CN106416654A - Corn southern leaf blight field natural inducement technology and application of technology - Google Patents
Corn southern leaf blight field natural inducement technology and application of technology Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
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Abstract
The invention relates to the technical field of crop disease research, in particular to a corn southern leaf blight field natural inducement technology and application of the technology. The technology basically includes the steps that plastic film mulching is conducted; individual core seedlings which all have 3-4 leaves and 1 core are planted in two rows, wherein the line spacing is 0.4-0.5 m; in the stage when each corn plant in the field has 7-8 leaves, fresh corn southern leaf blight diseased plants artificially inoculated in a greenhouse serve as a field inducement bacterium source and horizontally placed on moist surfaces between the corn plants, and field normal irrigation is used for keeping soil moist. The infected corn southern leaf blight diseased plants which are artificially inoculated serve as the inducement bacterium source, operation is easy, and the inducement bacterium source is easy to obtain. The structure of the inducement bacterium source is similar to that of a wild bipolaris maydis population, and the pathogenic ability is reliable. Good and stable pathogen conditions can be provided. The technology is applicable to corn southern leaf blight resistance screening of corn germplasm and also applicable to screening of disease prevention insecticides and technical research.
Description
Technical field
The present invention relates to corps diseases studying technological domain, and in particular to a kind of corn southern leaf blight natural induction in fields skill
Art and the application of the technology.
Background technology
Corn southern leaf blight (Southern corn leaf blight) is a kind of important disease of global Semen Maydiss, mainly
Harm blade, also endangers sheath, bract, fruit ear and seed.Corn southern leaf blight can be all fallen ill in the whole period of duration of Semen Maydiss, to take out hero
It is disease occurrence in peak period in front and back.Sick leaf is extended by plant lower to upper part, and when falling ill serious, blade is covered with scab, and impact is planted
Strain photosynthesis, cause blade withered ahead of time and the underproduction, can typically cause the susceptible variety underproduction more than 10%, the time seriously occurs
The underproduction 20%～80%, or even ruin kind of a total crop failure.The disease have occur universal, spread expansion rate fast the features such as.In recent years, with
Development, the renewal of corn variety and the change of cropping system that fresh edible maize plants industrialization, corn southern leaf blight is in China's jade
Rice main producing region occurs increasingly seriously, seriously to constrain the safety in production of China's Maize Industry.
Southern corn leaf blight [Bipolaris maydis (Nisikado et Miyake) Shoemake] is that one kind infects
The stronger pathogenic bacteria of ability and pathogenecity, pathogenic bacteria conidium sprouts 4-8h under optimum conditions, you can produces germ tube and invades
The epidermis cell of maize leaf, can cause after being inoculated with 24h in the quick death of mesophyll cell, 3-4d and can form scab, afterwards
Scab produces conidium, propagates by rainwater air-flow, carries out superinfection to the Semen Maydiss of field growing.Corn growth middle and late stage
Field precipitation is many, humidity height, easily causes the popular of helminthosporium maydis, and excessively dense planting ground, continuous cropping field morbidity is heavier.
It is control of leaf spot of corn method the most economic using the resistance of corn variety, and the use of chemical bactericide
It is then the maximally efficient method of control of leaf spot of corn.In corn variety resistance and antibacterial medicine efficacy screening field test,
Good stable onset condition, the accuracy of ability actual response result of the test is generally required, and field initially induces bacterium source
Quantity have close relationship with disease, which is also the seriously ill main cause in continuous corn cropping field.At present in Semen Maydiss product
In kind anti-helminthosporium maydis screening test, main using field natural occurrence method and indirect labor's inocalation method (will artificial culture disease
Bacterium spore Direct spraying is inoculated on each blade of milpa, or the sick leaf collected by the previous year is crushed, in corn core
The leaf phase imposes in lobus cardiacus or spreads fertilizer over the fields underproof Semen Maydiss in the ranks).Using field natural occurrence method, success of the test property and its result
Stability affected larger by the initial bacterium source in field, the same area difference plot occurring degree is not quite similar, generally before have in season
The continuous cropping field that corn southern leaf blight occurs is seriously ill.Milpa spray inoculation is carried out as inoculum using pathogenic bacteria spore, needs blade
Moisturizing is processed, and could meet spore germination infection condition, makes thus and easily all blades overweight, plant of morbidity for being jetted through pathogenic bacteria
Later stage long potential difference, can not embody the ability that anti-resistance to kind opposing disease extends vertically upward, it is possible to cause some to have originally
The performance of tolerance to diseases germplasm is susceptible, loses due value.Sick leaf crushing is collected using the previous year lobus cardiacus is imposed on as inoculum
In or to spread fertilizer over the fields underproof Semen Maydiss possible in theory in the ranks, but be subject to the previous year disease leaf occurring degree, storage condition and sick leaf
The impact of the factors such as spore quantity, conidium vitality, it is impossible to ensure the concordance of induction bacterium source and its pathogenecity per batch
Reliability, and fresh for the corn southern leaf blight of artificial vaccination diseased plant is initially induced bacterium source as field corn helminthosporium maydis, permissible
Solve this problem.But, if storage is more than two weeks after will be shady dry for fresh diseased plant, the spore quantity on sick leaf disease speckle and spore
Sub- vigor is all decreased obviously.Therefore, the selection of field corn helminthosporium maydis induction bacterium source and its Land use systems will directly affect disease
Generation development impact, there is no at present by the use of the fresh diseased plant of artificial vaccination as corn southern leaf blight field induce bacterium source report
Road.
