CN105441485A - Method for simultaneously preparing leaf application and root application agricultural biocontrol agents and application of leaf application and root application agricultural biocontrol agents - Google Patents
Method for simultaneously preparing leaf application and root application agricultural biocontrol agents and application of leaf application and root application agricultural biocontrol agents Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
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- 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
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- 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
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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
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/79—Paecilomyces
Abstract
The invention discloses a method for simultaneously preparing leaf application and root application agricultural biocontrol agents and an application of the leaf application and root application agricultural biocontrol agents. A product is acquired by liquid fermenting paecilomyces lilacinus YT08 with a preservation number of CGMCC No. 10026, after the fermentation, the fermenting liquid is subjected to the solid-liquid separation, a solid part containing thallus is directly used as the root application agricultural biocontrol agent, and the liquid part is used as the leaf application agricultural biocontrol agent. The preparation method is simple, and the production process is easy to control and suitable for the mass production of factories; and moreover, the leaf application and the root application agricultural biocontrol agents have a good inhibition effect respectively on phytopathogen and root-knot nematode.
Description
Technical field
The present invention relates to a kind of leaf with and root execute the method that agriculture biological prevention and control agent prepared simultaneously, and its application in pathogen of crop and Biological Control of Root-Knot Nematodes, belong to biological technical field.
Background technology
Nemas is one of the four large pathogenic organisms of farm crop, vegetables, fruit tree, forest, flowers and medicinal material plantation, and its harm is only second to fungi, exceedes bacterium and virus.The loss about 1,000 hundred million dollars that plant nematode causes world agriculture to produce every year, on Global Agriculture, most important harm nematode is root knot nematode and Cyst nematode, the comparatively serious nematode of China has root knot nematode, soybean cyst nematode Heterodera glycines, wheat anguina, sweet potato stem nematode, aphelenchoides besseyi, chestnut nematode, pine wood nematode etc., and only the loss of China's Agricultural planting oxyuriasis just reaches 60,000,000,000 yuan.The control of plant nematode is usually by chemical nematicides, cultivation technique, cultivation resistant variety and biological pesticide, but at present still without the chemical pesticide of special efficacy, and due to potential environmental problem, relating to human and animal's health, the use of nematocides is very restricted.
Paecilomyces lilacinus extensively can parasitize line eggs, and insecticidal effect is efficient, is considered to the root knot nematode biocontrol fungi most with application prospect, and this bacterium has the features such as effect is high, parasitic wide, easy cultivation, and particularly in control plant pathogeny nematode, effect is remarkable.As a kind of biotechnological formulation, Paecilomyces lilacinus has the features such as safety, efficient, the lasting period is long in nematicide process, and its nematicide activity is poor unlike chemical nematicides, and has the effect of Promoting plant growth.Paecilomyces lilacinus is except having nematicide effect, and all right parasitic Hemiptera, Homoptera, Isoptera and the insect such as Coleoptera and lepidopteran, have antagonistic action to phytopathogen in addition.The secondary metabolites of Paecilomyces lilacinus also has certain physiological potency, as produced similar indolylacetic acid product, promote root system of plant and plant strain growth, to Seed Germination and growth, there is promoter action, several functions enzyme can also be produced and there is certain chemical pesticide degradation effect.
Oligochitosan is marine organisms source pesticide of new generation, has toxicological harmless, free from environmental pollution, the feature that has drug effect and the dual biological regulation function of fertilizer efficiency concurrently, can induced activation plant immune system, improves plant virus resistance ability.Oligochitosan take chitosan as raw material, through the series product with certain polymerization degree and structure that enzymic degradation becomes.Oligochitosan can be used as plant-growth regulator, can the immune system response of stimulating plant, strengthens plant to the defence capability of disease and pest.Oligochitosan starts from the sixties in 20th century as the fundamental research of plant immunization incitant, reported that oligochitosan had good preventive and therapeutic effect to phytopathies such as tobacco mosaic virus disease, rice blast and banded sclerotial blight, nuclear disk mould pathogenic bacterias afterwards successively, research simultaneously finds that oligochitosan has good induced activation effect to several functions enzyme in plant materials, and plant protecting chemical can be induced to synthesize, and chitosan all has certain insecticidal activity to the insect such as lepidopteran and Homoptera in addition.The prevention effect of biocontrol effect in the farm crop such as tobacco, paddy rice, tomato, cotton, wheat, strawberry, rape, cucumber, soybean, capsicum, pawpaw of oligochitosan all has good preventive effect.Therefore to have broad-spectrum antibacterial active and nontoxic to people and animals for oligochitosan, and exploitation oligochitosan biogenic pesticide is the effective way reducing chemical pesticide amount of application.
