CN106929443B - Vibrio LX6-2 and application thereof in preparation of biological seaweed fertilizer - Google Patents

Vibrio LX6-2 and application thereof in preparation of biological seaweed fertilizer Download PDF

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CN106929443B
CN106929443B CN201710148058.4A CN201710148058A CN106929443B CN 106929443 B CN106929443 B CN 106929443B CN 201710148058 A CN201710148058 A CN 201710148058A CN 106929443 B CN106929443 B CN 106929443B
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seaweed
biological
fertilizer
fermentation
seaweed fertilizer
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CN106929443A (en
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孙杉杉
肖艳
杜迎辉
高小佳
杨娜
李丽艳
揣峻峰
徐延平
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LEADING BIO-AGRICULTURAL CO LTD
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Abstract

The invention relates to vibrio LX6-2 and application thereof in preparing biological seaweed fertilizer. The preparation steps of the biological seaweed fertilizer comprise vibrio LX6-2 slant culture, seed culture, preparation of seaweed digestive juice, fermentation of biological seaweed liquid and the like. The gibberellin content of the biological seaweed fertilizer is more than 5000ppm, and compared with the conventional chemical treatment method and other biological methods, the gibberellin content of the biological seaweed fertilizer is improved by 1-2 times, so that the growth and development of plants are effectively promoted, and the crop yield is improved. The composite biological seaweed fertilizer also has the soil improvement effects of promoting soil to form a granular structure and enhancing soil permeability, has the functions of obviously improving the drought resistance and stress resistance of plants and remarkable growth promoting effect, has good prevention effect on wheat take-all and pepper bacterial wilt, and has good prevention and control effects on rhizoctonia cerealis, rhizoctonia solani and the like.

Description

Vibrio LX6-2 and application thereof in preparation of biological seaweed fertilizer
[ technical field ] A method for producing a semiconductor device
The invention belongs to the technical field of biological fertilizers. More particularly, the invention relates to vibrio LX6-2 and application of the vibrio LX6-2 in preparation of biological seaweed fertilizers.
[ background of the invention ]
The seaweed is a marine organism with rich sources, and is widely applied to foods, industrial raw materials and botanical drugs. The application of seaweed in agriculture and animal husbandry has been known for hundreds of years. The seaweed fertilizer is a novel pure natural fertilizer without toxic and side effects, contains rich amino acids, mineral substances, polysaccharides, vitamins and physiological active substances, and has the effects of improving the yield of crops, improving the quality, enhancing the drought resistance, cold resistance, disease resistance and the like of the crops. Therefore, the production of organic fertilizer by using seaweed becomes one of the important hotspots of the research of the current novel fertilizer.
The seaweed fertilizer is prepared by using large fast-growing brown algae growing in the sea as a raw material and extracting effective components in the seaweed by a chemical, physical or biological method, and can promote plant growth, increase yield and improve the quality of agricultural products by applying to plants. The seaweed fertilizer is a natural organic fertilizer, and the fertilizer efficiency of the seaweed fertilizer depends on main active substances such as cytokinin, auxin, gibberellin, abscisic acid, ethylene, betaine, polyamine and the like contained in the seaweed fertilizer. The seaweed fertilizer contains a large amount of non-nitrogenous organic matters, a certain amount of amino acid, protein and trace nutrient elements, and also contains seaweed polysaccharide, alginic acid, highly unsaturated fatty acid which are specific to marine organisms and trace mineral elements such as zinc, bromine, iodine and the like which are required by terrestrial plant growth.
At present, the production methods of seaweed fertilizers at home and abroad generally comprise a chemical hydrolysis method (namely a potassium hydroxide hydrolysis method), a physical extraction method, a biological fermentation method (namely an enzyme degradation method) and the like. In addition, the fertilizer is obtained by extracting bioactive substances from seaweed industrial waste and then scientifically preparing the bioactive substances. The extraction method has great influence on the content of active substances and nutrient components in the product, and the difference is great when the content is the same but the activity is different.
At present, most of manufacturers at home and abroad mainly adopt a chemical hydrolysis method to produce the seaweed fertilizer, and the method has the biggest defect that the activity of endogenous substances of the seaweed is damaged by strong alkali at high temperature. The physical extraction method adopts a high-pressure low-temperature cooling process to achieve the aim of seaweed cell wall breaking, is superior to a chemical extraction method, but has great difficulty in physical purification technology and great difficulty in industrial implementation.
The biological fermentation method is to utilize various enzymes produced by microbes in the metabolic process of taking seaweed and the like as nutrients to degrade macromolecular substances forming the seaweed into micromolecular and water-soluble substances, and because the fermentation process has no strong alkali and high temperature of a chemical method and has no high pressure and low temperature of a physical method, the biological active substances and nutrient substances in the seaweed are completely reserved to the maximum extent and can be absorbed and utilized by plants. At present, the seaweed fertilizer produced by adopting a biological fermentation method is limited to laboratory research at home and abroad, and the research process and the application mechanism thereof are lack of deep system research, so the seaweed fertilizer still belongs to a blank in the aspect of industrialization technology.
The application method of the seaweed fertilizer mainly comprises three methods: (1) soil application: the seaweed fertilizer contains natural compounds such as seaweed polysaccharide which are natural soil regulators, so that heavy metal ions can be chelated, the durability and the effectiveness of active ingredients in soil can be increased, the formation of a soil aggregate structure can be promoted, the internal pore space of the soil can be improved, the proportion of solid, liquid and gas in the soil can be coordinated, the water retention rate and the air permeability of the soil can be improved, the natural colloid balance of the soil can be recovered, and soil organic matters can be increased. (2) Seed soaking: seed treatment has proven to be very effective in promoting early germination and improving early growth stress resistance. (3) And (3) foliage spraying: the foliar spraying is the most widely applied fertilizing mode, and can quickly supplement the marginal deficiency of the plants to the nutrient components, stimulate the development of rhizomes and the absorption of the nutrient components. Alginic acid in the seaweed fertilizer can reduce the surface tension of water, form a layer of film on the surface of plant leaves, increase the contact area, and enable water or water-soluble substances to easily permeate cell membranes on the surfaces of the leaves to enter plant cells, so that the plants can absorb the nutrient components in the seaweed extract most effectively.
Therefore, the seaweed fertilizer has the advantages of various application methods, simple and convenient use, wide application range and the like, so that more diversified seaweed products can be developed based on the seaweed fertilizer according to different requirements, and the seaweed fertilizer can meet the market requirements.
[ summary of the invention ]
[ problem to be solved ]
The invention aims to provide vibrio LX 6-2.
The invention also aims to provide application of the vibrio LX6-2 in preparing biological seaweed fertilizer.
[ solution ]
The invention is realized by the following technical scheme.
The invention relates to Vibrio (Vibrio sp.) LX6-2, which is preserved in the general microbiological culture Collection center of the institute of microbiology, China academy of sciences, China national institute of microbiology, No. 3, West Lu 1 institute of microbiology, Beijing, Tokyo, the area of rising, 3.23.2015, with the preservation number of CGMCC No. 10659.
The invention relates to application of vibrio LX6-2 in preparation of a biological seaweed fertilizer.