Content of the invention
It is an object of the invention to provide a kind of feasible, the efficient corn southern leaf blight natural induction in fields technology being suitable for of operation
And and the technology application, the technology can meet corn germplasm disease resistance screening and disease control research needed for morbidity bar
Part.
The purpose of the present invention is achieved through the following technical solutions：A kind of corn southern leaf blight natural induction in fields technology, it wraps
Include following steps：
A. the separation of corn southern leaf blight pathogen；
B. the activation of corn southern leaf blight pathogenic bacteria:
C. corn southern leaf blight pathogenic bacteria spore suspension is prepared:After southern corn leaf blight is activated, with the punching of a diameter of 5mm
Device cuts truffle in colony edge, and with Inoculating needle, truffle is moved to PDA culture medium surface, 25-28 DEG C of dark culturing 7-10d
Afterwards, conidium is obtained；In PDA culture medium surface injected clear water, and the spore on PDA culture medium surface is scraped, with clear after filtration
Water dilutes spore suspension, makes conidium final concentration be about 1-10 × 105Individual/mL；
D. the artificial spray inoculation of maize seedling:Maize planting susceptible variety in warm canopy, treats that maize seedling length, to 5-7 leaf, is sprayed
Corn southern leaf blight pathogenic bacteria spore suspension, per plant of milpa sprays spore suspension 15-25mL, the milpa after spray inoculation
With covered rearing with plastic film moisturizing process 24-48h, onset environment temperature 22-32 DEG C, after inoculation 7d, maize leaf is covered with scab, disease
Strain is uprooped, and induces bacterium source as field；
E. field corn helminthosporium maydis spontaneous induction：Semen Maydiss susceptible variety culturing and transplanting seedlings, covering with ground sheeting, duplicate rows single-strain planting 3-
4 leaf, 1 heart maize seedling, line space 0.4-0.5m, in field corn plant 7-8 leaf phase, the corn southern leaf blight that step d is uprooped
Diseased plant, 4-6 strain tiling is disposed across on the moisture in the soil face in the middle of milpa, combines field conditions normal management afterwards, normal pouring, is protected
Hold ground moistening.
Corn southern leaf blight of the present invention is caused by southern corn leaf blight (Bipolaris maydis).
Wherein, in step a, the separation method of corn southern leaf blight pathogen is：Corn southern leaf blight of the collection with classical symptom
Maize leaf is cut into single scab blade with shears by sick leaf standard specimen, with sterilizing tweezers, the blade for shearing is put into concentration for 75%
Ethanol in soak 25-30s, then proceed in 0.1% mercuric chloride solution process 2-3min, with aseptic water washing 3-5 time, use aseptic tweezer
Son proceeds to blade in PDA culture dish, puts 4 blades, cultivate in 25-28 DEG C in every ware, and after 4-6d, picking edge mycelia is to newly
PDA culture dish in, in 25-28 DEG C cultivate 4-6d, through single spore separation method after purification, single bacterium colony is proceeded to PDA culture medium inclined-plane
Upper culture 5-7d, 4 DEG C save backup.
In step b, the activation method of southern corn leaf blight is：The southern corn leaf blight of step a gained is proceeded to PDA culture
In base, 7-10d is cultivated in 25-28 DEG C.
Described Semen Maydiss susceptible variety is that Guangdong is sweet 22, the one kind in top grade, carbuncle 2 and agriculture section jade 368.