Summary of the invention
The present invention is directed to the deficiency of above-mentioned technical background and existence thereof, provide a kind of leaf with and root execute the method that agriculture biological prevention and control agent realizes preparing simultaneously, and its application in pathogen of crop and Biological Control of Root-Knot Nematodes.With Paecilomyces lilacinus YT08 for starting strain, through the method for liquid fermenting while, manufacture leaf executes agriculture biological prevention and control agent with root, its middle period has high inhibition effect with agriculture biological prevention and control agent to plant pathogenic fungi, and root is executed agriculture biological prevention and control agent and had stronger killing effect to root knot nematode.
Object of the present invention can be reached by following measure:
1. a strain has the Paecilomyces lilacinus of control tomato and Root of European Grape Root-knot evil, this bacterial strain has high yield chitosan enzyme viability, its preservation name is called: YT08, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date: on November 26th, 2014, preserving number is CGMCCNo.10026, Classification And Nomenclature: Paecilomyces lilacinus (Paecilomyceslilacinus).
2. actication of culture
Activation medium (g/L): peptone 3 ~ 5, yeast powder 1 ~ 3, glucose 10 ~ 20, powdered chitosan 2 ~ 5, potassium primary phosphate 1 ~ 3, magnesium sulfate 1 ~ 2, Sodium phosphate dibasic 1 ~ 3, pH7.0.Packing 100ml activation medium in 500ml Erlenmeyer flask, 121 DEG C of sterilizing 20min.Preserving picking 1 ring bacterial classification from test tube slant is inoculated in activation medium, 30 ~ 32 DEG C, to cultivate 30 ~ 36h under 180rpm muddy to thalline, obtained activated seed liquid.
3. liquid fermenting
(1) shake-flask culture
Adopt preferred substratum (g/L): glycerine 18 ~ 30, soy peptone 25 ~ 40, MnSO
41 ~ 3, powdered chitosan (deacetylation of more than 90%) 20 ~ 30, crab shell powder 10 ~ 15, NaCl2 ~ 5, CaCl
21 ~ 3, MgSO
41 ~ 3, FeSO
40.5 ~ 1.5, pH5 ~ 6.5, packing 200ml fermention medium in every 500ml Erlenmeyer flask, 121 DEG C of sterilizing 20min.According to 0.8 ~ 1.5%(v/v) inoculum size access activation seed culture fluid, after enter in the rotary shaker of 180rpm to cultivate, control temperature is 30 ~ 34 DEG C, fermentation 5-8d.
(2) fermentor cultivation
Adopt preferred substratum (g/L): glycerine 18 ~ 30, soy peptone 25 ~ 40, MnSO
41 ~ 3, powdered chitosan (deacetylation of more than 90%) 20 ~ 30, crab shell powder 10 ~ 15, NaCl2 ~ 5, CaCl
21 ~ 3, MgSO
41 ~ 3, FeSO
40.5 ~ 1.5.Fermention medium is loaded with 70% ratio; after 118 DEG C of steam sterilizing 20min; according to 0.8 ~ 1.5%(v/v under flame ring protective condition) ratio access activation bacterial classification; all the time pH5 ~ 6.5 are maintained by the pH regulating soda acid peristaltic pump to control in fermenting process; the heating unit of fermentor tank is utilized to regulate culture temperature at 30 ~ 34 DEG C; dissolved oxygen parameter makes it maintain more than 10% all the time by regulating into the sterile air amount of tank and the size of mixing speed, fermentation 4-7d.
