CN102234619B - Biocontrol bacteria coniothyrium minitans Cm2004 strain for preventing and treating sclerotiniose and preparation method and application thereof - Google Patents
Biocontrol bacteria coniothyrium minitans Cm2004 strain for preventing and treating sclerotiniose and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microbial pesticides and particularly relates to a biocontrol bacteria coniothyrium minitans Cm2004 strain for preventing and treating sclerotinia rot. The coniothyrium minitans Cm2004 strain is collected in the China General Microbiological Culture Collection Center (CGMCC) with the collection number of CGMCC No.3852. The invention further discloses a solid fermentation culturing method of a microbial agent and application of the microbial agent to the prevention and the treatment of sclerotinia rot of colza. The microbial agent has a good bio-control effect on sclerotinia rot.
Description
Technical field
The present invention relates to the microbial pesticide technical field, be specifically related to a kind of shield shell trichoderma strain with sclerotium disease biological and ecological methods to prevent plant disease, pests, and erosion function, the invention still further relates to the preparation method and application of this bacterial strain microbial inoculum.
Background technology
The sclerotium germ is a kind of global wide spectrum parasitics pathogenic fungi, adopts chemical can not reach this sick purpose of radical cure.In order to reduce pesticide residue, to avoid the germ resistance to strengthen, people place on hope the development and utilization aspect of biological and ecological methods to prevent plant disease, pests, and erosion agent more.The sclerotium germ is being play in the multiple-microorganism flora of biological inhibition effect, shield shell mould is to prevent and treat at present the microorganism that sclerotium disease has development potentiality most, it is the sclerotium of parasitic sclerotium germ formation optionally, the sclerotium and suppress its sprouting of surviving the winter that can decompose pathogenic bacteria in the soil effective, single-mindedly, and can in soil, survive for many years, this bacterium has that specificity is strong, long action time, to characteristics such as animal and plant no pathogenicities, be to prevent and treat the ideal biological control agent that sclerotium disease has development potentiality most.
The natural mode of life of solid fermentation and many microorganisms is closely similar, and low moisture content is conducive to acquisition and the drying of product in process of production; Secondly, the spore that produces by solid fermentation has better quality, than uvioresistant and resist drying, and has good vigor in the storage of preparation finished product.Therefore, the research of the mould solid fermentation method technology of shield shell is the mould large-scale production of shield shell and the requisite link of commercial applications.The mould solid fermentation level of existing shield shell only rests on the laboratory study level, and only for suitability for industrialized production provides indirect foundation, the result of study difficulty directly applies to suitability for industrialized production.
Summary of the invention
The objective of the invention is to screen a kind of shield shell trichoderma strain that sclerotium disease is had remarkable biological control effect, simultaneously, the present invention also provides the solid fermentation cultural method of this bacterial strain, and the method is simple to operate, is suitable for suitability for industrialized production; In addition, the present invention also provides the application of microbial preparation in the control sclerotinia rot of colza of this bacterium preparation.
The invention provides a kind of biocontrol fungus Coniothyrium minitans Campbell of preventing and treating sclerotium disease (
Coniothyrium minitans) the Cm2004 bacterial strain, in separation screening from the rape ground soil of Germany's generation sclerotium disease in 2003, and in introduction in 2004.This bacterial strain has been delivered China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preservation address on May 17th, 2010: the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is: CGMCC N0.3852.
Shield shell bacterium Cm2004 bacterial strain of the present invention has following biological characteristics:
1, cultivate to learn characteristic: bacterium colony is radial growth at the PDA flat board, the initial stage aerial hyphae be white in color, the bacterium colony surface is the fine hair shape subsequently, with the outward extending while of posterior border, begins to become tawny by white gradually from the bacterium colony center, is at last chocolate.The growth later stage forms a large amount of brown droplet-like pycnidiums, and how intensive pycnidium is in dull and stereotyped center amount, and diameter is large, and outwards radial the minimizing gradually diminishes;
2, morphological characteristic: the microscopically mycelia is netted, and barrier film is arranged, and forms pycnidium at mycelia proximal end place; Conidium is simple in structure, is oval or ellipse, monospore, and brown, shorter.
The present invention also provide a kind of by deposit number be the shield shell of CGMCC N0.3852 mould (
Coniothyrium minitans) microbiobacterial agent Cm2004 bacterial strain preparation, that sclerotium disease had the biological and ecological methods to prevent plant disease, pests, and erosion function.