The application of described corn southern leaf blight natural induction in fields technology：In disease resistance of the corn germplasm to corn southern leaf blight
Application in identification research.Disease Resistance Identification of the corn southern leaf blight natural induction in fields technology in corn germplasm to corn southern leaf blight
The concrete grammar of the application in research is：Different corn germplasm culturing and transplanting seedlings, covering with ground sheeting, 1 heart of duplicate rows single-strain planting 3-4 leaf jade
Meter Miao, line space 0.4-0.5m, in field corn plant 7-8 leaf phase, the corn southern leaf blight disease that step d is uprooped
Strain, 4-6 strain is disposed across on the moisture in the soil face in the middle of milpa, combines field normal management afterwards, normal pouring, keeps soil wet
Profit, investigates the incidence of each germplasm in Semen Maydiss milk stage.
The application of described corn southern leaf blight natural induction in fields technology：Screening control of leaf spot of corn medicament and
Application in the application method of research control of leaf spot of corn medicament.Corn southern leaf blight natural induction in fields technology is in screening preventing and treating
The concrete grammar of application in the application method of the medicament of corn southern leaf blight and research control of leaf spot of corn medicament is：Test
Semen Maydiss susceptible variety culturing and transplanting seedlings, covering with ground sheeting, 1 heart maize seedling of duplicate rows single-strain planting 3-4 leaf, line space 0.4-0.5m,
The field corn plant 7-8 leaf phase, the corn southern leaf blight diseased plant that step d is uprooped, 4-6 strain tiling is disposed across Semen Maydiss plant
On moisture in the soil face in the middle of strain, combine field normal management afterwards, normal pour, keep ground moistening, respectively at applying diseased plant 5d (8-9
Ye Qi, sick leaf rate reaches 20%) and 15d after (tasseling stage, sick leaf rate reaches 60%) carry out the chemical control of corn southern leaf blight, in Semen Maydiss
The incidence that milk stage investigation medicament is respectively processed.
For prior art, it is an advantage of the current invention that：A. the present invention is to infect the corn southern leaf blight of artificial vaccination
The diseased plant of pathogenic bacteria is as induction bacterium source, easy to operate, and induction bacterium source is readily available；B. bacterium source and field corn stigma pathogenic bacteria are induced
Population structure is similar, pathogenecity reliability；C. compared with for field natural occurrence method, the technology of the present invention can provide good, steady
Fixed pathogen condition；D., more manually for spraying inocalation method, the technology of the present invention can fully demonstrate anti-resistance to kind and resist disease
The ability for extending vertically upward, so as to effectively distinguish the disease resistance of kind；The technology that the present invention is provided is applicable not only to Semen Maydiss
The resistance screening of confrontation helminthosporium maydis is planted, is also applied for medicament and the technical research of controlling disease is screened, therefore the present invention has
Scientific and reasonable, simple, convenient and practical advantage.
Specific embodiment
With reference to embodiment, present invention is described in detail：
Embodiment one：A kind of corn southern leaf blight natural induction in fields technology, comprises the following steps：
A. the separation of corn southern leaf blight pathogen；
B. the activation of southern corn leaf blight；
C. corn southern leaf blight pathogenic bacteria spore suspension is prepared:After southern corn leaf blight is activated, with the punching of a diameter of 5mm
Device cuts truffle in colony edge, and with Inoculating needle, truffle is moved to PDA culture medium surface, after 28 DEG C of dark culturing 7d, obtains
Conidium；In PDA culture medium surface injected clear water, and the spore on PDA culture medium surface being scraped, after filtration, spore is diluted with clear water
Fullness over the chest during pregnancy supernatant liquid, makes conidium final concentration about 1 × 105Individual/mL；
D. the artificial spray inoculation of maize seedling:Maize planting susceptible variety top grade in warm canopy, treats that maize seedling length, to 6 leaves, is sprayed
Corn southern leaf blight pathogenic bacteria spore suspension is applied, per plant of milpa sprays spore suspension 20mL, the milpa after spray inoculation
With covered rearing with plastic film moisturizing process 24h, 25 DEG C of onset environment temperature, after inoculation 7d, maize leaf is covered with scab, and diseased plant connects root
Pulling up, bacterium source is induced as field；
E. field corn helminthosporium maydis spontaneous induction：Sweet 22 culturing and transplanting seedlings in test Semen Maydiss susceptible variety Guangdong, covering with ground sheeting, duplicate rows
4 leaf of single-strain planting, 1 heart maize seedling, line space 0.45m, in the 7 leaf phase of field corn plant, the Semen Maydiss that step d is uprooped are little
Pinta diseased plant, takes 4 plants of tilings respectively and is disposed across on the moisture in the soil face in the middle of milpa, combine field conditions normal management afterwards, just
Often pour, keep ground moistening.