4. product preparation: the centrifugal 5min of fermentation liquor 10000r/min of Paecilomyces lilacinus YT08, collect bacterial sediment thing and fermented supernatant fluid respectively, wherein fermented supernatant fluid collect filling after can directly use as the agriculture biological prevention and control agent of leaf, namely the bacterial sediment thing simultaneously comprising part non-degrade chitosan etc. executes the use of agriculture biological prevention and control agent as root.The root of preparation executes agro-ecology preparation is determined in unit fermentating liquid volume thalline content through weighing, and leaf measures the content of chitoanase vigor and oligochitosan respectively through DNS method with agriculture biological prevention and control agent simultaneously.
Chitoanase measuring method used: the agriculture biological prevention and control agent of leaf is diluted to suitable gradient, get 0.2mL and add water to 1ml, add 1mL colloid chitosan substrate solution (1% massfraction) again, insulation reaction 20min in 40 DEG C of water-baths, add DNS reagent 3mL, boiling water bath 5min, gets 4ml and reacts sample liquid centrifugal 10min under 5000rpm after being settled to 25ml.Measure OD value using control group as blank at 520nm place, according to DNS typical curve, calculate its enzyme activity.Blank group: 0.2mL is diluted to the agriculture biological prevention and control agent of leaf of identical gradient, adds water to 1ml, boiling water bath 5min deactivation, then adds chitosan solution and the DNS reagent of same volume, the same sample sets for the treatment of process.Enzyme work is calculated: the umol number of the glucosamine produced with unit volume (mL) unit time (min) hydrolysis is the definition unit of enzyme activity.
Standard curve making used: accurately take the glucosamine hydrochloride 0.13g that 100 DEG C are dried to constant weight, adding distil water dissolves and is settled to 100ml, is made into the solution of .6umol/mL concentration.In each 25mL colorimetric cylinder, add 1mL standard DAS, the ultrapure water of 1mL and the DNS nitrite ion of 3mL respectively by different concns gradient, boiling water bath reaction 5min, surveys OD value after being settled to 25ml under 520mn, and drawing standard curve.
Oligochitosan content assaying method used: in the accurate detectability of spectrophotometry, the agriculture biological prevention and control agent of leaf is diluted to suitable gradient, the dilution ultrapure water of posterior lobe with agriculture biological prevention and control agent 1mL, 1mL and the DNS nitrite ion of 3mL is added respectively in each 25mL colorimetric cylinder, boiling water bath reaction 5min, under 520mn, survey OD value after being settled to 25ml, according to the amount of typical curve determination oligochitosan and the Units of Account volume middle period by the oligochitosan total amount of agriculture biological prevention and control agent.
The beneficial effect of content of the present invention is:
1. Paecilomyces lilacinus YT08CGMCCNo.10026 is that a strain has the filamentous fungus of strong prevention effect to farm crop root knot nematode, surely can grow at plant root after being manured into soil, the protolysate enzymes such as a large amount of chitoanases, chitinase are produced by self-characteristic, root knot nematode inside is entered as infecting the factor, play the effect killing root knot nematode, the agriculture biological prevention and control agent that the Paecilomyces lilacinus YT08 thalline therefore after fermentation can be executed as root.
2. in fermention medium, add excessive powdered chitosan and crab shell powder, except a part is converted into oligochitosan, also have the undegradable chitosan of part, chitin as the cofactor of Biological Control of Root-Knot Nematodes, it can also suppress various plants pathogenic bacteria in addition, use is collected together with thalline, form the synergistic effect of biological control, as the agriculture biological prevention and control agent that root is executed together with Paecilomyces lilacinus YT08.
3. utilize Paecilomyces lilacinus YT08 high yield chitosan enzyme viability, in thalline fermenting process, add powdered chitosan induction produce chitoanase, degradation of chitosan in substratum is converted into again the lower molecular weight oligochitosan of high physiologically active by the chitoanase simultaneously generated, oligochitosan is present in fermented liquid in the mode of dissolving, and it is inhibited to various plants pathogenic bacteria, therefore oligochitosan can as biological pesticide, and the pathogenic bacteria for preventing and treating plant leaf surface after foliage spray endangers.