According to a preferred embodiment of the present invention, the preparation steps of the biological seaweed fertilizer are as follows:
A. slant culture of Vibrio LX6-2
Preparing a slant culture medium: preparing a pH7.0 aqueous solution containing 5g/l of sodium alginate, 3g/l of peptone, 3g/l of sodium chloride, 0.1g/l of magnesium sulfate, 2g/l of dipotassium hydrogen phosphate and 20g/l of agar powder, and then sterilizing at the temperature of 121 ℃ for 30min to obtain the slant culture medium;
inoculating a vibrio LX6-2 strain glycerol tube strain on an inclined plane of a test tube inclined plane solid culture medium, and then carrying out inclined plane culture for 2-3 d in a constant temperature incubator at the temperature of 28-30 ℃ to obtain an inclined plane culture;
B. seed culture
Preparing a seed culture medium: preparing a pH7.0 aqueous solution containing 5g/l of sodium alginate, 3g/l of peptone, 3g/l of sodium chloride, 0.1g/l of magnesium sulfate and 2g/l of dipotassium hydrogen phosphate, and then sterilizing at the temperature of 121 ℃ for 30min to obtain the seed culture medium;
b, taking a loop of the slant culture obtained in the step A by using an inoculating loop, inoculating the slant culture into the seed culture medium, and then culturing for 1-2 d in a constant temperature shaking table at the temperature of 28-30 ℃ and the rotating speed of 180rpm to obtain a seed culture;
C. preparation of seaweed digestive juice
Uniformly mixing the crushed dry seaweed raw material with a weak alkali aqueous solution, and then digesting for 1-2 hours at the temperature of 50-60 ℃ under the condition of stirring to obtain a seaweed digestive liquid;
D. fermentation of biological seaweed liquid
Adding culture medium components of peptone, sodium chloride, magnesium sulfate and dipotassium hydrogen phosphate into the seaweed digestive liquid obtained in the step C according to the ratio of 0.1-10 g/l peptone, 0.1-10 g/l sodium chloride, 0.1-10 g/l magnesium sulfate and 0.1-10 g/l dipotassium hydrogen phosphate in a fermentation tank filled with the seaweed digestive liquid;
and B, inoculating the seed culture obtained in the step B into a fermentation tank according to the volume of 1.5-2.5% of the seaweed digestive juice, stirring for 24 hours at a constant temperature under the conditions of the temperature of 28-30 ℃, the ventilation volume of 1: 1.0-1.2, the pressure of 0.04-0.06 MPa and the rotating speed of 100-200 rpm, and completely fermenting the seaweed digestive juice to obtain the biological seaweed fertilizer.
According to another preferred embodiment of the present invention, the biological seaweed fertilizer further comprises a fermentation broth selected from a macromolecular polyglutamic acid fermentation broth, a small polyglutamic acid fermentation broth, a pseudomonas fluorescens fermentation broth or a bacillus amyloliquefaciens fermentation broth.
According to another preferred embodiment of the invention, the macromolecular polyglutamic acid fermentation broth is obtained by fermenting and culturing bacillus subtilis (Bacillus subtilis) with the preservation number of CGMCC NO.2108 according to the method described in CN 101109010B.
According to another preferred embodiment of the present invention, the fermentation broth of the small molecule polyglutamic acid is obtained by LW fermentation culture of Bacillus licheniformis (B.licheniformis) with the preservation number of CGMCC NO.8821 according to the method described in CN 104694437A.
According to another preferred embodiment of the present invention, the P.fluorescens fermentation broth is obtained by fermentation culture of P.fluorescens F1 of Pseudomonas fluorescens with the preservation number of CGMCC No.8820 according to the method described in CN 104152380A.
According to another preferred embodiment of the present invention, the Bacillus amyloliquefaciens fermentation broth is obtained by fermenting and culturing Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LX-J1 with the preservation number of CGMCC No.11263 according to the method described in CN 105316266A.
According to another preferred embodiment of the invention, in the biological seaweed fertilizer containing the fermentation liquor, the effective viable bacteria of the bacillus subtilis CGMCC NO.2108, the bacillus licheniformis LW, the ultraviolet mutagenesis fluorescent pseudomonas F1 and the bacillus amyloliquefaciens LX-J1 are respectively more than 5 hundred million/ml.
According to another preferred embodiment of the invention, in the biological seaweed fertilizer containing the fermentation liquor, the content of the macromolecular polyglutamic acid fermentation liquor, the content of the micromolecular polyglutamic acid fermentation liquor, the content of the pseudomonas fluorescens fermentation liquor and the content of the bacillus amyloliquefaciens fermentation liquor are respectively 10-50 percent of the total volume of the biological seaweed fertilizer.
The present invention will be described in more detail below.
The invention relates to Vibrio (Vibrio sp.) LX6-2, which is preserved in the general microbiological culture Collection center of the institute of microbiology, China academy of sciences, China national institute of microbiology, No. 3, West Lu 1 institute of microbiology, Beijing, Tokyo, the area of rising, 3.23.2015, with the preservation number of CGMCC No. 10659.
The screening process of the strain will be described in more detail below.
The inventor separates 42 strains of bacteria from rotten kelp, and the plate screening experiment proves that 19 strains of bacteria can degrade alginic acid to different degrees under the plate culture condition, and then performs alginic acid degradation primary screening on the 19 strains of bacteria, wherein 7 strains of bacteria have better effect of degrading alginic acid, and the 7 strains of bacteria are Vibrio through 16S rDNA sequencing, and the strain numbers of the 7 strains are respectively listed in Table 1.
The detailed screening method is described as follows:
soaking herba Zosterae Marinae picked by seaside in seawater, standing at room temperature for 7 days, 15 days, and 30 days, respectively picking out rotten part of herba Zosterae Marinae, and transferring to plate culture medium (pH 7.0) composed of 5g/l sodium alginate, 3g/l peptone, 3g/l sodium chloride, 0.1g/l magnesium sulfate, 2g/l dipotassium hydrogen phosphate, and 20g/l agar powder. Culturing at constant temperature for 2-3 days to enrich the microbial flora on the kelp.
And selecting a ring of enriched lawn, carrying out streak culture and purification on the plate culture medium, and separating and purifying for multiple times to finally separate 42 strains of bacteria from the kelp with different rotting degrees in different periods.
And (3) carrying out a transparent circle degradation experiment on a flat plate, carrying out streak culture on the screened strains on the flat plate culture medium for 3-4 days, wherein 19 strains can degrade alginic acid to different degrees, and carrying out preliminary screening to obtain better degradation effect of 7 strains, so that 16S rDNA sequencing is carried out on the 7 strains to determine that the 7 strains are vibrios.
The 16S rDNA sequencing conditions were as follows:
preparation of sample total DNA: the extraction is carried out according to the conventional bacterial DNA extraction method.
PCR primers: forward primer 27F, reverse primer 1492R;
and (3) PCR reaction system: volume of the reaction system: 20 μ L, reaction composition: mu.L of DNA template, 10. mu.L of 2 XMMastarmix, 0.4. mu.L of 27F, 0.4. mu.L of 1492R, and ultrapure water to make up to 20. mu.L.
