CN108641966B - A kind of microorganism Biological agents - Google Patents

A kind of microorganism Biological agents Download PDF

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Publication number
CN108641966B
CN108641966B CN201810470343.2A CN201810470343A CN108641966B CN 108641966 B CN108641966 B CN 108641966B CN 201810470343 A CN201810470343 A CN 201810470343A CN 108641966 B CN108641966 B CN 108641966B
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bacillus amyloliquefaciens
biological agents
liquid
rhodopseudomonas palustris
long shoot
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CN108641966A (en
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刘文波
秦春秀
缪卫国
靳鹏飞
林春花
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Hainan University
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Hainan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/10—Animals; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Abstract

The present invention provides a kind of microorganism Biological agents, the microorganism Biological agents are liquid, its effective component is made of long shoot trichoderma bacterium and its fermentating metabolism product, bacillus amyloliquefaciens and its fermentating metabolism product, Rhodopseudomonas palustris and its fermentating metabolism product, small radix bupleuri alkali and chitosan, and the concentration of small radix bupleuri alkali is 0.1-0.8wt% in the microorganism Biological agents, the concentration of chitosan be 0.5-1wt%, long shoot trichoderma bacterium, bacillus amyloliquefaciens and Rhodopseudomonas palustris total viable count >=5 × 107cfu/ml.Effective component of the present invention belongs to the active constituent of different role mechanism, more preferable to tea zonate spot preventive effect under synergistic effect, and each component belongs to active constituent green, environmental-friendly, nonhazardous, having drug effect and the dual biological regulation function of fertilizer efficiency concurrently.