4. Paecilomyces lilacinus YT08 also can produce numerous secondary metabolites in fermentation culture process, it is dissolved in water more, crop growth can be promoted, and to the suppression of phytopathogen, there is certain synergism, therefore the oligochitosan of these components and generation forms synergistic effect, for preventing and treating the pathogenic bacteria harm of plant leaf surface after foliage spray.
Outstanding advantage of the present invention is:
1. utilize Paecilomyces lilacinus YT08CGMCCNo.10026 high yield chitoanase and part chitin enzyme viability, powdered chitosan and crab shell powder is added in fermentation production process, realize in a kind of zymotechnique, prepare root to execute and use agriculture biological prevention and control agent with leaf simultaneously, save facility investment, reduce production site, reduce production cost.
2. cultivate easily, working method is simply efficient, and makes not easily microbiological contamination in its culturing process to most pathogenic bacteria is inhibited due to Paecilomyces lilacinus YT08.
3. the root prepared in the present invention is executed and is used agriculture biological prevention and control agent with leaf, has high inhibition effect respectively, wide market to root knot nematode and phytopathogen.
Embodiment
Be described in further detail the present invention below in conjunction with specific embodiment, these embodiments are used for understanding instead of limiting the scope of the invention.
Embodiment 1 shake-flask culture is prepared leaf simultaneously and is executed agriculture biological prevention and control agent with root
Preserving picking 1 ring bacterial classification from test tube slant is inoculated in activation medium, 30 DEG C, to cultivate 32h under 180rpm muddy to thalline, obtained activated seed liquid.Activation medium (g/L): peptone 3, yeast powder 1, glucose 10, powdered chitosan 2, potassium primary phosphate 1, magnesium sulfate 1, Sodium phosphate dibasic 1, pH7.0.Packing 100ml activation medium in 500ml Erlenmeyer flask, 121 DEG C of sterilizing 20min.
Adopt preferred substratum (g/L): glycerine 18, soy peptone 25, MnSO
41, powdered chitosan (deacetylation of more than 90%) 20, crab shell powder 10, NaCl2, CaCl
21, MgSO
41, FeSO
40.5, pH5, packing 200ml fermention medium in every 500ml Erlenmeyer flask, 121 DEG C of sterilizing 20min.According to 0.8%(v/v) inoculum size access activation seed culture fluid, after enter in the rotary shaker of 180rpm to cultivate, control temperature is 30 DEG C, fermentation 6d.
The centrifugal 5min of fermentation liquor 10000r/min of Paecilomyces lilacinus YT08, collect bacterial sediment thing and fermented supernatant fluid respectively, wherein fermented supernatant fluid collect filling after can directly use as the agriculture biological prevention and control agent of leaf, comprise simultaneously part non-degrade chitosan bacterial sediment thing namely as root execute agriculture biological prevention and control agent use.In often liter of fermented liquid after fermentation stops, the root of preparation is executed agriculture biological prevention and control agent and is determined that through weighing weight in wet base is 142g, and leaf is 75.1U/mL with chitoanase vitality test in agriculture biological prevention and control agent, and measuring its oligochitosan content through DNS method is 7.2g.
Embodiment 2 fermentor tank (10L) cultivation is prepared leaf simultaneously and is executed agriculture biological prevention and control agent with root
Activation medium (g/L): peptone 5, yeast powder 3, glucose 20, powdered chitosan 5, potassium primary phosphate 3, magnesium sulfate 2, Sodium phosphate dibasic 3, pH7.0.Packing 100ml activation medium in 500ml Erlenmeyer flask, 121 DEG C of sterilizing 20min.Preserving picking 1 ring bacterial classification from test tube slant is inoculated in activation medium, 32 DEG C, to cultivate 36h under 180rpm muddy to thalline, obtained activated seed liquid.
Adopt preferred substratum (g/L): glycerine 30, soy peptone 40, MnSO
43, powdered chitosan (deacetylation of more than 90%) 30, crab shell powder 15, NaCl5, CaCl
23, MgSO
43, FeSO
41.5.Fermention medium is loaded with 70% ratio; liquid amount is 7L; after 118 DEG C of steam sterilizing 20min; according to 1.5%(v/v under flame ring protective condition) ratio access activation bacterial classification; all the time pH6.5 is maintained by the pH regulating soda acid peristaltic pump to control in fermenting process; utilize the heating unit of fermentor tank to regulate culture temperature at 34 DEG C, dissolved oxygen parameter makes it maintain more than 10% all the time by regulating into the sterile air amount of tank and the size of mixing speed, fermentation 7d.