PCR amplification procedure: 2min at 95 ℃; 30 cycles (84 ℃ for 1min, 52 ℃ for 1min, 65 ℃ for 8 min); 18min at 65 ℃.
The amplified product is detected to be qualified by 0.7 percent agarose gel electrophoresis and then sent to Beijing Sanbo Polygala company for sequencing. After the detected DNA sequence is artificially corrected, the BIAST software in NCBI is used for on-line comparison, and through comparison, the similarity between the identified 9 bacterial strains and vibrio is 99%, so that the 9 bacterial strains are judged to be vibrio.
In order to select the strain more suitable for processing the seaweed leaching liquor, a comparative experiment of seaweed leaching liquor processing is carried out, and the experimental method comprises the following steps:
firstly preparing seaweed leach liquor. Weighing a proper amount of sargassum, adding water which is about 10-20 times of the volume of the sargassum, adding a small amount of sodium carbonate alkali to process the sargassum into slurry paste, and then adding peptone, sodium chloride, magnesium sulfate and dipotassium hydrogen phosphate into the slurry according to the ratio of 3g/l peptone, 3g/l sodium chloride, 0.1g/l magnesium sulfate and 2g/l dipotassium hydrogen phosphate to obtain the seaweed leaching liquor.
And then preparing a seed culture solution. Respectively inoculating 7 strains obtained by primary screening into a seed culture medium containing 5g/l of sodium alginate, 3g/l of peptone, 3g/l of sodium chloride, 0.1g/l of magnesium sulfate and 2g/l of dipotassium hydrogen phosphate in a shake flask with pH value of 7.0, and then placing the seed culture medium in a constant temperature shaking table to culture for 1-2 days at the temperature of 28-30 ℃ and the rotating speed of 180rpm to obtain a seed culture;
thirdly, respectively inoculating the strain seed cultures into the seaweed leaching solution according to 2% of the volume of the seaweed extracting solution, stirring at constant temperature and uniform speed at 28-30 ℃, sampling at different times, measuring the viscosity by adopting a Ubbelohde viscometer method, and the experimental results are listed in Table 1:
table 1: degradation effect of seaweed leaching liquor of 7 vibrio strains
Figure BDA0001243880800000061
The experimental results in Table 1 clearly show that the Vibrio LX6-2 strain (⑥ -2) has obvious advantages, and the viscosity of the extracting solution can be reduced to below 60 seconds within 24 hours.
The vibrio LX6-2 has the following biological characteristics:
morphological characteristics:
the bacillus genus negative bacillus, terminal flagellum, can move without spore by microscope and plate colony observation. Under the condition of liquid culture, the strain is in a thin rod shape and has no obvious bending radian. Under the condition of a solid culture plate, when the culture is carried out for 2 to 3 days, the bacterial colony is in a circular bulge shape, the diameter is 0.5 to 1.0 mm, the edge is neat, the bacterial colony is in a light yellow semitransparent shape, and the surface is wet.
Combining the morphological characteristics of the strain and the sequencing result of 16SrDNA, the strain is named as Vibrio sp LX6-2, which has been preserved by the China general microbiological culture Collection center of the institute of microbiology, China academy of sciences, China institute of microbiology, No. 3, West Lu 1, Beijing, the sunward, 3.23 days in 2015, 3.23.3.3.3.
The invention also relates to application of the vibrio LX6-2 in preparation of biological seaweed fertilizers.
According to the invention, the preparation steps of the biological seaweed fertilizer are as follows:
A. slant culture of Vibrio LX6-2
Preparing a slant culture medium: preparing a pH7.0 aqueous solution containing 5g/l of sodium alginate, 3g/l of peptone, 3g/l of sodium chloride, 0.1g/l of magnesium sulfate, 2g/l of dipotassium hydrogen phosphate and 20g/l of agar powder, and then sterilizing at the temperature of 121 ℃ for 30min to obtain the slant culture medium;
inoculating a vibrio LX6-2 strain glycerol tube strain on an inclined plane of a test tube inclined plane solid culture medium, and then carrying out inclined plane culture for 2-3 d in a constant temperature incubator at the temperature of 28-30 ℃ to obtain an inclined plane culture;
the peptone used in the preparation of the slant culture is, for example, a product of tryptone under the trade name manufactured by Obotaxy Biotechnology Co., Ltd, Beijing; the agar powder is, for example, a product of the bio-technology limited company of obozoxin, beijing under the trade name of agar powder.
In this step, the slant culture apparatus used is a product currently marketed, for example, a constant temperature incubator manufactured by Shanghai laboratory instruments Co., Ltd; the equipment used for sterilization is currently marketed products, such as portable pressure steam sterilizers manufactured by Jiangyin Binjiang medical equipment, Inc. or autoclave products of Xiansheng instruments, Equipment, Inc.
B. Seed culture
Preparing a seed culture medium: preparing a pH7.0 aqueous solution containing 5g/l of sodium alginate, 3g/l of peptone, 3g/l of sodium chloride, 0.1g/l of magnesium sulfate and 2g/l of dipotassium hydrogen phosphate, and then sterilizing at the temperature of 121 ℃ for 30min to obtain the seed culture medium;
b, taking a loop of the slant culture obtained in the step A by using an inoculating loop, inoculating the slant culture into the seed culture medium, and then culturing for 1-2 d in a constant temperature shaking table at the temperature of 28-30 ℃ and the rotating speed of 180rpm to obtain a seed culture;
the sodium alginate used in this step is a product currently marketed, for example by the company obozin under the trade name sodium alginate, other reagents as described above or as commonly used in the chemical technology field. The equipment used in this step is as described above and will not be described in detail here.
C. Preparation of seaweed digestive juice
Uniformly mixing the crushed dry seaweed raw material with a weak alkali aqueous solution, and then digesting for 1-2 hours at the temperature of 50-60 ℃ under the condition of stirring to obtain a seaweed digestive liquid;
the preparation method of the seaweed digestive juice is described in paragraph 0029-0039 of the patent application Specification of CN 201510287164.1 entitled "an active seaweed fertilizer and a production method thereof".
In the present invention, the seaweed is one or more kinds selected from gulfweed, kelp or Ascophyllum nodosum. Gulfweed, kelp and Ascophyllum are all products sold in the market at present.
The dried seaweed material is pulverized using equipment commonly used in the art of food processing, such as the pulverizing equipment sold under the trade name SCF biaxial micronizer by Shanghai Tri-powder Equipment Co., Ltd. The particle size of the dried seaweed material is not particularly critical, but a suitable particle size is advantageous for the subsequent digestion process. Generally, the dried seaweed has a particle size of 50 to 200 mesh.
According to the invention, the weak base is potassium carbonate, potassium bicarbonate, sodium carbonate or sodium bicarbonate, preferably potassium carbonate, potassium bicarbonate or sodium carbonate, more preferably potassium carbonate or potassium bicarbonate.
The concentration of the weak base aqueous solution used in the invention is 0.001-0.004 g/ml. It is not suitable if the concentration of the weak alkaline aqueous solution is too high or too low, because if the concentration of the weak alkaline aqueous solution is higher than 0.004g/ml, the pH of the solution is too high, which destroys the activity of natural nutrients in the seaweed; if the concentration of the weak alkali aqueous solution is lower than 0.001g/ml, the swelling effect of the weak alkali on the seaweed is limited, the subsequent enzymolysis process is influenced, and the extraction rate of active substances is reduced.