Description

A kind of microorganism Biological agents
Technical field
The invention belongs to microbial control technical fields, and in particular to a kind of microorganism Biological agents.
Background technique
It is extremely sick that tea zonate spot is also known as tea top drying.It is distributed in each area Chan Cha in the whole nation.Main cause harm blade and young sprout.Blade is caught an illness Tender leaf, Cheng Ye, old leaf are shown in morbidity, and the small scab of yellow green is first born on blade tip or leaf margin, after be extended to it is round to ellipse Or the big scab of irregular shape brown, the scab on Cheng Ye and old leaf have apparent concentric wheel stripe, become greyish white among later period scab Color, the big black small grain point occurred in wheel line shape arrangement of humidity, the i.e. fructification of pathogen.From blade tip to leaf margin when tender leaf is caught an illness Gradual change dark brown, scab is irregular, shrivelled shape, the scattered coal dirt shape dot in scab front, and without wheel line on scab, scab is mostly often Mutually fusion causes blade to be largely covered with brown withered spot.Tender tip tip of catching an illness first is fallen ill, and rear blackening is withered, is continued extension downwards and is drawn Cause branch withered, blade largely falls off when occurring serious or cuttage seeding is dead in flakes.
Germ is overwintering on sick leaf or the sick tip with mycelium or aceravlus, and next spring condition generates mitogenetic spore when being suitable for Son invades from tea tree tender leaf or at leaf wound, causes to fall ill through 7-14 days incobations, generate new scab, form son when humidity is big Entity releases mature understanding and rainwater splashing is borrowed to propagate, repeatedly infect again.
The disease belongs to high temperature and humidity type disease, and 25-28 DEG C of temperature, relative humidity 85% one 87% is conducive to morbidity.Summer, autumn two Ji Fasheng weight.The tea place susceptible disease adopted, more than mechanical picking, trimming, summer cuttage seeding and Pests of Tea-Plants is smoothed out with the fingers in production.Tea place draining It is bad, transplant overstocked cuttage nursery severe disease.Disease-resistant sex differernce is obvious between kind.Phoenix narcissus, Hunan wave be green, big-leaf species in yunnan Susceptible disease.
Currently used control method is that after tea picking or early stage sprays 50% benomyl wettable powder 1500 in time Times liquid or more than 50% mould clever (ten thousand mould spirit 2#) 1000 times of liquid of wettable powder, 25% carbendazol wettable powder 500 times of liquid, 80% 1500 times of liquid of difoltan wettable powder, 75% Bravo wettable powder, 600 times of liquid, 36% thiophanate-methyl suspending agent 700 Times liquid was prevented and treated 1 time every 7-14 days, continuous prevention and treatment 2-3 times.It is anti-that the chronic administration of chemical pesticide brings pesticide residue, pathogen The advantages of pharmacological property enhances and the serious problems such as environmental pollution, prevents and treats crop disease using antagonistic microbe and its active material is gradually Show, the research of green biological prevention and control agent has become hot spot.
Summary of the invention
It is an object of the invention to overcome deficiency in the prior art, a kind of microorganism Biological agents are provided, to tea wheel Pinta bacterium has good antagonism, environmental-friendly, nonhazardous.
The first aspect of the invention is to provide a kind of microorganism Biological agents, and the microorganism Biological agents are liquid Body, effective component is by long shoot trichoderma bacterium and its fermentating metabolism product, bacillus amyloliquefaciens and its fermentating metabolism product, marsh Red pseudomonas and its fermentating metabolism product, small radix bupleuri alkali and chitosan composition, and it is small in the microorganism Biological agents The concentration of radix bupleuri alkali is 0.1-0.8wt%, and the concentration of chitosan is 0.5-1wt%, long shoot trichoderma bacterium, bacillus amyloliquefaciens and Total viable count >=5 × 10 of Rhodopseudomonas palustris7cfu/ml。
Preferably, the concentration of small radix bupleuri alkali is 0.3-0.6wt%, the concentration of chitosan in the microorganism Biological agents For 0.6-0.8wt%.
Preferably, long shoot trichoderma bacterium, the red false unit cell of bacillus amyloliquefaciens and marsh in the microorganism Biological agents The viable count ratio of bacterium is (1-10) ︰ (0.1-10) ︰ (0.1-10).
It is further preferred that long shoot trichoderma bacterium, bacillus amyloliquefaciens and marsh are red in the microorganism Biological agents The viable count ratio of pseudomonad is (3-8) ︰ (3-8) ︰ (3-8).
The second aspect of the invention is to provide microorganism Biological agents described in a kind of first aspect of the present invention Preparation method, comprising the following steps: long shoot trichoderma bacterium, bacillus amyloliquefaciens and Rhodopseudomonas palustris are inoculated into training respectively Liquid seed culture and liquid fermentation and culture are carried out in nutrient solution, obtain long shoot trichoderma fermented liquid, bacillus amyloliquefaciens fermentation Liquid and Rhodopseudomonas palustris fermentation liquid；Then long shoot trichoderma fermented liquid, bacillus amyloliquefaciens fermentation liquid and marsh is red Pseudomonad fermentation liquid be uniformly mixed with small radix bupleuri alkali, chitosan to get.