Correspondingly, the present invention also provide this shield shell mould (
Coniothyrium minitans) preparation method of Cm2004 microbial inoculum, may further comprise the steps:
A, bacterial strain activation
The mould Cm2004 inoculation of shield shell on the PDA of pH5.5～6.5 culture medium flat plate, was cultivated 14～17 days for 18 ℃～23 ℃, get the activation conidium of the mould Cm2004 bacterial strain of shield shell;
B, spore suspension preparation
Conidium with aseptic water washing A step obtains makes spore suspension;
C, solid fermentation are cultivated
The cavings substratum is put into solid-state fermenter, sterilization, the spore suspension of B step preparation is inoculated in the solid-state fermenter, leave standstill in ventilation under 18 ℃～23 ℃, matrix relative humidity 90～95%, pH5.5～6.5 conditions and to stir 2～3 minutes every 48 hours after cultivating 72 hours, under dark condition, cultivated 20～25 days, be put in the air-dry culture material in shady and cool place, namely get the mould microbial inoculum of described shield shell.
The equipment that preparation method of the present invention uses is simple, can finish the preparation of the mould Cm2004 microbial inoculum of shield shell with solid-state fermenter; Simple to operate, only need to grasp aseptic technique and just can finish fermentation; And with low cost; Be suitable for the suitability for industrialized production of the mould Cm2004 of shield shell.
Preferably, the concentration of B step miospore suspension is 1 * 10
6～5 * 10
6Individual spore/ml.
The method of preferably, sterilizing in the C step is sterilization 60 minutes under 121 ℃, 0.15MPa.
Spore suspension is 1:8～1:10 with the volume mass ratio of cavings substratum when preferably, inoculating in the C step.
Preferably, each component of cavings substratum and weight percentages of components are as follows in the C step:
Semen Maydis powder 7～14%, straw 20～30%, cavings 5～7%, surplus are fermented nutritive liquid;
Or wheat bran 7～14%, straw 20～30%, cavings 5～7%, surplus are fermented nutritive liquid;
Or Semen Maydis powder 7～14%, rape straw 20～30%, cavings 5～7%, surplus are fermented nutritive liquid;
Or Semen Maydis powder 7～14%, corn cob 20～30%, cavings 5～7%, surplus are fermented nutritive liquid;
Or Semen Maydis powder 7～14%, wheat straw 20～30%, cavings 5～7%, surplus are fermented nutritive liquid.
Each component of cavings substratum and the most preferred weight percent of component in the C step: Semen Maydis powder 7%, straw 30%, cavings 5%, surplus are nutritive medium.
Preferably, each component of fermented nutritive liquid and component proportion are as follows: CaCl
20.03～0.05g, MgCl
24.0～5.0g, urea 3.0～3.5g, liquid microelement 8～12ml, Zulkovsky starch 30.0～40.0g, KH
2PO
40.8～1.2g replenishes distilled water to 1000ml, transfers pH to 5.5～6.5; Wherein liquid microelement consists of: ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O.
Fermentation raw material adopts agriculture common materials or the agricultural wastes such as Semen Maydis powder, straw, cavings in the inventive method, and it is very extensive to originate, cheap, and sporulation quantity reached 5.58 * 10 after fermentation was finished
9Individual/g, and collect easily, the stirring and washing culture material can separate conidium after fermentation was finished, and was suitable for suitability for industrialized production.
The present invention also provide the shield shell mould (
Coniothyrium minitans) application of microbial preparation in the control sclerotinia rot of colza of Cm2004 bacterial strain preparation.
This microbial inoculum of evidence is 55% to the sclerotinite mycelial growth inhibition rate, and the sclerotium that can suppress more than 45% forms; Indoor sclerotium parasitism causes rotten rate and reaches 97%; Field sclerotium parasitism causes rotten rate and reaches 92%.
Description of drawings
Fig. 1 is the mould inhibition figure to the inhibition of sclerotinite mycelial growth and sclerotium formation of shield shell provided by the invention.
Fig. 2 is the apothecium formational situation design sketch that under the mould indoor conditions of shield shell provided by the invention the sclerotium parasitism is caused rotten test, row is the clear water contrast on the picture, the sclerotium apothecium formational situation of namely processing without shield shell mould, row is the sclerotium apothecium formational situation through the mould processing of shield shell under the picture.
Fig. 3 is that sclerotium causes the apothecium design sketch of formation in the rotten clear water contrast of testing at the field parasitism.