The centrifugal 5min of fermentation liquor 10000r/min of Paecilomyces lilacinus YT08, collect bacterial sediment thing and fermented supernatant fluid respectively, wherein fermented supernatant fluid collect filling after can directly use as the agriculture biological prevention and control agent of leaf, comprise simultaneously part non-degrade chitosan bacterial sediment thing namely as root execute agriculture biological prevention and control agent use.In often liter of fermented liquid after fermentation stops, the root of preparation is executed agriculture biological prevention and control agent and is determined weight in wet base 203g through weighing, and leaf is 93.5U/mL with chitoanase vitality test in agriculture biological prevention and control agent, and measuring its oligochitosan content through DNS method is 8.4g.
Embodiment 3 fermentor tank (50L) cultivation is prepared leaf simultaneously and is executed agriculture biological prevention and control agent with root
Activation medium (g/L): peptone 4, yeast powder 2, glucose 15, powdered chitosan 3, potassium primary phosphate 2, magnesium sulfate 2, Sodium phosphate dibasic 2, pH7.0.Packing 100ml activation medium in 500ml Erlenmeyer flask, 121 DEG C of sterilizing 20min.Preserving picking 1 ring bacterial classification from test tube slant is inoculated in activation medium, 30 DEG C, to cultivate 34h under 180rpm muddy to thalline, obtained activated seed liquid.
Adopt preferred substratum (g/L): glycerine 20, soy peptone 30, MnSO
42, powdered chitosan (deacetylation of more than 90%) 25, crab shell powder 12, NaCl4, CaCl
22, MgSO
42, FeSO
41.Fermention medium is loaded with 70% ratio; liquid amount is 35L; after 118 DEG C of steam sterilizing 20min; according to 1%(v/v under flame ring protective condition) ratio access activation bacterial classification; all the time pH6 is maintained by the pH regulating soda acid peristaltic pump to control in fermenting process; utilize the heating unit of fermentor tank to regulate culture temperature at 32 DEG C, dissolved oxygen parameter makes it maintain more than 10% all the time by regulating into the sterile air amount of tank and the size of mixing speed, fermentation 5d.
The centrifugal 5min of fermentation liquor 10000r/min of Paecilomyces lilacinus YT08, collect bacterial sediment thing and fermented supernatant fluid respectively, wherein fermented supernatant fluid collect filling after can directly use as the agriculture biological prevention and control agent of leaf, comprise simultaneously part non-degrade chitosan bacterial sediment thing namely as root execute agriculture biological prevention and control agent use.In often liter of fermented liquid after fermentation stops, the root of preparation is executed agriculture biological prevention and control agent and is determined weight in wet base 185g through weighing, and leaf is 91.8U/mL with chitoanase vitality test in agriculture biological prevention and control agent, and measuring its oligochitosan content through DNS method is 8.6g.
Embodiment 4 executes the result of use evaluation of agriculture biological prevention and control agent
In embodiment 1, the root of preparation executes agriculture biological prevention and control agent called after PL-1, and in embodiment 2, the root of preparation executes agriculture biological prevention and control agent called after PL-2, and in embodiment 3, the root of preparation executes agriculture biological prevention and control agent called after PL-3.
Cultivation matrix is that cultivation matrix used all exists without nematode after testing without sick soil, fertilizer and sand according to 9:1:3 ratio mixed preparing.The tomato seedling of picking about 12cm length is cultivated in the flowerpot of 30 × 30cm, the strain of every basin 1, totally 60 strains.All apply in the cultivation matrix of 60 above-mentioned basin tomato seedlings 200 2 age tomato root-knot eelworm, wherein 30 basins apply 3g root respectively and execute agriculture biological prevention and control agent, are labeled as PL-1, PL-2 and PL-3 root and execute agriculture biological prevention and control agent and respectively account for 10 basins; Other 30 basins apply 3g again and wet vinasse as blank.Routine Management, after 60 days, calculates prevention effect.