Wherein, about the culture medium of seed culture, the present invention is not particularly limited herein, for long shoot trichoderma bacterium, as long as It is the culture medium that seed culture can be carried out to long shoot trichoderma bacterium, for bacillus amyloliquefaciens, as long as can be to solution starch Bacillus carries out the culture medium of seed culture, for Rhodopseudomonas palustris, as long as can be to Rhodopseudomonas palustris The culture medium of seed culture is carried out, those skilled in the art can reasonably select according to strain property.
Wherein, about the culture medium of fermented and cultured, the present invention is not particularly limited herein, for long shoot trichoderma bacterium, as long as It is the culture medium that fermented and cultured can be carried out to long shoot trichoderma bacterium, for bacillus amyloliquefaciens, as long as can be to solution starch Bacillus carries out the culture medium of fermented and cultured, for Rhodopseudomonas palustris, as long as can be to Rhodopseudomonas palustris The culture medium of fermented and cultured is carried out, those skilled in the art can reasonably select according to strain property.
The third aspect of the invention is to provide microorganism Biological agents described in first aspect of the present invention and is preventing and treating Application in tea zonate spot.
Beneficial effects of the present invention:
The present invention provides a kind of microorganism Biological agents, effective component is produced by long shoot trichoderma bacterium and its fermentating metabolism Object, bacillus amyloliquefaciens and its fermentating metabolism product, Rhodopseudomonas palustris and its fermentating metabolism product, small radix bupleuri alkali and shell Glycan composition, belongs to the active constituent of different role mechanism, more preferable to tea zonate spot preventive effect under synergistic effect, and each component belongs to In active constituent green, environmental-friendly, nonhazardous, having drug effect and the dual biological regulation function of fertilizer efficiency concurrently.
Specific embodiment
In order to keep the present invention easier to understand, the present invention will be further explained by examples below.It should be understood that These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.
Embodiment 1
Long shoot trichoderma bacterium, bacillus amyloliquefaciens and Rhodopseudomonas palustris are inoculated into respectively in culture solution and carry out liquid It is red false single to obtain long shoot trichoderma fermented liquid, bacillus amyloliquefaciens fermentation liquid and marsh for seed culture and liquid fermentation and culture Born of the same parents' fermented liquid；Then by long shoot trichoderma fermented liquid, bacillus amyloliquefaciens fermentation liquid and Rhodopseudomonas palustris fermentation liquid It is mixed to get mixed fermentation liquid in proportion, then weighs small radix bupleuri alkali, chitosan in right amount and is added in mixed fermentation liquid, mixing is equal Even, so that it is 0.3wt% that small radix bupleuri alkali, which is concentration, in total liquid, the concentration of chitosan is 0.8wt%, the work of long shoot trichoderma bacterium Bacterium number is 3 × 107Cfu/ml, bacillus amyloliquefaciens viable count be 8 × 107Cfu/ml, the viable count of Rhodopseudomonas palustris It is 3 × 107cfu/ml。
Embodiment 2
Long shoot trichoderma bacterium, bacillus amyloliquefaciens and Rhodopseudomonas palustris are inoculated into respectively in culture solution and carry out liquid It is red false single to obtain long shoot trichoderma fermented liquid, bacillus amyloliquefaciens fermentation liquid and marsh for seed culture and liquid fermentation and culture Born of the same parents' fermented liquid；Then by long shoot trichoderma fermented liquid, bacillus amyloliquefaciens fermentation liquid and Rhodopseudomonas palustris fermentation liquid It is mixed to get mixed fermentation liquid in proportion, then weighs small radix bupleuri alkali, chitosan in right amount and is added in mixed fermentation liquid, so that total liquid It is 0.6wt% that the content of small radix bupleuri alkali, which is concentration, in body, and the concentration of chitosan is 0.6wt%, and the viable count of long shoot trichoderma bacterium is 8 ×107Cfu/ml, bacillus amyloliquefaciens viable count be 3 × 107Cfu/ml, the viable count of Rhodopseudomonas palustris is 8 × 107cfu/ml。
Embodiment 3