Wherein, in step a, the separation method of corn southern leaf blight pathogen is：Corn southern leaf blight of the collection with classical symptom
Maize leaf is cut into single scab blade with shears by sick leaf standard specimen, with sterilizing tweezers, the blade for shearing is put into concentration for 75%
Ethanol in soak 30s, then proceed in 0.1% mercuric chloride solution process 2min, with aseptic water washing 3 times, with aseptic nipper by leaf
Piece, proceeds in PDA culture dish, puts 4 blades, cultivate in 28 DEG C in every ware, and after 5d, picking edge mycelia is to new PDA culture dish
In, 5d being cultivated in 28 DEG C, through single spore separation method after purification, single bacterium colony is proceeded to culture 7d on PDA culture medium inclined-plane, 4 DEG C of preservations
Standby.
In step b, the activation method of southern corn leaf blight is：The southern corn leaf blight of step a gained is proceeded to PDA culture
In base, 7d is cultivated in 28 DEG C.
Embodiment two：A kind of corn southern leaf blight natural induction in fields technology, comprises the following steps：
A. the separation of corn southern leaf blight pathogen；
B. the activation of southern corn leaf blight；
C. corn southern leaf blight pathogenic bacteria spore suspension is prepared:After southern corn leaf blight is activated, with the punching of a diameter of 5mm
Device cuts truffle in colony edge, and with Inoculating needle, truffle is moved to PDA culture medium surface, after 28 DEG C of dark culturing 7d, obtains
Conidium；In PDA culture medium surface injected clear water, and the spore on PDA culture medium surface being scraped, after filtration, spore is diluted with clear water
Fullness over the chest during pregnancy supernatant liquid, makes conidium final concentration about 1 × 105Individual/mL；
D. the artificial spray inoculation of maize seedling:Maize planting susceptible variety top grade in warm canopy, treats that maize seedling length, to 6 leaves, is sprayed
Corn southern leaf blight pathogenic bacteria spore suspension is applied, per plant of milpa sprays spore suspension 20mL, the milpa after spray inoculation
With covered rearing with plastic film moisturizing process 24h, 25 DEG C of onset environment temperature, after inoculation 7d, maize leaf is covered with scab, and diseased plant connects root
Pulling up, bacterium source is induced as field；
E. field corn helminthosporium maydis spontaneous induction：Sweet 22 culturing and transplanting seedlings in test Semen Maydiss susceptible variety Guangdong, covering with ground sheeting, duplicate rows
4 leaf of single-strain planting, 1 heart maize seedling, line space 0.45m, in the 7 leaf phase of field corn plant, the Semen Maydiss that step d is uprooped are little
Pinta diseased plant, takes 2 plants of tilings respectively and is disposed across on the moisture in the soil face in the middle of milpa, combine field conditions normal management afterwards, just
Often pour, keep ground moistening.
Wherein, in step a, the separation method of corn southern leaf blight pathogen is：Corn southern leaf blight of the collection with classical symptom
Maize leaf is cut into single scab blade with shears by sick leaf standard specimen, with sterilizing tweezers, the blade for shearing is put into concentration for 75%
Ethanol in soak 30s, then proceed in 0.1% mercuric chloride solution process 2min, with aseptic water washing 3 times, with aseptic nipper by leaf
Piece, proceeds in PDA culture dish, puts 4 blades, cultivate in 28 DEG C in every ware, and after 5d, picking edge mycelia is to new PDA culture dish
In, 5d being cultivated in 28 DEG C, through single spore separation method after purification, single bacterium colony is proceeded to culture 7d on PDA culture medium inclined-plane, 4 DEG C of preservations
Standby.
In step b, the activation method of southern corn leaf blight is：The southern corn leaf blight of step a gained is proceeded to PDA culture
In base, 7d is cultivated in 28 DEG C.
Embodiment three：A kind of corn southern leaf blight natural induction in fields technology, comprises the following steps：
A. the separation of corn southern leaf blight pathogen；
B. the activation of southern corn leaf blight；
C. corn southern leaf blight pathogenic bacteria spore suspension is prepared:After southern corn leaf blight is activated, with the punching of a diameter of 5mm
Device cuts truffle in colony edge, and with Inoculating needle, truffle is moved to PDA culture medium surface, after 28 DEG C of dark culturing 7d, obtains
Conidium；In PDA culture medium surface injected clear water, and the spore on PDA culture medium surface being scraped, after filtration, spore is diluted with clear water
Fullness over the chest during pregnancy supernatant liquid, makes conidium final concentration about 1 × 105Individual/mL；
D. the artificial spray inoculation of maize seedling:Maize planting susceptible variety top grade in warm canopy, treats that maize seedling length, to 6 leaves, is sprayed
Corn southern leaf blight pathogenic bacteria spore suspension is applied, per plant of milpa sprays spore suspension 20mL, the milpa after spray inoculation
With covered rearing with plastic film moisturizing process 24h, 25 DEG C of onset environment temperature, after inoculation 7d, maize leaf is covered with scab, and diseased plant connects root
Pulling up, bacterium source is induced as field；
E. field corn helminthosporium maydis spontaneous induction：Sweet 22 culturing and transplanting seedlings in test Semen Maydiss susceptible variety Guangdong, covering with ground sheeting, duplicate rows
4 leaf of single-strain planting, 1 heart maize seedling, line space 0.45m, in the 7 leaf phase of field corn plant, the Semen Maydiss that step d is uprooped are little
Pinta diseased plant, takes 6 plants of tilings respectively and is disposed across on the moisture in the soil face in the middle of milpa, combine field conditions normal management afterwards, just
Often pour, keep ground moistening.