According to the invention, the mass ratio of the seaweed to the weak base is 5-8: 1. Preferably, the mass ratio of the seaweed to the weak base is 6-8: 1. More preferably, the mass ratio of the seaweed to the weak base is 7-8: 1.
When weak base is used for digestion, the seaweed powder particles need to be continuously stirred so as to be fully contacted with a weak base aqueous solution, and the digestion reaction is complete.
In this step, if the digestion reaction temperature is lower than 50 ℃, the digestion reaction is not completely carried out, the swelling effect of the seaweed cell wall is limited, and the dissolution of nutrient substances in the seaweed is not facilitated; if the digestion reaction temperature is higher than 60 ℃, the activity of the effective components in the seaweed is damaged, and the use effect of the seaweed fertilizer is influenced. Therefore, the temperature of the digestion reaction is 50-60 ℃, preferably 52-58 ℃, and more preferably 54-56 ℃.
Under the condition that the digestion reaction temperature is 50-60 ℃, if the digestion time is less than 1h, the digestion reaction is incomplete, the swelling effect of the cell walls of the seaweed is limited, and the dissolution of nutrient substances in the seaweed is not facilitated; if the digestion time is longer than 2 hours, the digestion reaction tends to be gentle, which causes great waste of energy and increases the cost. Therefore, the digestion reaction time is 1-2 h, preferably 1.2-1.8 h, and more preferably 1.4-1.6 h.
D. Fermentation of biological seaweed liquid
Adding culture medium components of peptone, sodium chloride, magnesium sulfate and dipotassium hydrogen phosphate into the seaweed digestive liquid obtained in the step C according to the ratio of 0.1-10 g/l peptone, 0.1-10 g/l sodium chloride, 0.1-10 g/l magnesium sulfate and 0.1-10 g/l dipotassium hydrogen phosphate in a fermentation tank filled with the seaweed digestive liquid;
and B, inoculating the seed culture obtained in the step B into a fermentation tank according to the volume of 1.5-2.5% of the seaweed digestive juice, stirring for 24 hours at a constant temperature under the conditions of the temperature of 28-30 ℃, the ventilation volume of 1: 1.0-1.2, the pressure of 0.04-0.06 MPa and the rotating speed of 100-200 rpm, and completely fermenting the seaweed digestive juice to obtain the biological seaweed fertilizer.
The high performance liquid chromatography is adopted for detection, the gibberellin content of the biological seaweed fertilizer prepared by fermenting the LX6-2 strain is more than 5000ppm, and compared with the conventional chemical treatment method and other biological methods, the gibberellin content is improved by 1-2 times, the original active substances in seaweed are protected to the greatest extent, the growth and development of plants are promoted more effectively, and the crop yield is improved.
According to the invention, the biological seaweed fertilizer also contains a fermentation broth selected from a macromolecular polyglutamic acid fermentation broth, a small-molecular polyglutamic acid fermentation broth, a pseudomonas fluorescens fermentation broth or a bacillus amyloliquefaciens fermentation broth.
In the invention, the biological seaweed fertilizer containing macromolecular polyglutamic acid fermentation liquor, micromolecular polyglutamic acid fermentation liquor, pseudomonas fluorescens fermentation liquor or bacillus amyloliquefaciens fermentation liquor is also called as a composite biological seaweed fertilizer.
The macromolecular polyglutamic acid fermentation broth is obtained by fermenting and culturing Bacillus subtilis with the preservation number of CGMCC NO.2108 according to the method described in CN 101109010B.
The micromolecular polyglutamic acid fermentation broth is obtained by fermenting and culturing Bacillus licheniformis (B.licheniformis) LW with the preservation number of CGMCC NO.8821 according to the method described in CN 104694437A.
The Pseudomonas fluorescens fermentation liquor is obtained by performing fermentation culture on Pseudomonas fluorescens (P.florescens) F1 with the preservation number of CGMCC No.8820 according to the method described in CN 104152380A.
The bacillus amyloliquefaciens fermentation liquid is obtained by fermenting and culturing bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LX-J1 with the preservation number of CGMCC No.11263 according to the method described in CN 105316266A.
In the invention, the effective viable bacteria of the bacillus subtilis CGMCC NO.2108, the bacillus licheniformis LW, the ultraviolet mutagenesis fluorescent pseudomonas F1 and the bacillus amyloliquefaciens LX-J1 in the biological seaweed fertilizer containing the fermentation liquor are respectively more than 5 hundred million/ml by adopting a flat viable bacteria counting method which is well known by technical personnel in the technical field.
According to the invention, in the biological seaweed fertilizer containing the fermentation liquor, the contents of the macromolecular polyglutamic acid fermentation liquor, the micromolecular polyglutamic acid fermentation liquor, the pseudomonas fluorescens fermentation liquor and the bacillus amyloliquefaciens fermentation liquor are respectively 10-50 percent of the total volume of the biological seaweed fertilizer.
In addition, the biological seaweed fertilizer is a compound biological seaweed fertilizer obtained by matching the mother liquor with the other functional microbial bacterial liquid, has the effects of other functional microbial fertilizers besides the effects of the biological seaweed fertilizer, is richer and more specific in function, can meet crops with different requirements, and effectively improves the fertilizing efficiency.
The composite biological seaweed fertilizer is obtained by mixing 10-50% of the total volume of the biological seaweed fertilizer with bacillus subtilis CGMCC No.2108 fermentation liquor, the polyglutamic acid content of the composite biological seaweed fertilizer is 3-15 g/l by adopting a CTAB method, the effective viable count of the bacillus subtilis is more than or equal to 5 hundred million/ml by adopting a flat viable count method well known by technical personnel in the technical field, and the composite biological seaweed fertilizer has the functions and effects of promoting soil to form a granular structure and enhancing the soil permeability.
The composite biological seaweed fertilizer is obtained by mixing the biological seaweed fertilizer with bacillus licheniformis CGMCC No.8821 fermentation liquor according to 10-50 percent of the total volume of the composite biological seaweed fertilizer, the content of micromolecular polyglutamic acid of the composite biological seaweed fertilizer is 4-20 g/l by adopting a CTAB method for detecting, and the effective viable count of the bacillus licheniformis is more than or equal to 5 hundred million/ml by adopting a flat viable count method well known by technical personnel in the technical field.
The composite biological seaweed fertilizer is obtained by mixing 10-50% of the total volume of the biological seaweed fertilizer with pseudomonas fluorescens CGMCC No.8820 fermentation broth, and the composite biological seaweed fertilizer is detected by adopting a flat viable count method well known by technical personnel in the technical field, wherein the effective viable count of the pseudomonas fluorescens is more than or equal to 5 hundred million/ml, and the composite biological seaweed fertilizer has the functional effects of the biological seaweed fertilizer and also has good control effect on wheat take-all and pepper bacterial wilt;
the composite biological seaweed fertilizer is obtained by mixing 10-50% of the total volume of the biological seaweed fertilizer with bacillus amyloliquefaciens CGMCC No.11263 fermentation liquor, and the composite biological seaweed fertilizer is detected by a flat viable count method well known by technical personnel in the technical field, wherein the number of effective viable starch-decomposing bacteria is more than or equal to 5 hundred million/ml, and the composite biological seaweed fertilizer has good control effects on rhizoctonia cerealis, rhizoctonia solani, pythium ultimum, sclerotium solani, potato black hemorrhoid and peanut southern blight besides the functional effects of the biological seaweed fertilizer.