To be sclerotium cause in the rotten test apothecium design sketch that the sclerotium through the mould processing of shield shell forms at the field parasitism to Fig. 4.
Embodiment
For making the purpose, technical solutions and advantages of the present invention clearer, embodiment of the present invention is described further in detail below in conjunction with accompanying drawing.
Embodiment 1
1. the separation screening of the mould Cm2004 bacterial strain of shield shell of the present invention
Gather the soil sample of German sclerotinia rot of colza spot 5～10cm soil layer, Indoor Natural is air-dry, pulverizes; At PDA flat board inoculation sclerotinite mycelia piece, cultivated 3-4 days, and after mycelia is covered with flat board, spread the air-dry soil sample 6-8 rickle that pulverizes along dull and stereotyped periphery, dull and stereotyped inner ring 4-6 rickle, every rickle 2-3mg for 20 ℃.Place 1-2 under the room temperature after the month, isolated the mould Cm2004 bacterial strain of shield shell in the rotten sclerotium from infecting.
2. the purifying of the mould Cm2004 bacterial strain of shield shell
Get 1ml spore diluent (10 spores/ml) on the PDA flat board, 20 ℃ of lower cultivations 10 days, the mycelia piece that picking list bacterium colony forms transfers to that PDA is dull and stereotyped to be cultivated 15 days, obtains the strain excellent Cm2004 that sclerotinite is had better inhibition.
Through selecting good shield shell trichoderma strain Cm2004 with testing sieves such as sclerotinite face-off cultivation, sporulation quantity mensuration.
The mould Cm2004 bacterial strain of the shield shell of purifying is delivered China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preservation address on May 17th, 2010: the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is: CGMCC N0.3852.
Embodiment 2
The preparation method of shield shell mould (Coniothyrium minitans) Cm2004 microbial inoculum is as follows:
A, bacterial strain activation
On the PDA of pH5.5 flat board, 20 ℃ of lower cultivations 15 days get the activation conidium of shield shell trichoderma strain Cm2004 with the bacterial classification inoculation of the mould Cm2004 bacterial strain of shield shell;
B, spore suspension preparation
With the conidium that aseptic water washing A step obtains, with the blood counting chamber counting, making spore concentration is 1 * 10
6The spore suspension of individual spore/ml;
C, solid fermentation are cultivated
Preparation cavings substratum: Semen Maydis powder 700g, straw 3000g, cavings 500g, fermented nutritive liquid 5800g, wherein every liter of fermented nutritive liquid is composed of the following components: CaCl
20.03g, MgCl
25.0g, urea 3.0g, liquid microelement (contains ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O) 8ml, Zulkovsky starch 30.0g, KH
2PO
41.2g, replenish distilled water to 1000ml, transfer pH to 5.5.
The cavings substratum is put into solid-state fermenter, sterilization is 60 minutes under 121 ℃, 0.15MPa, the spore suspension of B step preparation is inoculated in the solid-state fermenter, spore suspension is 1:8 with the volume mass of cavings substratum than (mL/g) during inoculation, after the inoculation, in 20 ℃, matrix relative humidity 90%, cultivate under pH5.5, the air flow 20NL/min condition, leave standstill and cultivated 72 hours, stirred 2 minutes every 48 hours afterwards, under dark condition, cultivated 21 days, finish the fermenting process of the mould microbial inoculum preparation of shield shell; Put tank and locate air-dry culture material in the cool place, obtain microbial inoculum.
Embodiment 3
The preparation method of shield shell mould (Coniothyrium minitans) Cm2004 microbial inoculum is as follows:
A, bacterial strain activation
On the PDA of pH6.0 flat board, 18 ℃ of lower cultivations 17 days get the activation conidium of shield shell trichoderma strain Cm2004 with the bacterial classification inoculation of the mould Cm2004 bacterial strain of shield shell;
B, spore suspension preparation
With the conidium that aseptic water washing A step obtains, with the blood counting chamber counting, making spore concentration is 2 * 10
6The spore suspension of individual spore/ml;
C, solid fermentation are cultivated
Preparation cavings substratum: Semen Maydis powder 1000g, straw 2500g, cavings 700g, fermented nutritive liquid 5800g, wherein every liter of fermented nutritive liquid is composed of the following components: CaCl
20.04g, MgCl
24.0g, urea 3.5g, liquid microelement (contains ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O) 12ml, Zulkovsky starch 40.0g, KH
2PO
41.2g, replenish distilled water to 1000ml, transfer pH to 6.0.