Wherein, in step a, the separation method of corn southern leaf blight pathogen is：Corn southern leaf blight of the collection with classical symptom
Maize leaf is cut into single scab blade with shears by sick leaf standard specimen, with sterilizing tweezers, the blade for shearing is put into concentration for 75%
Ethanol in soak 30s, then proceed in 0.1% mercuric chloride solution process 2min, with aseptic water washing 3 times, with aseptic nipper by leaf
Piece, proceeds in PDA culture dish, puts 4 blades, cultivate in 28 DEG C in every ware, and after 5d, picking edge mycelia is to new PDA culture dish
In, 5d being cultivated in 28 DEG C, through single spore separation method after purification, single bacterium colony is proceeded to culture 7d on PDA culture medium inclined-plane, 4 DEG C of preservations
Standby.
In step b, the activation method of southern corn leaf blight is：The southern corn leaf blight of step a gained is proceeded to PDA culture
In base, 7d is cultivated in 28 DEG C.
Knowable to the morbidity effect of embodiment one, embodiment two, embodiment three is contrasted：Corn southern leaf blight field nature
Induction technology can improve the natural occurrence effect of field corn helminthosporium maydis, when the diseased plant that field is discharged reaches 4 plants and 6 plants, after 5d
The corn disease leaf rate of (8 leaf phase of Semen Maydiss) all reaches 20%, and when the diseased plant for discharging is 2 plants, the rarely seen fragmentary disease of the milpa after 5d
Speckle, sick leaf rate about 5%.The corn southern leaf blight diseased plant that tiling is disposed across in the middle of milpa on moisture in the soil face is more, the effect of natural occurrence
Fruit also can be more preferable, but is considered based on workload, and avoids plant early growth morbidity overweight, hence it is evident that impact plant late growing stage
Phenomenon, the corn southern leaf blight diseased plant 4-6 strain of the traverse that therefore selects to tile on moisture in the soil face in the middle of the milpa is more suitable.
Example IV：A kind of corn southern leaf blight natural induction in fields technology, comprises the following steps：
A. the separation of corn southern leaf blight pathogen；
B. the activation of southern corn leaf blight；
C. corn southern leaf blight pathogenic bacteria spore suspension is prepared:After southern corn leaf blight is activated, with the punching of a diameter of 5mm
Device cuts truffle in colony edge, and with Inoculating needle, truffle is moved to PDA culture medium surface, after 25 DEG C of dark culturing 10d, obtains
Conidium；In PDA culture medium surface injected clear water, and the spore on PDA culture medium surface being scraped, after filtration, spore is diluted with clear water
Fullness over the chest during pregnancy supernatant liquid, makes conidium final concentration be about 5 × 105/mL；
D. the artificial spray inoculation of maize seedling:Maize planting susceptible variety carbuncle 2 in warm canopy, treats maize seedling length to 5 leaves,
Corn southern leaf blight pathogenic bacteria spore suspension is sprayed, per plant of milpa sprays spore suspension 15mL, and the Semen Maydiss after spray inoculation are planted
Strain covered rearing with plastic film moisturizing process 48h, 22 DEG C of onset environment temperature, after inoculation 7d, maize leaf is covered with scab, and diseased plant is even
Having eradicated, bacterium source has been induced as field；
E. field corn helminthosporium maydis spontaneous induction：Test Semen Maydiss susceptible variety top grade culturing and transplanting seedlings, covering with ground sheeting, duplicate rows list
Strain plantation 3 leaf, 1 heart maize seedling, line space 0.4m, in 8 leaf phase of field corn plant, the corn southern leaf blight that step d is uprooped
Diseased plant, 5 plants of tilings are disposed across on the moisture in the soil face in the middle of milpa, combine field conditions normal management afterwards, and normal pouring keeps
Ground moistening.