[ advantageous effects ]
The invention has the beneficial effects that: compared with the conventional chemical treatment method and other biological methods, the gibberellin content of the biological seaweed fertilizer prepared by fermenting the LX6-2 strain is increased by 1-2 times, so that the growth and development of plants are effectively promoted and the crop yield is increased.
The compound biological seaweed fertilizer obtained by matching the mother liquor with the macromolecular polyglutamic acid fermentation liquor, the micromolecular polyglutamic acid fermentation liquor, the pseudomonas fluorescens fermentation liquor or the bacillus amyloliquefaciens fermentation liquor has the functions of promoting soil to form a granular structure and enhancing soil permeability, has the functions of obviously improving the drought resistance and stress resistance of plants and remarkable growth promoting effect, has good prevention and treatment effects on wheat take-all and pepper bacterial wilt and has good prevention and treatment effects on rhizoctonia cerealis, rhizoctonia solani, pythium ultimum, sclerotium solani, and peanut southern blight.
[ detailed description ] embodiments
The invention will be better understood from the following examples.
Example 1: preparation of the biological seaweed fertilizer
The implementation steps of this example are as follows:
A. slant culture of Vibrio LX6-2
Preparing a slant culture medium: preparing a pH7.0 aqueous solution containing 5g/l of sodium alginate, 3g/l of peptone, 3g/l of sodium chloride, 0.1g/l of magnesium sulfate, 2g/l of dipotassium hydrogen phosphate and 20g/l of agar powder, and then sterilizing at the temperature of 121 ℃ for 30min to obtain the slant culture medium;
inoculating a vibrio LX6-2 strain glycerol tube strain on an inclined plane of a test tube inclined plane solid culture medium, and then carrying out inclined plane culture for 2d in a constant temperature incubator at the temperature of 28 ℃ to obtain an inclined plane culture;
B. seed culture
Preparing a seed culture medium: preparing a pH7.0 aqueous solution containing 5g/l of sodium alginate, 3g/l of peptone, 3g/l of sodium chloride, 0.1g/l of magnesium sulfate and 2g/l of dipotassium hydrogen phosphate, and then sterilizing at the temperature of 121 ℃ for 30min to obtain the seed culture medium;
taking a loop of the slant culture obtained in the step A by using an inoculating loop, inoculating the slant culture into the seed culture medium, and then culturing for 1d in a constant temperature shaking table at the temperature of 29 ℃ and the rotating speed of 180rpm to obtain a seed culture;
C. preparation of seaweed digestive juice
Uniformly mixing the crushed dry sargassum raw material with a sodium carbonate aqueous solution with the concentration of 0.0034g/ml according to the mass ratio of the seaweed to the weak base of 6:1, and then digesting for 1.8 hours at the temperature of 58 ℃ under the stirring condition to obtain a seaweed digestive juice;
D. fermentation of biological seaweed liquid
In a fermentation tank filled with the seaweed digestive juice, adding the culture medium components of peptone, sodium chloride, magnesium sulfate and dipotassium hydrogen phosphate into the seaweed digestive juice obtained in the step C according to the ratio of 0.1g/l peptone, 3g/l sodium chloride, 6g/l magnesium sulfate and 0.1g/l dipotassium hydrogen phosphate; then the
And D, inoculating the seed culture obtained in the step B into a fermentation tank according to the volume of 2.2 percent of the seaweed digestive juice, stirring for 24 hours at the constant temperature of 29 ℃, the ventilation quantity of 1:1.0 and the pressure of 0.04MPa and the rotating speed of 100rpm, and completely fermenting the seaweed digestive juice to obtain the biological seaweed fertilizer.
Example 2: preparation of the biological seaweed fertilizer
The implementation steps of this example are as follows:
A. slant culture of Vibrio LX6-2
Preparing a slant culture medium: preparing a pH7.0 aqueous solution containing 5g/l of sodium alginate, 3g/l of peptone, 3g/l of sodium chloride, 0.1g/l of magnesium sulfate, 2g/l of dipotassium hydrogen phosphate and 20g/l of agar powder, and then sterilizing at the temperature of 121 ℃ for 30min to obtain the slant culture medium;
inoculating a vibrio LX6-2 strain glycerol tube strain on an inclined plane of a test tube inclined plane solid culture medium, and then carrying out inclined plane culture in a constant temperature incubator at the temperature of 29 ℃ for 3d to obtain an inclined plane culture;
B. seed culture
Preparing a seed culture medium: preparing a pH7.0 aqueous solution containing 5g/l of sodium alginate, 3g/l of peptone, 3g/l of sodium chloride, 0.1g/l of magnesium sulfate and 2g/l of dipotassium hydrogen phosphate, and then sterilizing at the temperature of 121 ℃ for 30min to obtain the seed culture medium;
taking a loop of the slant culture obtained in the step A by using an inoculating loop, inoculating the slant culture into the seed culture medium, and then culturing for 2d in a constant temperature shaking table at the temperature of 28 ℃ and the rotating speed of 180rpm to obtain a seed culture;
C. preparation of seaweed digestive juice
Uniformly mixing the crushed dry sargassum raw material with a sodium bicarbonate aqueous solution with the concentration of 0.002g/ml according to the mass ratio of the seaweed to the weak base of 7:1, and then digesting for 2.0 hours at the temperature of 52 ℃ under the condition of stirring to obtain a seaweed digestive juice;
D. fermentation of biological seaweed liquid
In a fermentation tank filled with the seaweed digestive juice, adding peptone, sodium chloride, magnesium sulfate and dipotassium hydrogen phosphate medium components into the seaweed digestive juice obtained in the step C according to the ratio of 2g/l peptone, 0.1g/l sodium chloride, 4g/l magnesium sulfate and 5g/l dipotassium hydrogen phosphate; then the
And D, inoculating the seed culture obtained in the step B into a fermentation tank according to the volume of 1.5 percent of the seaweed digestive juice, stirring for 24 hours at the constant temperature of 28 ℃, the ventilation quantity of 1:1.1, the pressure of 0.05MPa and the rotation speed of 200rpm, and completely fermenting the seaweed digestive juice to obtain the biological seaweed fertilizer.