The cavings substratum is put into solid-state fermenter, sterilization is 60 minutes under 121 ℃, 0.15MPa, the spore suspension of B step preparation is inoculated in the solid-state fermenter, spore suspension is 1:9 with the volume mass of cavings substratum than (mL/g) during inoculation, after the inoculation, in 18 ℃, matrix relative humidity 92%, pH6.5, air flow are to cultivate under the 20NL/min condition, leave standstill to cultivate after 72 hours and stirred 3 minutes every 48 hours, under dark condition, cultivated 23 days, finish the fermenting process of the mould microbial inoculum preparation of shield shell; Put tank and locate air-dry culture material in the cool place, obtain microbial inoculum.
Embodiment 4
The preparation method of shield shell mould (Coniothyrium minitans) Cm2004 microbial inoculum is as follows:
A, bacterial strain activation
On the PDA of pH6.5 flat board, 23 ℃ of lower cultivations 14 days get the activation conidium of shield shell trichoderma strain Cm2004 with the bacterial classification inoculation of the mould Cm2004 bacterial strain of shield shell;
B, spore suspension preparation
With the conidium that aseptic water washing A step obtains, with the blood counting chamber counting, making spore concentration is 5 * 10
6The spore suspension of individual spore/ml;
C, solid fermentation are cultivated
Preparation cavings substratum: Semen Maydis powder 1400g, straw 2000g, cavings 600g, fermented nutritive liquid 7800g, wherein every liter of fermented nutritive liquid is composed of the following components: CaCl
20.05g, MgCl
24.5g, urea 3.3g, liquid microelement (contains ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O) 10ml, Zulkovsky starch 35.0g, KH
2PO
41.0g, replenish distilled water to 1000ml, transfer pH to 6.5.
The cavings substratum is put into solid-state fermenter, sterilization is 60 minutes under 121 ℃, 0.15MPa, the spore suspension of B step preparation is inoculated in the solid-state fermenter, spore suspension is 1:10 with the volume mass of cavings substratum than (mL/g) during inoculation, after the inoculation, in 23 ℃, matrix relative humidity 95%, pH6.0, air flow are to cultivate under the 20NL/min condition, leave standstill to cultivate after 72 hours and stirred 3 minutes every 48 hours, under dark condition, cultivated 25 days, finish the fermenting process of the mould microbial inoculum preparation of shield shell; Put tank and locate air-dry culture material in the cool place, obtain microbial inoculum.
Embodiment 5
The difference of present embodiment and embodiment 2 is: preparation cavings substratum: wheat bran 700g, straw 3000g, cavings 500g, fermented nutritive liquid 5800g, wherein every liter of fermented nutritive liquid is composed of the following components: CaCl
20.03g, MgCl
25.0g, urea 3.0g, liquid microelement (contains ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O) 8ml, Zulkovsky starch 30.0g, KH
2PO
41.2g, replenish distilled water to 1000ml, transfer pH to 5.5.
Rest part is all identical with embodiment 2.
Embodiment 6
The difference of present embodiment and embodiment 3 is: preparation cavings substratum: wheat bran 1000g, straw 2500g, cavings 700g, fermented nutritive liquid 5800g, wherein every liter of fermented nutritive liquid is composed of the following components: CaCl
20.04g, MgCl
24.0g, urea 3.5g, liquid microelement (contains ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O) 12ml, Zulkovsky starch 40.0g, KH
2PO
41.2g, replenish distilled water to 1000ml, transfer pH to 6.0.
Rest part is all identical with embodiment 3.
Embodiment 7
The difference of present embodiment and embodiment 4 is: preparation cavings substratum: wheat bran 1400g, straw 2000g, cavings 600g, fermented nutritive liquid 7800g, wherein every liter of fermented nutritive liquid is composed of the following components: CaCl
20.05g, MgCl
24.5g, urea 3.3g, liquid microelement (contains ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O) 10ml, Zulkovsky starch 35.0g, KH
2PO
41.0g, replenish distilled water to 1000ml, transfer pH to 6.5.
Rest part is all identical with embodiment 4.