Wherein, in step a, the separation method of corn southern leaf blight pathogen is：Corn southern leaf blight of the collection with classical symptom
Maize leaf is cut into single scab blade with shears by sick leaf standard specimen, with sterilizing tweezers, the blade for shearing is put into concentration for 75%
Ethanol in soak 25s, then proceed in 0.1% mercuric chloride solution process 2min, with aseptic water washing 5 times, with aseptic nipper by leaf
Piece, proceeds in PDA culture dish, puts 4 blades, cultivate in 25 DEG C in every ware, and after 6d, picking edge mycelia is to new PDA culture dish
In, 4d being cultivated in 25 DEG C, through single spore separation method after purification, single bacterium colony is proceeded to culture 6d on PDA culture medium inclined-plane, 4 DEG C of preservations
Standby.
In step b, the activation method of southern corn leaf blight is：The southern corn leaf blight of step a gained is proceeded to PDA culture
In base, 8d is cultivated in 25 DEG C.
Embodiment five：A kind of corn southern leaf blight natural induction in fields technology, comprises the following steps：
A. the separation of corn southern leaf blight pathogen；
B. the activation of southern corn leaf blight；
C. corn southern leaf blight pathogenic bacteria spore suspension is prepared:After southern corn leaf blight is activated, with the punching of a diameter of 5mm
Device cuts truffle in colony edge, and with Inoculating needle, truffle is moved to PDA culture medium surface, after 27 DEG C of dark culturing 7d, obtains
Conidium；In PDA culture medium surface injected clear water, and the spore on PDA culture medium surface being scraped, after filtration, spore is diluted with clear water
Fullness over the chest during pregnancy supernatant liquid, makes conidium final concentration about 10 × 105Individual/mL；
D. the artificial spray inoculation of maize seedling:In warm canopy, maize planting susceptible variety agriculture section jade 368, treats maize seedling length to 7
Leaf, sprays corn southern leaf blight pathogenic bacteria spore suspension, and per plant of milpa sprays spore suspension 25mL, the Semen Maydiss after spray inoculation
Plant covered rearing with plastic film moisturizing process 36h, 32 DEG C of onset environment temperature, after inoculation 7d, maize leaf is covered with scab, diseased plant
Uprooping, bacterium source is induced as field；
E. field corn helminthosporium maydis spontaneous induction：Test Semen Maydiss susceptible variety agriculture section 368 culturing and transplanting seedlings of jade, covering with ground sheeting,
4 leaf of duplicate rows single-strain planting, 1 heart maize seedling, line space 0.5m, in 8 leaf phase of field corn plant, the Semen Maydiss that step d is uprooped
Helminthosporium maydis diseased plant, 5 plants of tilings are disposed across on the moisture in the soil face in the middle of milpa, are combined field conditions normal management afterwards, are normally poured
Fill, keep ground moistening.
Wherein, in step a, the separation method of corn southern leaf blight pathogen is：Corn southern leaf blight of the collection with classical symptom
Maize leaf is cut into single scab blade with shears by sick leaf standard specimen, with sterilizing tweezers, the blade for shearing is put into concentration for 75%
Ethanol in soak 28s, then proceed in 0.1% mercuric chloride solution process 3min, with aseptic water washing 4 times, with aseptic nipper by leaf
Piece, proceeds in PDA culture dish, puts 4 blades, cultivate in 27 DEG C in every ware, and after 4d, picking edge mycelia is to new PDA culture dish
In, 6d being cultivated in 27 DEG C, through single spore separation method after purification, single bacterium colony is proceeded to culture 5d on PDA culture medium inclined-plane, 4 DEG C of preservations
Standby.
In step b, the activation method of southern corn leaf blight is：The southern corn leaf blight of step a gained is proceeded to PDA culture
In base, 10d is cultivated in 27 DEG C.
Embodiment six：The corn southern leaf blight natural induction in fields technology of the present invention and field natural occurrence method, plant spore
The induced effectiveness contrast test of spray inoculation method
When the induced effectiveness contrast test of embodiment six is carried out, the corn southern leaf blight natural induction in fields that inventor selects
Technology is the corn southern leaf blight natural induction in fields technology described in embodiment one, and concrete induced effectiveness contrast test method is as follows：
A. the separation of corn southern leaf blight pathogen：In step a, the separation method of corn southern leaf blight pathogen is：Collection has
Maize leaf is cut into single scab blade with shears, will be sheared with sterilizing tweezers by the corn southern leaf blight disease leaf standard specimen of classical symptom
Blade be put in the ethanol that concentration is 75% leaching 30s, then proceed to process 2min in 0.1% mercuric chloride solution, rushed with sterilized water
Washing 3 times, with aseptic nipper by blade, proceed in PDA culture dish, 4 blades is put in every ware, cultivate in 28 DEG C, picking side after 5d
Edge mycelia is cultivated 5d in 28 DEG C, through single spore separation method after purification, single bacterium colony is proceeded to PDA culture medium in new PDA culture dish
6d is cultivated on inclined-plane, 4 DEG C save backup.