Example 3: preparation of the biological seaweed fertilizer
The implementation steps of this example are as follows:
A. slant culture of Vibrio LX6-2
Preparing a slant culture medium: preparing a pH7.0 aqueous solution containing 5g/l of sodium alginate, 3g/l of peptone, 3g/l of sodium chloride, 0.1g/l of magnesium sulfate, 2g/l of dipotassium hydrogen phosphate and 20g/l of agar powder, and then sterilizing at the temperature of 121 ℃ for 30min to obtain the slant culture medium;
inoculating a vibrio LX6-2 strain glycerol tube strain on an inclined plane of a test tube inclined plane solid culture medium, and then carrying out inclined plane culture for 2d in a constant temperature incubator at the temperature of 30 ℃ to obtain an inclined plane culture;
B. seed culture
Preparing a seed culture medium: preparing a pH7.0 aqueous solution containing 5g/l of sodium alginate, 3g/l of peptone, 3g/l of sodium chloride, 0.1g/l of magnesium sulfate and 2g/l of dipotassium hydrogen phosphate, and then sterilizing at the temperature of 121 ℃ for 30min to obtain the seed culture medium;
b, taking a loop of the slant culture obtained in the step A by using an inoculating loop, inoculating the slant culture into the seed culture medium, and then culturing for 1d in a constant temperature shaking table at the temperature of 30 ℃ and the rotating speed of 180rpm to obtain a seed culture;
C. preparation of seaweed digestive juice
Uniformly mixing the crushed dried kelp raw material with a potassium bicarbonate aqueous solution with the concentration of 0.001g/ml according to the mass ratio of the seaweed to the weak base of 5:1, and then digesting for 1.0h at the temperature of 50 ℃ under the stirring condition to obtain a seaweed digestive juice;
D. fermentation of biological seaweed liquid
Adding peptone, sodium chloride, magnesium sulfate and dipotassium hydrogen phosphate medium components into the seaweed digestive liquid obtained in the step C according to the ratio of 10g/l peptone, 7g/l sodium chloride, 0.1g/l magnesium sulfate and 10g/l dipotassium hydrogen phosphate in a fermentation tank filled with the seaweed digestive liquid; then the
And D, inoculating the seed culture obtained in the step B into a fermentation tank according to the volume of 1.8 percent of the seaweed digestive juice, stirring for 24 hours at the constant temperature of 30 ℃, the ventilation volume of 1:1.2, the pressure of 0.06MPa and the rotation speed of 140rpm, and completely fermenting the seaweed digestive juice to obtain the biological seaweed fertilizer.
Example 4: preparation of the biological seaweed fertilizer
The implementation steps of this example are as follows:
A. slant culture of Vibrio LX6-2
Preparing a slant culture medium: preparing a pH7.0 aqueous solution containing 5g/l of sodium alginate, 3g/l of peptone, 3g/l of sodium chloride, 0.1g/l of magnesium sulfate, 2g/l of dipotassium hydrogen phosphate and 20g/l of agar powder, and then sterilizing at the temperature of 121 ℃ for 30min to obtain the slant culture medium;
inoculating a vibrio LX6-2 strain glycerol tube strain on an inclined plane of a test tube inclined plane solid culture medium, and then carrying out inclined plane culture in a constant temperature incubator at the temperature of 29 ℃ for 3d to obtain an inclined plane culture;
B. seed culture
Preparing a seed culture medium: preparing a pH7.0 aqueous solution containing 5g/l of sodium alginate, 3g/l of peptone, 3g/l of sodium chloride, 0.1g/l of magnesium sulfate and 2g/l of dipotassium hydrogen phosphate, and then sterilizing at the temperature of 121 ℃ for 30min to obtain the seed culture medium;
taking a loop of the slant culture obtained in the step A by using an inoculating loop, inoculating the slant culture into the seed culture medium, and then culturing for 2d in a constant temperature shaking table at the temperature of 29 ℃ and the rotating speed of 180rpm to obtain a seed culture;
C. preparation of seaweed digestive juice
Uniformly mixing the crushed dry paulownia raw material and 0.004g/ml potassium carbonate aqueous solution according to the mass ratio of the seaweed to the weak base of 8:1, and then digesting for 1.2 hours at the temperature of 60 ℃ under the condition of stirring to obtain a seaweed digestive juice;
D. fermentation of biological seaweed liquid
Adding peptone, sodium chloride, magnesium sulfate and dipotassium hydrogen phosphate medium components into the seaweed digestive liquid obtained in the step C according to the ratio of 8g/l peptone, 10g/l sodium chloride, 10g/l magnesium sulfate and 5g/l dipotassium hydrogen phosphate in a fermentation tank filled with the seaweed digestive liquid; then the
And D, inoculating the seed culture obtained in the step B into a fermentation tank according to the volume of 2.5 percent of the seaweed digestive juice, stirring for 24 hours at the constant temperature of 29 ℃, the ventilation quantity of 1:1.1, the pressure of 0.05MPa and the rotation speed of 160rpm, and completely fermenting the seaweed digestive juice to obtain the biological seaweed fertilizer.
The gibberellin contents of the bio-algal fertilizers prepared in examples 1-4 and the commercially available bio-enzymatic treatment liquid algal fertilizer products from a company and the commercially available chemical production liquid algal fertilizer control products were measured by high performance liquid chromatography, and the results are shown in Table 1.
Table 1: analysis result of gibberellin content in seaweed fertilizer produced by different methods
Gibberellins (ppm)
Seaweed liquid fertilizer product produced by biological enzyme method of certain manufacturer 2284.535
Seaweed liquid fertilizer product produced by chemical method of certain manufacturer 2564.006
Seaweed liquid fertilizer product prepared in example 1 5503.142
Seaweed liquid fertilizer product prepared in example 2 5211.688
Seaweed liquid fertilizer product prepared in example 3 5475.269
Seaweed liquid fertilizer product prepared in example 4 5383.867
The results shown in Table 1 show that compared with conventional chemical treatment process and other biological process, the gibberellin content of the product of the present invention is increased by 1-2 times, so that the active matter in seaweed may be protected to the maximum extent, and the growth of plant and crop yield may be raised effectively.
Test example 1: application test of composite biological seaweed fertilizer containing bacillus subtilis
The composite biological seaweed fertilizer contains 50 percent of the biological seaweed fertilizer and 50 percent of bacillus subtilis CGMCC No.2108 fermentation liquor by volume.
The bacillus subtilis CGMCC No.2108 is a strain for highly producing the macromolecular polyglutamic acid, and the macromolecular polyglutamic acid has obvious effect on improving the soil aggregate structure.
The biological seaweed fertilizer prepared in example 1 was used in this experimental example;
the bacillus subtilis CGMCC No.2108 fermentation broth used in the experimental example is prepared by adopting a fermentation method described in CN101109010B owned by the advanced biological agriculture GmbH company and the invention name of 'a strain for producing gamma-polyglutamic acid and a culture method thereof';
the extraction and purification method is described in CN201510127399.4, entitled Bacillus licheniformis and its application in producing gamma-polyglutamic acid, and the polyglutamic acid is extracted from the fermentation liquor and purified.
I. Effect of polyglutamic acid on soil aggregate structure
The soil sample is air-dried, large pieces of soil are broken off by hands when the soil sample is wet, plant root systems and small stones are removed, and the soil sample with the weight of 700g is accurately weighed.
3 treatments are set, and the mass concentrations of the macromolecular polyglutamic acid are respectively 0% (ck), 0.3% and 0.6%. The weighed air-dried soil sample and the macromolecular polyglutamic acid are uniformly mixed and put into a pot, 150ml of water is poured, the operation is repeated for 2 times, the granular structure of the soil after 10 months of treatment is detected according to the standard method of a Shavenov classification method (wet sieve method), and the detection results are listed in table 2.