Embodiment 8
The difference of present embodiment and embodiment 2 is: preparation cavings substratum: Semen Maydis powder 700g, rape straw 3000g, cavings 500g, fermented nutritive liquid 5800g, wherein every liter of fermented nutritive liquid is composed of the following components: CaCl
20.03g, MgCl
25.0g, urea 3.0g, liquid microelement (contains ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O) 8ml, Zulkovsky starch 30.0g, KH
2PO
41.2g, replenish distilled water to 1000ml, transfer pH to 5.5.
Rest part is all identical with embodiment 2.
Embodiment 9
The difference of present embodiment and embodiment 3 is: preparation cavings substratum: Semen Maydis powder 1000g, rape straw 2500g, cavings 700g, fermented nutritive liquid 5800g, wherein every liter of fermented nutritive liquid is composed of the following components: CaCl
20.04g, MgCl
24.0g, urea 3.5g, liquid microelement (contains ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O) 12ml, Zulkovsky starch 40.0g, KH
2PO
41.2g, replenish distilled water to 1000ml, transfer pH to 6.0.
Rest part is all identical with embodiment 3.
Embodiment 10
The difference of present embodiment and embodiment 4 is: preparation cavings substratum: Semen Maydis powder 1400g, rape straw 2000g, cavings 600g, fermented nutritive liquid 7800g, wherein every liter of fermented nutritive liquid is composed of the following components: CaCl
20.05g, MgCl
24.5g, urea 3.3g, liquid microelement (contains ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O) 10ml, Zulkovsky starch 35.0g, KH
2PO
41.0g, replenish distilled water to 1000ml, transfer pH to 6.5.
Rest part is all identical with embodiment 4.
Embodiment 11
The difference of present embodiment and embodiment 2 is: preparation cavings substratum: Semen Maydis powder 700g, corn cob 3000g, cavings 500g, fermented nutritive liquid 5800g, wherein every liter of fermented nutritive liquid is composed of the following components: CaCl
20.03g, MgCl
25.0g, urea 3.0g, liquid microelement (contains ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O) 8ml, Zulkovsky starch 30.0g, KH
2PO
41.2g, replenish distilled water to 1000ml, transfer pH to 5.5.
Rest part is all identical with embodiment 2.
Embodiment 12
The difference of present embodiment and embodiment 3 is: preparation cavings substratum: Semen Maydis powder 1000g, corn cob 2500g, cavings 700g, fermented nutritive liquid 5800g, wherein every liter of fermented nutritive liquid is composed of the following components: CaCl
20.04g, MgCl
24.0g, urea 3.5g, liquid microelement (contains ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O) 12ml, Zulkovsky starch 40.0g, KH
2PO
41.2g, replenish distilled water to 1000ml, transfer pH to 6.0.
Rest part is all identical with embodiment 3.
Embodiment 13
The difference of present embodiment and embodiment 4 is: preparation cavings substratum: Semen Maydis powder 1400g, corn cob 2000g, cavings 600g, fermented nutritive liquid 7800g, wherein every liter of fermented nutritive liquid is composed of the following components: CaCl
20.05g, MgCl
24.5g, urea 3.3g, liquid microelement (contains ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O) 10ml, Zulkovsky starch 35.0g, KH
2PO
41.0g, replenish distilled water to 1000ml, transfer pH to 6.5.
Rest part is all identical with embodiment 4.
Embodiment 14
The difference of present embodiment and embodiment 2 is: preparation cavings substratum: Semen Maydis powder 700g, wheat straw 3000g, cavings 500g, fermented nutritive liquid 5800g, wherein every liter of fermented nutritive liquid is composed of the following components: CaCl
20.03g, MgCl
25.0g, urea 3.0g, liquid microelement (contains ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O) 8ml, Zulkovsky starch 30.0g, KH
2PO
41.2g, replenish distilled water to 1000ml, transfer pH to 5.5.
Rest part is all identical with embodiment 2.
Embodiment 15
The difference of present embodiment and embodiment 3 is: preparation cavings substratum: Semen Maydis powder 1000g, wheat straw 2500g, cavings 700g, fermented nutritive liquid 5800g, wherein every liter of fermented nutritive liquid is composed of the following components: CaCl
20.04g, MgCl
24.0g, urea 3.5g, liquid microelement (contains ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O) 12ml, Zulkovsky starch 40.0g, KH
2PO
41.2g, replenish distilled water to 1000ml, transfer pH to 6.0.
Rest part is all identical with embodiment 3.