B. the activation of southern corn leaf blight：In step b, the activation method of southern corn leaf blight is：Jade by step a gained
Stigma pathogenic bacteria proceeds in PDA culture medium rice, cultivates 7d in 28 DEG C.
C. corn southern leaf blight pathogenic bacteria spore suspension is prepared:After southern corn leaf blight is activated, with the punching of a diameter of 5mm
Device cuts truffle in colony edge, and with Inoculating needle, truffle is moved to PDA culture medium surface, after 28 DEG C of dark culturing 7d, obtains
Conidium；In PDA culture medium surface injected clear water, and the spore on PDA culture medium surface being scraped, after filtration, spore is diluted with clear water
Fullness over the chest during pregnancy supernatant liquid, makes conidium final concentration be about 1 × 105/mL；
D. the artificial spray inoculation of maize seedling:Maize planting susceptible variety Guangdong sweet 22 in warm canopy, treats maize seedling length to 6 leaves,
Corn southern leaf blight pathogenic bacteria spore suspension is sprayed, per plant of milpa sprays spore suspension 20mL, and the Semen Maydiss after spray inoculation are planted
Strain covered rearing with plastic film moisturizing process 24h, 25 DEG C of onset environment temperature, after inoculation 7d, maize leaf is covered with scab, and diseased plant is even
Having eradicated, bacterium source has been induced as field；
E. field corn plantation：Sweet 22 culturing and transplanting seedlings in test Semen Maydiss susceptible variety Guangdong, covering with ground sheeting, duplicate rows single-strain planting 4
1 heart maize seedling of leaf, line space 0.45m, often process and 8 young plants are planted, 3 groups of repetitions, bulk testing divides 3 fields to carry out, and 3 fields are entered
Row carries out plant spore spray inoculation, the corn southern leaf blight natural induction in fields technology of the present invention and natural occurrence respectively, its
The test field of middle natural occurrence selected from upper one year have corn southern leaf blight occur continuous cropping field, plant spore spray inoculation, this
The experimental plot of bright corn southern leaf blight natural induction in fields technology and natural occurrence is separated by more than 50m.Plant spacing is about
3500 plants/acre, field normal management.
F. field corn helminthosporium maydis spontaneous induction：The 7 leaf phase of field corn plant and the male initial stage is being taken out, respectively by step d even
The corn southern leaf blight diseased plant that has eradicated, 4 plants of tilings are disposed across on the moisture in the soil face in the middle of milpa, are normally poured with reference to field afterwards
Fill, holding ground moistening is made, remaining same land for growing field crops of management.
Field corn plant stigma pathogenic bacteria spray inoculation：Take out the male initial stage in field corn plant, the about 11-13 leaf phase, spray
Corn southern leaf blight pathogenic bacteria spore suspension, the spray spore suspension about 50mL per milpa, postvaccinal milpa divides two groups,
One group processes 24h with covered rearing with plastic film moisturizing, and another group of unused covered rearing with plastic film moisturizing is processed, the same field of remaining rich water quality management
Between corn southern leaf blight spontaneous induction, i.e., normally pour with reference to field, make holding ground moistening, remaining same land for growing field crops of management.
G. the vaned incidence of institute under each process milpa fringe Top-three Leaves of milk stage investigation and fringe, calculates the state of an illness
Index.
Sick leaf grade scale is with reference to [1248 2-2006 of People's Republic of China (PRC) agricultural industry criteria NY/T《Semen Maydiss are disease-resistant
Worm property identification technology specification part 2：The anti-helminthosporium maydis identification technology specification of Semen Maydiss]
0 grade, disease-free spot on blade；
1 grade, on blade, scab accounts for leaf area less equal than 5%；
3 grades, on blade, scab accounts for leaf area 6%-10%；
5 grades, on blade, scab accounts for leaf area 11%-30%；
7 grades, on blade, scab accounts for leaf area 31%-70%；
9 grades, blade is withered.