Tomato root-knot eelworm access approaches used is as follows: the root system gathering root knot nematode diseased plant tomato in warmhouse booth, first rinse well with water, add after being cut into segment in chlorine bleach liquor, sealing shake is after 3 minutes, first cross 200 mesh sieves and use distilled water flushing, after 500 mesh sieves, after repeatedly rinsing with distilled water, be collected in aseptic small beaker.The root-knot nematode egg collected is hatched 3 days at 26 DEG C, namely obtains 2 instar larvaes of tomato root-knot eelworm.
According to root knot raw number coincident with severity degree of condition is divided into 5 grades: without root knot is 0 grade, what root knot accounted for 1%-25% of full root system is 1 grade, what root knot accounted for 26%-50% of full root system is 2 grades, and what root knot accounted for 51%-75% of full root system is 3 grades, and what root knot accounted for 76%-100% of full root system is 4 grades.The calculation formula of disease index and prevention effect is as follows:
Results from pot experiment test is as shown in table 1, be labeled as PL-1, PL-2 and PL-3 root and execute the prevention effect of agriculture biological prevention and control agent control tomato root-knot eelworm for being respectively 85.6%, 85.9% and 86.3%, three groups of prevention effect for test preparation group are substantially identical, and the root for the treatment of group tomato heavy rate of increase, plant height rate of increase and over-ground part weight rate of increase are all significantly higher than control group.
The prevention effect of agriculture biological prevention and control agent to tomato root-knot eelworm executed by table 1 piece
The inhibition evaluation of agriculture biological prevention and control agent to phytopathogen of embodiment 5 leaf
The agriculture biological prevention and control agent called after LBC-1 of leaf of preparation in embodiment 1, the agriculture biological prevention and control agent called after LBC-2 of leaf of preparation in embodiment 2, the agriculture biological prevention and control agent called after LBC-3 of leaf of preparation in embodiment 3.
Adopt and suppress mycelial growth rate method to measure the leaf inhibition of agriculture biological prevention and control agent to phytopathogen.Added in the solid PDA medium of sterilizing thawing with agriculture biological prevention and control agent by leaf, every 100mL substratum adds 5mL, and does blank with the sterilized water of 5mL, and mixing is down flat plate.First solid PDA medium is adopted by three kind of plant pathogenic bacterias (apple anthrax-bacilus, watermelon Fusarium oxysporum and tomato gray mould bacterium) to activate respectively, to the bacterium colony forming more than diameter 1cm, then supply activated the colony edge trying agriculture pathogenic bacteria with the punch tool of diameter 0.5cm, prepare bacterium cake by concentric(al) circles and be inoculated in add the agriculture biological prevention and control agent of leaf solid PDA flat board on, 3 repetitions are established in each process, in 28 DEG C of constant temperature culture 5d, right-angled intersection method is adopted to measure colony diameter.
Bacteriostasis rate (%)=(contrast net growth-process net growth)/contrast net growth * 100%
Net growth=tri-time repeat mean value (cm)-0.5cm
Leaf with the result of use evaluation of agriculture biological prevention and control agent in table 2.As can be seen from Table 2, preparation three kinds of leaves all have stronger restraining effect with agriculture biological prevention and control agent to apple anthrax-bacilus, watermelon Fusarium oxysporum and tomato gray mould bacterium, the leaf adopting different culture condition to prepare with agriculture biological prevention and control agent to the inhibition of phytopathogen slightly difference.
The inhibition evaluation of agriculture biological prevention and control agent to phytopathogen of table 2 leaf
In sum, the invention has the advantages that the agriculture biological prevention and control agent of leaf and Gen Shi agricultural biological prevention and control agent have high inhibiting rate to phytopathogen and root knot nematode respectively, preparation method is simple and Controlling Technology condition is relatively easy, be applicable to large-scale industrialized production, its commercial crop plant pathogenic bacteria control and root knot nematode disease prevent and treat in have broad application prospects.