Embodiment 16
The difference of present embodiment and embodiment 4 is: preparation cavings substratum: Semen Maydis powder 1400g, wheat straw 2000g, cavings 600g, fermented nutritive liquid 7800g, wherein every liter of fermented nutritive liquid is composed of the following components: CaCl
20.05g, MgCl
24.5g, urea 3.3g, liquid microelement (contains ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O) 10ml, Zulkovsky starch 35.0g, KH
2PO
41.0g, replenish distilled water to 1000ml, transfer pH to 6.5.
Rest part is all identical with embodiment 4.
Embodiment 17
The mould inhibition to the inhibition of sclerotinite mycelial growth and sclerotium formation of shield shell:
A, get on the PDA flat board and to cultivate 3 days, diameter is that the sclerotinite mycelia piece of 6mm size is placed on the PDA flat board of diameter 90mm, getting Coniothyrium minitans suspension streak culture (contrast replaces spore suspension with clear water) at a distance of mycelia piece 30mm place, cultivate respectively after 3,6,9 days under stereoscopic microscope observed and recorded sclerotinite mycelial growth, sclerotium quantity of formation and by parasitic situation, as shown in Figure 1.
The shield shell is mould to cause rotten effect to the sclerotium parasitism:
A, 20 ℃ indoor, contain the 500g sterile soil with the basin alms bowl of diameter 15cm, put into 100 sclerotium (a), adopt respectively soil pouring and spray method with 10ml spore suspension (10
6Individual spore/ml) imposes on soil surface (contrast is processed with clear water), and waters with the 100ml sterilized water, checks in the soil sclerotium situation of rotting after 30 days, records the sclerotium number (b) that can find in the soil; Tell afterwards half sclerotium separation and Culture, the sclerotium number (c) that forms mycelia can be sprouted in record; Half stays in soil, observes the apothecium formational situation, and record can form apothecium sclerotium number (d), and calculating the sclerotium rotting rate by following formula is 97%.
The sclerotium rotting rate=(a-b-c-d)/a * 100%.
B, on rape ground, 100 sclerotium (A) are put into 1 square metre of scope, adopt the soil spray method with 100ml spore suspension (10
6Individual spore/ml) impose on soil surface (contrast is processed with clear water) is checked the rotten situation of sclerotium in the soil, the sclerotium number (B) that can find in the record soil after 50 days; Take out afterwards half sclerotium separation and Culture, the sclerotium number (C) that forms mycelia can be sprouted in record, and half stays in soil, observes the apothecium formational situation, and record forms apothecium sclerotium number (D), and calculating the sclerotium rotting rate by following formula is 92%.
The sclerotium rotting rate=(A-B-C-D)/A * 100%.
By above-mentioned experimental technique repeatedly revision test prove that this bacterium is 55.2% to the sclerotinite mycelial growth inhibition rate, it is over half that the sclerotium quantity of formation reduces, indoor sclerotium parasitism causes rotten rate and reaches 97%; Field sclerotium parasitism causes rotten rate and reaches 92%.Each sclerotium can be sprouted at least and form 1-2 apothecium, form at most 10 apotheciums, apothecium quantity in the clear water contrast is 46.3/square metre, and the apothecium of field after the soil spray method is processed is 12.3/square metre, and the apothecium quantity of formation has reduced 73.4%.
The above only is preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. biocontrol fungus Coniothyrium minitans Campbell (Coniothyrium minitans) Cm2004 bacterial strain of preventing and treating sclerotium disease is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and deposit number is: CGMCC N0.3852.
2. one kind is the microbiobacterial agent of shield shell mould (Coniothyrium minitans) the Cm2004 bacterial strain preparation of CGMCC N0.3852 by deposit number claimed in claim 1.