Table 2: improvement effect of pure polyglutamic acid on soil aggregate structure
Figure BDA0001243880800000161
As can be seen from the results shown in Table 2, pure polyglutamic acid has a significant effect on promoting the structure of soil aggregates.
II. Action of composite biological seaweed fertilizer on soil aggregate structure
A soil sample to be tested is taken, and one 1000g soil sample is accurately weighed. The composite biological seaweed fertilizer obtained by mixing 50 percent of the biological seaweed fertilizer and 50 percent of bacillus subtilis CGMCC No.2108 fermentation liquor by volume contains 16g/l of polyglutamic acid. The compound biological seaweed fertilizer is diluted by 100 times by water, the diluted compound biological seaweed fertilizer is poured into a soil sample according to the proportion of 300ml each time, the compound biological seaweed fertilizer is continuously poured for 5 times in a half month, the soil aggregate structure after 10 months of treatment is detected according to the standard method of a Schvedorf classification method (wet sieve method) by taking clear water as a control, and the detection result is listed in a table 3.
Table 3: improvement effect of composite biological seaweed fertilizer on soil aggregate structure
Name (R) >5mm(％) 2～5mm(％) 1～2mm(％) 0.5～1mm(％) 0.25～0.5mm(％) Collection (%)
ck 1.25 5.998 11.164 16.768 20.818 55.998
Composite biological seaweed fertilizer 2.426 7.944 11.954 17.744 19.798 59.866
As can be seen from the results shown in Table 2, the composite biological seaweed fertilizer of the present invention has a significant improvement effect on the granular structure of soil.
Test example 2: wheat take-all prevention and control test of composite biological seaweed fertilizer containing pseudomonas fluorescens
The composite biological seaweed fertilizer contains 80 percent of the biological seaweed fertilizer and 20 percent of pseudomonas fluorescens CGMCC No.8820 fermentation liquor by volume, and the pseudomonas fluorescens CGMCC No.8820 is 7.2 hundred million/ml when the composite biological seaweed fertilizer is detected by a flat viable count method well known by technical personnel in the technical field.
Wherein, the biological seaweed fertilizer used in the experimental example is the biological seaweed fertilizer prepared in example 2;
the fermentation broth of pseudomonas fluorescens CGMCC No.8820 used in the experimental example is prepared by adopting the fermentation method described in CN 201410386364 of advanced biological agriculture GmbH and the invention name of 'one strain of ultraviolet induced pseudomonas fluorescens and application thereof'.
Test site: in the areas pacified by Qinhuang island, the attack of take-all disease is serious.
Test method, the test set up three treatments:
CK: base fertilizer
Treatment 1: base fertilizer and biological seaweed fertilizer
And (3) treatment 2: base fertilizer and composite biological seaweed fertilizer containing pseudomonas fluorescens
Wherein: the base fertilizer has N amount of 23kg and P amount per mu2O5Amount of 15kg, K2The amount of O was 23 kg.
Fertilizing for 2 times from the emergence stage of the wheat to the tillering stage, wherein the dosage is 20L/mu each time, irrigating, investigating at the heading stage of the wheat, and observing the growth condition and the morbidity condition of the take-all disease of the wheat during the period.
The results of the investigation are shown in Table 4.
Table 4: wheat growth situation survey with different treatments
Figure BDA0001243880800000181
The incidence of take-all disease of 200 wheat plants was investigated, and the average control effect was calculated according to the following formula.
Control effect (%) - (control disease index-treatment group disease index)/control disease index X100%
The results of the investigation are as follows: the control treated wheat take-all disease index is 97%, the treatment 1 is irrigated by the biological seaweed fertilizer, the wheat take-all disease index is 47%, only the biological seaweed fertilizer is used for promoting growth, the disease resistance of plants is improved, and the disease prevention effect reaches 51.5%; treatment 2 the disease index of the wheat take-all disease using the composite biological seaweed fertilizer is 10 percent, and the disease prevention effect reaches 89.7 percent.
The investigation results show that the compound biological seaweed fertilizer containing pseudomonas fluorescens has obvious promotion effect on plant growth, also has good prevention and control effect, improves the fertilizer application efficiency and reduces the fertilizer cost.
Test example 3: application test of pakchoi containing bacillus licheniformis composite biological seaweed fertilizer
The composite biological seaweed fertilizer contains 70 percent of the biological seaweed fertilizer and 30 percent of bacillus licheniformis CGMCC No.8821 fermentation liquor by volume, and the bacillus licheniformis is 8.5 hundred million/ml when the composite biological seaweed fertilizer is detected by adopting a flat viable count method well known by technical personnel in the technical field;
wherein, the biological seaweed fertilizer used in the experimental example is the biological fermentation liquid prepared in example 3;
the compound biological seaweed fertilizer used in the experimental example is a fermentation broth prepared by adopting a fermentation culture method described in CN201510127399.4, the name of the invention, Bacillus licheniformis and the application thereof in the production of gamma-polyglutamic acid, which is a member of the advanced biological agriculture Limited company;
the test is carried out in a foam box, and the influence of the fertilizer on the drought resistance of the pakchoi is verified through water control.
The test method comprises the following steps:
the test set up four treatments, 2 in parallel per group;
treatment 1: water is not controlled (water is irrigated by clear water, normal water supply is carried out, and the water holding capacity is 65% -75%)
And (3) treatment 2: controlling water (irrigating with clear water, moderate drought, 40% -50% water holding capacity)
And (3) treatment: controlling water (irrigating the biological seaweed fertilizer of the invention, moderate drought, 40-50% of water holding capacity)
And (4) treatment: controlling water (irrigating the composite biological seaweed fertilizer of the invention, moderate drought, 40-50% of water holding capacity)
Each foam box is filled with 9kg of soil; designing water control amount to be moderate drought (40-50% of the maximum water holding capacity of the soil), controlling water after first fertilization in a seedling stage, measuring the soil water content by a digital moisture meter every night at 17: 00-18: 00 after natural drought reaches a set soil water content standard range, replenishing water and controlling water, and fertilizing 2 times (20L/mu dosage) in the whole growth period for 35 days.
The test results are shown in Table 5.
Table 5: the growth of each treated wheat was investigated after the end of the growing period:
process 1 Treatment 2 Treatment 3 Treatment 4
Height cm of plant 27.78 18.56 29.16 30.8
Fresh weight g/plant 16.95 5.36 20.96 28.17
The test results show that the drought resistance and stress resistance of the plant can be obviously improved by adding the composite biological seaweed fertilizer containing the bacillus licheniformis fermentation liquor, and the plant can still keep good growth tendency under the drought and water-deficient conditions.
Test example 4: plate bacteriostasis test of composite biological seaweed fertilizer containing bacillus amyloliquefaciens
The composite biological seaweed fertilizer contains 60 percent of the biological seaweed fertilizer and 40 percent of bacillus amyloliquefaciens CGMCC No.8821 fermentation liquor by volume, and the effective viable count of the bacillus amyloliquefaciens is 8.3 hundred million/ml by adopting a flat viable count method well known by technical personnel in the technical field for detection;
wherein, the compound biological seaweed fertilizer used in the experimental example is the fermentation liquor prepared in example 4;
the bacillus amyloliquefaciens fermentation liquid used in the test example is prepared by adopting a preparation method described in CN 201510894025, the name of the invention, Bacillus amyloliquefaciens LX-J1 and application thereof, of the advanced biological agriculture GmbH.