Long shoot trichoderma bacterium, bacillus amyloliquefaciens and Rhodopseudomonas palustris are inoculated into respectively in culture solution and carry out liquid It is red false single to obtain long shoot trichoderma fermented liquid, bacillus amyloliquefaciens fermentation liquid and marsh for seed culture and liquid fermentation and culture Born of the same parents' fermented liquid；Then by long shoot trichoderma fermented liquid, bacillus amyloliquefaciens fermentation liquid and Rhodopseudomonas palustris fermentation liquid It is mixed to get mixed fermentation liquid in proportion, then weighs small radix bupleuri alkali, chitosan in right amount and is added in mixed fermentation liquid, so that total liquid It is 0.1wt% that the content of small radix bupleuri alkali, which is concentration, in body, and the concentration of chitosan is 1wt%, the viable count of long shoot trichoderma bacterium is 1 × 107Cfu/ml, bacillus amyloliquefaciens viable count be 1 × 107Cfu/ml, the viable count of Rhodopseudomonas palustris is 10 × 107cfu/ml。
Embodiment 4
Long shoot trichoderma bacterium, bacillus amyloliquefaciens and Rhodopseudomonas palustris are inoculated into respectively in culture solution and carry out liquid It is red false single to obtain long shoot trichoderma fermented liquid, bacillus amyloliquefaciens fermentation liquid and marsh for seed culture and liquid fermentation and culture Born of the same parents' fermented liquid；Then by long shoot trichoderma fermented liquid, bacillus amyloliquefaciens fermentation liquid and Rhodopseudomonas palustris fermentation liquid It is mixed to get mixed fermentation liquid in proportion, then weighs small radix bupleuri alkali, chitosan in right amount and is added in mixed fermentation liquid, so that total liquid It is 0.8wt% that the content of small radix bupleuri alkali, which is concentration, in body, and the concentration of chitosan is 0.5wt%, and the viable count of long shoot trichoderma bacterium is 10×107Cfu/ml, bacillus amyloliquefaciens viable count be 10 × 107Cfu/ml, the viable count of Rhodopseudomonas palustris are 1 ×107cfu/ml。
Embodiment 5
Long shoot trichoderma bacterium, bacillus amyloliquefaciens and Rhodopseudomonas palustris are inoculated into respectively in culture solution and carry out liquid It is red false single to obtain long shoot trichoderma fermented liquid, bacillus amyloliquefaciens fermentation liquid and marsh for seed culture and liquid fermentation and culture Born of the same parents' fermented liquid；Then by long shoot trichoderma fermented liquid, bacillus amyloliquefaciens fermentation liquid and Rhodopseudomonas palustris fermentation liquid It is mixed to get mixed fermentation liquid in proportion, then weighs small radix bupleuri alkali, chitosan in right amount and is added in mixed fermentation liquid, so that total liquid It is 0.8wt% that the content of small radix bupleuri alkali, which is concentration, in body, and the concentration of chitosan is 1wt%, the viable count of long shoot trichoderma bacterium is 3 × 107Cfu/ml, bacillus amyloliquefaciens viable count be 10 × 107Cfu/ml, the viable count of Rhodopseudomonas palustris is 8 × 107cfu/ml。
The suppression experimental study of 6 Pestallozzia theae of embodiment
The preparation of PDA culture medium: weighing peeled potatoes 200g, and stripping and slicing boils 30min, then takes juice with filtered through gauze, Again plus 20.0g glucose and 20.0g agar, water is supplied after dissolving to 1000mL, is dispensed into the triangular flask of 10 150mL, 121 DEG C Sterilize 30min.
Aseptically, it is cooled to 45 DEG C or so of PDA culture medium after taking 20mL sterilized, culture dish is added, be made PDA plate.After placing for 24 hours, pathogen Pestallozzia theae mycelia, 25 DEG C of culture 4d are inoculated in plate center.In the wheel grown In pinta bacterium plate, bacterium colony round block is cut in colony edge with diameter 1cm punch, it is spare.
The Biological agents of Example 1-5 respectively, diluting fermentation liquid to cell concentration with sterile water is 5 × 105cfu/ The Biological agents dilution of 100 μ L is added into PDA plate, is smoothened with spreader for the dilution of ml, 4 weights of each processing It is multiple.The zonate spot bacterium fungus block cut is seeded in the plate center coated, there is a face contact plate for mycelia.25 DEG C of culture 4d Afterwards, zonate spot bacterium colony diameter (unit cm) is measured with ruler " crossing method ", as a result takes its average value.Separately take corresponding reality The mixed fermentation liquid of example is applied, 4000r/min is centrifuged 30min, and supernatant filters by 0.22 μ L film, filters out thallus, obtain sterile hair Ferment supernatant, as control.Bacteriostasis rate is calculated as follows: bacteriostasis rate=(DControl-DProcessing)/DControl× 100%.It the results are shown in Table 1。
Inhibitory effect of 1 present invention of table to zonate spot bacterium
Bacteriostasis rate
Embodiment 1 97.5%
Embodiment 2 96.8%
Embodiment 3 93.7%
Embodiment 4 95.1%
Embodiment 5 92.7%
As seen from the above table, the present invention has good inhibiting effect for zonate spot bacterium.
Specific embodiments of the present invention are described in detail above, but it is merely an example, the present invention is simultaneously unlimited It is formed on particular embodiments described above.To those skilled in the art, any couple of present invention carries out equivalent modifications and Substitution is also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by equal transformation and Modification, all should be contained within the scope of the invention.

