CN102234619A - 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, the invention still further relates to the preparation method and the application of this bacterial strain microbial inoculum with sclerotium disease biological and ecological methods to prevent plant disease, pests, and erosion function.
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.In the sclerotium germ being played biological inhibiting multiple microorganism species, shield shell mould is to prevent and treat the microorganism that sclerotium disease has development potentiality most at present, it can be optionally parasitic the sclerotium that forms of sclerotium germ, 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 desirable biocontrol agent that sclerotium disease has development potentiality most.
The natural mode of life of solid fermentation and many microorganisms is closely similar, and low in process of production moisture content helps the acquisition and the drying of product; 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 mould solid fermentation method Study on Technology of shield shell is 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 this 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 microorganisms shield shell of preventing and treating sclerotium disease mould (
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 on 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 chocolate at last.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 big, 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, promptly 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 it is 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.
Preferably, the method for 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 set of dispense are such as following: 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 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 design sketch to inhibition of sclerotinite mycelial growth and sclerotium formation of shield shell provided by the invention.
Fig. 2 is for causing the apothecium formation situation design sketch of rotten test to the sclerotium parasitism under the mould indoor conditions of shield shell provided by the invention, row is the clear water contrast on the picture, promptly the sclerotium apothecium of handling without shield shell mould forms situation, and picture row down forms situation for the sclerotium apothecium through the mould processing of shield shell.
Fig. 3 forms apothecium design sketch for sclerotium causes in the rotten clear water contrast of testing at the field parasitism.
Fig. 4 causes in the rotten test apothecium design sketch that the sclerotium through the mould processing of shield shell forms for sclerotium at the field parasitism.
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 wind is done, and pulverizes; Inoculation sclerotinite mycelia piece was cultivated 3-4 days for 20 ℃ on the PDA flat board, treat that mycelia is covered with flat board after, 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.Place 1-2 under the room temperature after the month, isolated the mould Cm2004 bacterial strain of shield shell in the septic sclerotium from infecting.
2. the purifying of the mould Cm2004 bacterial strain of shield shell
(10 spores/ml) were cultivated 10 days down for 20 ℃ on the PDA flat board, and 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 effect to get 1ml spore diluent.
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
The bacterial classification inoculation of the mould Cm2004 bacterial strain of shield shell on the PDA of pH5.5 flat board, was cultivated 15 days down for 20 ℃, the activation conidium of shield shell trichoderma strain Cm2004;
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, in the sterilization 60 minutes down of 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 jar 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
The bacterial classification inoculation of the mould Cm2004 bacterial strain of shield shell on the PDA of pH6.0 flat board, was cultivated 17 days down for 18 ℃, the activation conidium of shield shell trichoderma strain Cm2004;
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, in the sterilization 60 minutes down of 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 jar 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
The bacterial classification inoculation of the mould Cm2004 bacterial strain of shield shell on the PDA of pH6.5 flat board, was cultivated 14 days down for 23 ℃, the activation conidium of shield shell trichoderma strain Cm2004;
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, in the sterilization 60 minutes down of 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 jar 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 effect of shield shell to inhibition of sclerotinite mycelial growth and sclerotium formation:
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 that observed and recorded sclerotinite mycelial growth, sclerotium form quantity 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 soil pouring and spray method with 10ml spore suspension (10 respectively
6Individual spore/ml) imposes on soil surface (contrast is handled with clear water), and waters with the 100ml sterilized water, checks in the soil sclerotium situation of rotting after 30 days, writes down the sclerotium number (b) that can find in the soil; Tell half sclerotium separation and Culture afterwards, the sclerotium number (c) that forms mycelia can be sprouted in record; Half stays in soil, observes apothecium and forms 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 handled 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 half sclerotium separation and Culture afterwards, the sclerotium number (C) that forms mycelia can be sprouted in record, and half stays in soil, observes apothecium and form 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, sclerotium forms quantity and reduces over halfly, 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 10 apotheciums at most, apothecium quantity in the clear water contrast is 46.3/square metre, and the apothecium of field after the soil spray method is handled is 12.3/square metre, and apothecium forms quantity and reduced 73.4%.
The above only is preferred embodiment of the present invention, and is in order to restriction the present invention, within the spirit and principles in the present invention not all, any modification of being done, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. biocontrol microorganisms shield shell mould (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 the described deposit number of claim 1.
3. the preparation method of the described shield shell of 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, dark condition was cultivated 20～25 days down, was put in the air-dry culture material in shady and cool place, obtained the mould Cm2004 microbial inoculum of described shield shell.
4. method according to claim 3 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: spore suspension is 1:8～1:10 with the volume mass ratio of cavings substratum when inoculating in the described C step.
7. method according to claim 3 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 7～14%, 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.
8. method according to claim 7 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.
9. according to claim 7 or 8 described methods, it is characterized in that: each component of described fermented nutritive liquid and set of dispense are such as following: 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.
10. the application of the described shield shell of claim 1 mould (Coniothyrium minitans) Cm2004 bacterial strain in preparation control sclerotinia rot of colza microbial preparation.
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| CN104744160A (en) * | 2015-03-12 | 2015-07-01 | 四川省农业科学院植物保护研究所 | Biocompound fertilizer for preventing and controlling sclerotinia and application method thereof |
| CN104759445A (en) * | 2015-03-12 | 2015-07-08 | 四川省农业科学院植物保护研究所 | Harmless treatment method of fungus residue in coniothyrium minitans fermentation technology |
| CN116064241A (en) * | 2022-11-03 | 2023-05-05 | 云南省林业和草原科学院 | Shell mould YAFEF037 strain and separation method and application thereof |
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| 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 |
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| 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 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104744160A (en) * | 2015-03-12 | 2015-07-01 | 四川省农业科学院植物保护研究所 | Biocompound fertilizer for preventing and controlling sclerotinia and application method thereof |
| CN104759445A (en) * | 2015-03-12 | 2015-07-08 | 四川省农业科学院植物保护研究所 | Harmless treatment method of fungus residue in coniothyrium minitans fermentation technology |
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| CN116064241A (en) * | 2022-11-03 | 2023-05-05 | 云南省林业和草原科学院 | Shell mould YAFEF037 strain and separation method and application thereof |
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