Claims (3)
1. a leaf with and root execute the method and application thereof that agriculture biological prevention and control agent prepared simultaneously, it is characterized in that: be that the bacterial strain being CGMCCNo.10026 by preserving number obtains after liquid fermenting, all have high inhibition effect to phytopathogen and root knot nematode respectively, its preparation method comprises the steps:
(1) actication of culture: preserve picking 1 ring bacterial classification from test tube slant and be inoculated in activation medium, 30 ~ 32 DEG C, to cultivate 30 ~ 36h under 180rpm muddy to thalline, obtained activated seed liquid; Described activation medium (g/L): peptone 3 ~ 5, yeast powder 1 ~ 3, glucose 10 ~ 20, powdered chitosan 2 ~ 5, potassium primary phosphate 1 ~ 3, magnesium sulfate 1 ~ 2, Sodium phosphate dibasic 1 ~ 3, pH7.0;
(2) shake-flask culture liquid fermentation: the preferred fermention medium of packing 200ml in every 500ml Erlenmeyer flask, 121 DEG C of sterilizing 20min, according to 0.8 ~ 1.5%(v/v) inoculum size access activation seed culture fluid, after enter in the rotary shaker of 180rpm to cultivate, control temperature is 30 ~ 34 DEG C, fermentation 5-8d;
(3) fermentor cultivation liquid fermenting: load preferred fermention medium with 70% ratio, after 118 DEG C of steam sterilizing 20min, according to 0.8 ~ 1.5%(v/v) ratio access activation bacterial classification, control fermentation pH in pH5 ~ 6.5, regulate temperature at 30 ~ 34 DEG C, dissolved oxygen maintains more than 10%, fermentation 4-7d;
(4) product preparation: the centrifugal 5min of fermentation liquor 10000r/min of Paecilomyces lilacinus YT08, collect bacterial sediment thing and fermented supernatant fluid respectively, wherein fermented supernatant fluid collect filling after can directly use as the agriculture biological prevention and control agent of leaf, the bacterial sediment thing simultaneously comprising partly non-degrade chitosan etc. is root and executes agriculture biological prevention and control agent.
2. leaf as claimed in claim 1 with and root execute the method and application thereof that agriculture biological prevention and control agent prepared simultaneously, it is characterized in that, the preferred fermention medium (g/L) of employing is: glycerine 18 ~ 30, soy peptone 25 ~ 40, MnSO
41 ~ 3, powdered chitosan 20 ~ 30, crab shell powder 10 ~ 15, NaCl2 ~ 5, CaCl
21 ~ 3, MgSO
41 ~ 3, FeSO
40.5 ~ 1.5.
3. leaf as claimed in claim 1 with and root execute the method and application thereof that agriculture biological prevention and control agent prepared simultaneously, it is characterized in that, the inducing substrate of employing is powdered chitosan (its deacetylation is more than 90%) and crab shell powder.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105671024A (en) * | 2016-04-21 | 2016-06-15 | 鲁东大学 | Method for producing chitosanase by utilizing paecilomyces lelacinus YT08 |
| CN105713886A (en) * | 2016-04-21 | 2016-06-29 | 山东省林木种质资源中心 | Preparation method and application of Paecilomyces lilacinus chitosanase |
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| WO2015069938A1 (en) * | 2013-11-06 | 2015-05-14 | The Texas A & M University System | Fungal endophytes for improved crop yields and protection from pests |
| CN104818216A (en) * | 2015-01-20 | 2015-08-05 | 鲁东大学 | Paecilomyces lilacinus for prevention and control of meloidogyne diseases of tomato and grape |
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| WO2014086753A3 (en) * | 2012-12-03 | 2014-08-14 | Bayer Cropscience Ag | Composition comprising biological control agents |
| WO2015069938A1 (en) * | 2013-11-06 | 2015-05-14 | The Texas A & M University System | Fungal endophytes for improved crop yields and protection from pests |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105671024A (en) * | 2016-04-21 | 2016-06-15 | 鲁东大学 | Method for producing chitosanase by utilizing paecilomyces lelacinus YT08 |
| CN105713886A (en) * | 2016-04-21 | 2016-06-29 | 山东省林木种质资源中心 | Preparation method and application of Paecilomyces lilacinus chitosanase |
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| Publication number | Publication date |
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| CN105441485B (en) | 2019-12-17 |
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