Claims (5)

1. a kind of microorganism Biological agents, which is characterized in that the microorganism Biological agents be liquid, effective component by Long shoot trichoderma bacterium and its fermentating metabolism product, bacillus amyloliquefaciens and its fermentating metabolism product, Rhodopseudomonas palustris and its Fermentating metabolism product, small radix bupleuri alkali and chitosan composition, and the concentration of small radix bupleuri alkali is in the microorganism Biological agents 0.1-0.8wt%, the concentration of chitosan are 0.5-1wt%, long shoot trichoderma bacterium, bacillus amyloliquefaciens and Rhodopseudomonas palustris Total viable count >=5 × 107cfu/ml；The long shoot trichoderma bacterium, bacillus amyloliquefaciens and Rhodopseudomonas palustris viable bacteria Number ratio is (1-10) ︰ (0.1-10) ︰ (0.1-10).
2. microorganism Biological agents according to claim 1, which is characterized in that small in the microorganism Biological agents The concentration of radix bupleuri alkali is 0.3-0.6wt%, and the concentration of chitosan is 0.6-0.8wt%.
3. microorganism Biological agents according to claim 2, which is characterized in that long in the microorganism Biological agents The viable count ratio of branch Trichoderma, bacillus amyloliquefaciens and Rhodopseudomonas palustris is (3-8) ︰ (3-8) ︰ (3-8).
4. a kind of preparation method of microorganism Biological agents of any of claims 1 or 2, which is characterized in that including following step It is rapid: long shoot trichoderma bacterium, bacillus amyloliquefaciens and Rhodopseudomonas palustris are inoculated into progress liquid seed in culture solution respectively Culture and liquid fermentation and culture, obtain long shoot trichoderma fermented liquid, bacillus amyloliquefaciens fermentation liquid and Rhodopseudomonas palustris Fermentation liquid；Then by long shoot trichoderma fermented liquid, bacillus amyloliquefaciens fermentation liquid and Rhodopseudomonas palustris fermentation liquid with it is small Radix bupleuri alkali, chitosan be uniformly mixed to get；Wherein the concentration of the small radix bupleuri alkali is 0.1-0.8wt%, the concentration of chitosan For 0.5-1wt%, long shoot trichoderma bacterium, bacillus amyloliquefaciens and Rhodopseudomonas palustris total viable count >=5 × 107cfu/ ml；The viable count ratio of the long shoot trichoderma bacterium, bacillus amyloliquefaciens and Rhodopseudomonas palustris is (1-10) ︰ (0.1- 10) ︰ (0.1-10).
5. application of the microorganism Biological agents of any of claims 1 or 2 in prevention and treatment tea zonate spot.
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CN110564646B (en) * 2019-09-20 2020-05-05 湖北省生物农药工程研究中心 Bacillus belgii CY30 and application thereof in prevention and treatment of tea round spot
CN113308382A (en) * 2021-05-14 2021-08-27 海南大学 Biocontrol bacterium preparation for enhancing stress resistance of crops
CN114606136A (en) * 2022-02-25 2022-06-10 赤天化科技集团有限公司 Compound microbial agent for preventing and treating fungal diseases of fruits and vegetables as well as preparation method and application thereof

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CN107129358A (en) * 2017-05-11 2017-09-05 北京中农富源生物工程技术有限公司 A kind of complex microorganism preparations that crop root part can be promoted to develop
CN107549212A (en) * 2017-11-01 2018-01-09 海南大学 A kind of botanical fungicide and preparation method thereof
CN108409485A (en) * 2018-04-03 2018-08-17 山东沃地丰生物肥料有限公司 A kind of preparation method of composite microbe fertilizer and the composite microbe fertilizer of preparation

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