The plate bacteriostasis test is as follows:
preparing a PDA solid culture medium: boiling peeled potato in water at a ratio of 200g/L for 30min, cooling, filtering with 2 layers of gauze, adding sucrose and agar into the filtrate according to a ratio of 20g/L sucrose and 20g/L agar, and sterilizing to obtain the PDA solid culture medium.
Inoculating sclerotium graminearum, rhizoctonia solani, pythium ultimum, sclerotium rolfsii and potato black hemorrhoid bacteria to the prepared PDA solid culture medium respectively, and culturing at 25 deg.C for 4-5 days until pathogenic fungi overgrow the whole culture dish.
And (3) determination of antagonistic effect:
firstly, taking pathogenic bacteria cake with the diameter of about 6mm from a culture dish by using a puncher, placing the pathogenic bacteria cake in the center of a PDA flat plate, punching holes at the positions of about 150mm on the left side and the right side of the PDA flat plate respectively, and adding 20 mu l of the composite biological seaweed fertilizer containing the bacillus amyloliquefaciens; meanwhile, the compound biological seaweed fertilizer is not added as a reference, the culture is carried out for 4-5 days at the temperature of 25 ℃, the bacteriostatic diameter of the compound biological seaweed fertilizer to each pathogenic bacterium is recorded when the reference grows over the whole culture dish, the test result is shown in the table 6, and the corresponding bacteriostatic rate is calculated according to the following formula:
the colony growth inhibition rate (control colony net growth diameter-treated colony net growth diameter)/control colony net growth diameter × 100%
Table 6: the composite biological seaweed fertilizer has the antibacterial effect
Pathogenic bacteria Grain silk core Rhizoctonia solani Pythium ultimum Southern blight of peanut Black nevus disease of potato
Rate of inhibition of bacteria 87.6％ 69.8％ 88.7％ 76.4％ 72.9％
The results in table 6 clearly show that the bacillus amyloliquefaciens LX-J1 compounded with the biological seaweed fertilizer of the invention has strong inhibition effect on rhizoctonia cerealis, rhizoctonia solani, pythium ultimum, peanut southern blight and potato black hemorrhoid, that is, the compounded functional seaweed fertilizer has the basic effect of the biological seaweed fertilizer and can effectively prevent and treat plant diseases.

Claims (5)

1. Vibrio (Vibrio sp.) LX6-2 has been deposited at the general microbiological culture Collection center of the institute of microbiology, China institute of microbiology, national institute of sciences, No. 3, West Lu 1, Beijing, the sunward, 3.23.2015, and has a collection number of CGMCC No. 10659.
2. The use of Vibrio maritima LX6-2 according to claim 1 in the preparation of biological seaweed fertilizer.
3. Use according to claim 2, characterized in that the biological seaweed fertilizer is prepared by the following steps:
A. slant culture of Vibrio LX6-2
Preparing a slant culture medium: preparing a pH7.0 aqueous solution containing 5g/L sodium alginate, 3g/L peptone, 3g/L sodium chloride, 0.1g/L magnesium sulfate, 2g/L dipotassium hydrogen phosphate and 20g/L agar powder, and then sterilizing at the temperature of 121 ℃ for 30min to obtain the slant culture medium;
inoculating a vibrio LX6-2 strain glycerol tube strain on an inclined plane of an inclined plane culture medium, and then carrying out inclined plane culture for 2-3 d in a constant temperature incubator at the temperature of 28-30 ℃ to obtain an inclined plane culture;
B. seed culture
Preparing a seed culture medium: preparing a pH7.0 aqueous solution containing 5g/L sodium alginate, 3g/L peptone, 3g/L sodium chloride, 0.1g/L magnesium sulfate and 2g/L dipotassium hydrogen phosphate, and then sterilizing at the temperature of 121 ℃ for 30min to obtain the seed culture medium;
b, taking a loop of the slant culture obtained in the step A by using an inoculating loop, inoculating the slant culture into the seed culture medium, and then culturing for 1-2 d in a constant temperature shaking table at the temperature of 28-30 ℃ and the rotating speed of 180rpm to obtain a seed culture;
C. preparation of seaweed digestive juice
Uniformly mixing a crushed and dried seaweed raw material with a weak alkali aqueous solution, and then digesting for 1-2 hours at the temperature of 50-60 ℃ under the condition of stirring to obtain a seaweed digestive liquid;
D. fermentation of biological seaweed liquid
Adding peptone, sodium chloride, magnesium sulfate and dipotassium hydrogen phosphate medium components into the seaweed digestive liquid obtained in the step C according to the ratio of 0.1-10 g/L peptone, 0.1-10 g/L sodium chloride, 0.1-10 g/L magnesium sulfate and 0.1-10 g/L dipotassium hydrogen phosphate in a fermentation tank filled with the seaweed digestive liquid;
and B, inoculating the seed culture obtained in the step B into a fermentation tank according to the volume of 1.5-2.5% of the seaweed digestive juice, stirring for 24 hours at a constant temperature under the conditions of the temperature of 28-30 ℃, the ventilation volume of 1: 1.0-1.2, the pressure of 0.04-0.06 MPa and the rotating speed of 100-200 rpm, and completely fermenting the seaweed digestive juice to obtain the biological seaweed fertilizer.
4. The use of claim 2, wherein the biological seaweed fertilizer further comprises a fermentation broth selected from the group consisting of a fermentation broth of macromolecular polyglutamic acid, a fermentation broth of small molecular polyglutamic acid, a fermentation broth of Pseudomonas fluorescens, and a fermentation broth of Bacillus amyloliquefaciens, the macromolecular polyglutamic acid fermentation broth is obtained by fermenting and culturing bacillus subtilis with the preservation number of CGMCC NO.2108, the micromolecular polyglutamic acid fermentation broth is obtained by fermenting and culturing Bacillus licheniformis LW with the preservation number of CGMCC NO.8821, the pseudomonas fluorescens fermentation liquid is obtained by fermenting and culturing the pseudomonas fluorescens F1 with the preservation number of CGMCCNo.8820 in an ultraviolet mutation type, the bacillus amyloliquefaciens fermentation liquid is obtained by fermenting and culturing bacillus amyloliquefaciens LX-J1 with the preservation number of CGMCC No. 11263; in the biological seaweed fertilizer containing the fermentation liquor, the effective viable bacteria number of the bacillus subtilis, the bacillus licheniformis LW, the ultraviolet mutagenesis fluorescent pseudomonas F1 and the bacillus amyloliquefaciens LX-J1 is more than 5 hundred million/ml respectively.
5. The use of claim 4, wherein the contents of the macromolecular polyglutamic acid fermentation broth, the micromolecular polyglutamic acid fermentation broth, the pseudomonas fluorescens fermentation broth and the bacillus amyloliquefaciens fermentation broth in the biological seaweed fertilizer containing the fermentation broth are respectively 10-50% of the total volume of the biological seaweed fertilizer.
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