<212> DNA

<213> Flavobacterium acidicum (Flavobacterium acidificum)

<400> 1

cggtgtgtac aaggcccggg aacgtattca ccgtggcatt ctgatccacg attactagcg 60

attccgactt cacggagtcg agttgcagac tccgatccgg actacgacgc actttatgag 120

gtccgcttgc tctcgcgagg tcgcttctct ttgtatgcgc cattgtagca cgtgtgtagc 180

cctactcgta agggccatga tgacttgacg tcatccccac cttcctccgg tttatcaccg 240

gcagtctcct ttgagttccc gaccgaatcg ctggcaacaa aggataaggg ttgcgctcgt 300

tgcgggactt aacccaacat ttcacaacac gagctgacga cagccatgca gcacctgtct 360

cacggttccc gaaggcacta aggcatctct gccgaattcc gtggatgtca agagtaggta 420

aggttcttcg cgttgcatcg aattaaacca catgctccac cgcttgtgcg ggcccccgtc 480

aattcatttg agttttaacc ttgcggccgt actccccagg cggtcgactt aacgcgttag 540

ctccggaagc cactcctcaa gggaacaacc ctccaagtcg acatcgttta cggcgtggac 600

taccagggta tctaatcctg tttgctcccc acgctttcgc accctgagcg tcagtcttcg 660

tccagggggc cgccttcgcc accggtattc ctccagatct ctacgcattt caccgctaca 720

cctggaattc tacccccctc tacgagactc aagcctgcca gtttcaaatg cagttcccag 780

gttaagcccg gggatttcac atctgactta acagaccgcc tgcgtgcgct ttacgcccag 840

taattccgat taacgcttgc accctccgta ttaccgcggc tgctggcacg gagttagccg 900

gtgcttcttc tgcgggtaac gtcaatcagt aaggttatta acctcattgc cttcctcccc 960

gctgaaagta ctttacaacc cgaaggcctt cttcatacac gcggcatggc tgcatcaggc 1020

ttgcgcccat tgtgcaatat tccccactgc tgcctcccgt aggagtctgg accgtgtctc 1080

agttccagtg tggctggtca tcctctcaga ccagctaggg atcgtcgcct aggtgggccg 1140

ttaccccgcc tactagctaa tcccatctgg gttcatccga tagtgagagg cccgaaggtc 1200

cccctctttg gtcttgcgac gttatgcggt attagccacc gtttccagtg gttatccccc 1260

tctatcgggc agatccccag acatta 1286

Claims

1. Flavobacterium acidicum, characterized in that: the acid Flavobacterium is Flavobacterium acidificium 237T2, and the registration number of the acid Flavobacterium is CGMCC No.19869 in the common microorganism center of China Committee for culture Collection of microorganisms.

2. The microbial inoculum, the microbial ecological agent containing the microbial inoculum or the biological fertilizer containing the microbial inoculum are characterized in that: the microbial agent contains the flavobacterium acidi of claim 1 or/and metabolites of the flavobacterium acidi.

3. The microbial inoculum, probiotic or biofertilizer of claim 2, wherein: the microbial inoculum is any one of the following microbial inocula:

a1, bacterial agent for improving saline-alkali soil or alkaline soil,

a9, a bacterial agent for improving the photosynthetic performance of plants,

a11, a bacterial agent for increasing the net photosynthetic rate of plants,

a12, a microbial inoculum for improving the stomatal conductance of plants,

a13, a microbial inoculum for improving plant transpiration rate,

a15, a bacterial agent for increasing the chlorophyll content of plants,

a16, a bacterial agent for producing auxin,

a17, bacterial agent for promoting plant growth,

a18, microbial inoculum for improving crop yield,

a19, microbial inoculum for increasing plant biomass.

4. The culture of Flavobacterium acidocaldarium of claim 1, which is a product obtained by culturing the Flavobacterium acidocaldarium of claim 1 in a microorganism medium.

5. The culture of claim 4, wherein: the culture has at least one of the following functions:

b1, improving saline-alkali soil or alkaline soil,

b2, reducing the pH value of saline-alkali soil or alkaline soil,

b7, reducing Na content in saline-alkali soil or alkaline soil⁺Absorbing and improving K in saline-alkali soil by plants⁺The absorption is carried out by the absorption device,

b9, improving the photosynthetic performance of the plant,

b11, improving the net photosynthetic rate of plants,

b12, improving the conductivity of the plant stomata,

b13, improving the plant transpiration rate,

b14, improving the water utilization efficiency of the plant leaves,

b15, increasing the chlorophyll content of the plant,

b16, producing the auxin,

b17, promoting the growth of plants,

b18, improving the yield of the crops,

and B19, increasing the biomass of the plant.

6. The use of Flavobacterium acidocaldarium and/or the metabolite of Flavobacterium acidocaldarium and/or any one of the following applications of the culture of claim 4 or 5:

u16, use in the production of auxin or use in the preparation of an auxin-producing product,

7. The microbial inoculum of claim 2 or 3 for any one of the following uses:

c16, use in the production of auxin or use in the preparation of an auxin-producing product,

8. The method for culturing F.acidocaldarius according to claim 1, comprising the step of culturing the F.acidocaldarius in a medium for culturing the microorganism.

9. A method for producing the microbial agent according to claim 2 or 3, comprising a step of obtaining the microbial agent by using the Flavobacterium acidicum according to claim 1 and/or the metabolite of the Flavobacterium acidicum according to claim 1 as a component of the microbial agent.