3. the preparation method of a shield shell claimed in claim 2 mould (Coniothyrium minitans) Cm2004 microbial inoculum is characterized in that: may further comprise the steps:
A, bacterial strain activation
The mould Cm2004 inoculation of shield shell on the PDA of pH5.5～6.5 culture medium flat plate, was cultivated 14～17 days for 18 ℃～23 ℃, get the activation conidium of the mould Cm2004 bacterial strain of shield shell;
B, spore suspension preparation
Conidium with aseptic water washing A step obtains makes spore suspension;
C, solid fermentation are cultivated
The cavings substratum is put into solid-state fermenter, sterilization, the spore suspension of B step preparation is inoculated in the solid-state fermenter, in under 18 ℃～23 ℃, matrix relative humidity 90～95%, pH5.5～6.5 conditions, after ventilation leaves standstill and cultivate 72 hours, stirred 2～3 minutes every 48 hours, cultivated under the dark condition 20～25 days, and be put in the air-dry culture material in shady and cool place, obtain the mould Cm2004 microbial inoculum of described shield shell;
Wherein, each component of cavings substratum and weight percentages of components are as follows in the described C step:
Semen Maydis powder 7～14%, straw 20～30%, cavings 5～7%, surplus are fermented nutritive liquid;
Or wheat bran 7～14%, straw 20～30%, cavings 5～7%, surplus are fermented nutritive liquid;
Or Semen Maydis powder 7～14%, rape straw 20～30%, cavings 5～7%, surplus are fermented nutritive liquid;
Or Semen Maydis powder 7～14%, corn cob 20～30%, cavings 5～7%, surplus are fermented nutritive liquid;
Or Semen Maydis powder 7～14%, wheat straw 20～30%, cavings 5～7%, surplus are fermented nutritive liquid;
Each component of described fermented nutritive liquid and component proportion are as follows: CaCl
20.03～0.05g, MgCl
24.0～5.0g, urea 3.0～3.5g, liquid microelement 8～12ml, Zulkovsky starch 30.0～40.0g, KH
2PO
40.8～1.2g replenishes distilled water to 1000ml, transfers pH to 5.5～6.5; Wherein liquid microelement consists of: ZnSO
47H
2O, MnCl
24H
2O, NaMo
42H
2O, CoCl
26H
2O, CuSO
46H
2O.
4. method according to claim 3, it is characterized in that: the concentration of described B step miospore suspension is 1 * 10
6～5 * 10
6Individual spore/ml.
5. method according to claim 3 is characterized in that: the method for sterilizing in the described C step is sterilization 60 minutes under 121 ℃, 0.15MPa.
6. method according to claim 3 is characterized in that: in the described C step during inoculation volume mass of spore suspension and cavings substratum than being 1:8～1:10.
7. method according to claim 3, it is characterized in that: each component of cavings substratum and weight percentages of components are as follows in the described C step: Semen Maydis powder 7%, straw 30%, cavings 5%, surplus are fermented nutritive liquid.
8. the application of shield shell claimed in claim 1 mould (Coniothyrium minitans) Cm2004 bacterial strain in preparation control sclerotinia rot of colza microbial preparation.
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| CN104744160B (en) * | 2015-03-12 | 2017-06-16 | 四川省农业科学院植物保护研究所 | A kind of biological compound fertilizer and its application process for preventing and treating sclerotiniose |
| CN104759445B (en) * | 2015-03-12 | 2016-06-29 | 四川省农业科学院植物保护研究所 | A kind of shield shell mould fermentation technique harmless treatment dreg method |
| CN116491524B (en) * | 2022-06-13 | 2024-07-05 | 四川省农业科学院植物保护研究所 | Application and method for controlling mulberry sclerotinia by using coniothyrium minitans |
| CN116064241B (en) * | 2022-11-03 | 2023-07-21 | 云南省林业和草原科学院 | Shell mould YAFEF037 strain and separation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002087344A1 (en) * | 2001-04-27 | 2002-11-07 | Uws Ventures Limited | Biological control of soil dwelling pests |
| CN101603009A (en) * | 2009-06-05 | 2009-12-16 | 华中农业大学 | One strain prevents and treats the mould ZS-1SB of biocontrol microorganisms shield shell and the preparation method and the application of sclerotium disease |
| CN101851600A (en) * | 2010-03-09 | 2010-10-06 | 成都趋元科技有限责任公司 | Method for producing coniothyrium minitans conidium |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002087344A1 (en) * | 2001-04-27 | 2002-11-07 | Uws Ventures Limited | Biological control of soil dwelling pests |
| CN101603009A (en) * | 2009-06-05 | 2009-12-16 | 华中农业大学 | One strain prevents and treats the mould ZS-1SB of biocontrol microorganisms shield shell and the preparation method and the application of sclerotium disease |
| CN101851600A (en) * | 2010-03-09 | 2010-10-06 | 成都趋元科技有限责任公司 | Method for producing coniothyrium minitans conidium |
Non-Patent Citations (1)
| Title |
|---|
| 董晓军.菌核病生防菌小盾壳霉的固体发酵及几个菌株的生物学特性研究.《四川农业大学硕士学位论文》.2008,第13页. * |
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