Result of the test (is shown in Table 1):
Compared with natural occurrence matched group, using the corn southern leaf blight natural induction in fields technology of the present invention, substantially can carry
The natural occurrence effect of corn southern leaf blight between high fields, and effect stability of falling ill, are wherein induced with the milpa 7-8 leaf phase, morbidity
Effect is significantly better than the induced effectiveness for taking out the male initial stage.And pathogenic bacteria spore spray inoculation method is adopted, need cover film moisturizing to process
Preferable morbidity effect can just be caused, but the method process is more tall and big due to field plant, thin film moisturizing processes operation more
Difficulty, is such as provided without thin film moisturizing process, is easily affected by the inoculation amblent air temperature of the same day or second day, it is impossible to ensure to send out per batch
The stability of sick effect.In addition, with plant spore spray inoculation method, processing in combination with thin film moisturizing, easily making to be sprayed with pathogenic bacteria
Blade overweight, the plant later stage long potential difference of morbidity, can not embody the ability that anti-resistance to kind opposing disease extends vertically upward, it is possible to
Cause some originally susceptible with the performance of tolerance to diseases germplasm, lose due value.
Using the corn southern leaf blight natural induction in fields technology of the present invention, sick leaf is extended by plant lower to upper part, the state of an illness
Development trend is with natural occurrence method, and wherein with the plant radical leaves state of an illness most heavy (having 9,7,5 grades), the middle part blade state of an illness is secondly
(5,3,1 grade), the upper blade state of an illness is most light (3,1,0 grade).Using pathogenic bacteria spore spray inoculation method, the sick level of plant disease leaf is more
Close, its reason is consistent relevant by bacterium with each position blade of plant.
1 different tests method of table compares to the induced effectiveness of corn southern leaf blight
* wherein, described diseased plant induction is the corn southern leaf blight natural induction in fields technology of the present invention, in diseased plant induction and
In 3 test fields that spore spray inoculation is processed, its inoculation time is all spaced 1d.
In same column, after numerical value, different lowercase letter indication differences are notable (P=0.05)；Different capitalizations represent difference pole
Significantly (P=0.01).
Embodiment seven：Disease Resistance Identification of the corn southern leaf blight natural induction in fields technology in corn germplasm to corn southern leaf blight
Application in research
1. materials and methods：What this test was selected has top grade, Guangdong sweet 22 for examination corn variety, and banyan is sweet No. 1, forever precious No. 7, beautiful
Rice kind plantation random alignment, in the Semen Maydiss 7-8 leaf phase, using to top grade, Guangdong sweet 22, banyan is sweet No. 1, forever precious No. 7 this 4 kinds different
Corn germplasm carries out the natural induction in fields of corn southern leaf blight, and concrete grammar is as follows：
A. the separation of corn southern leaf blight pathogen：Corn southern leaf blight disease leaf standard specimen of the collection with classical symptom, uses shears
Maize leaf is cut into single scab blade, with sterilizing tweezers, the blade for shearing is put into leaching 30s in the ethanol that concentration is 75%, then
Process 2min is proceeded in 0.1% mercuric chloride solution, with aseptic water washing 3 times, with aseptic nipper by blade, proceeds to PDA culture dish
In, 4 blades being put in every ware, cultivates in 28 DEG C, after 5d, picking edge mycelia cultivates 5d in 28 DEG C in new PDA culture dish,
Through single spore separation method after purification, single bacterium colony is proceeded to culture 7d on PDA culture medium inclined-plane, 4 DEG C save backup.
B. the activation of southern corn leaf blight：The southern corn leaf blight of step a gained is proceeded in PDA culture medium, in 28 DEG C
Culture 7d.
C. corn southern leaf blight pathogenic bacteria spore suspension is prepared:After southern corn leaf blight is activated, with the punching of a diameter of 5mm
Device cuts truffle in colony edge, and with Inoculating needle, truffle is moved to PDA culture medium surface, after 28 DEG C of dark culturing 7d, obtains
Conidium；In PDA culture medium surface injected clear water, and the spore on PDA culture medium surface being scraped, after filtration, spore is diluted with clear water
Fullness over the chest during pregnancy supernatant liquid, makes conidium final concentration about 1 × 105Individual/mL；
D. the artificial spray inoculation of maize seedling:Maize planting susceptible variety top grade in warm canopy, treats that maize seedling length, to 6 leaves, is sprayed
Corn southern leaf blight pathogenic bacteria spore suspension is applied, per plant of milpa sprays spore suspension 20mL, the milpa after spray inoculation
With covered rearing with plastic film moisturizing process 24h, 25 DEG C of onset environment temperature, after inoculation 7d, maize leaf is covered with scab, and diseased plant connects root
Pulling up, bacterium source is induced as field；
E. field corn helminthosporium maydis spontaneous induction：Sweet 22 culturing and transplanting seedlings in test Semen Maydiss susceptible variety Guangdong, covering with ground sheeting, duplicate rows
4 leaf of single-strain planting, 1 heart maize seedling, line space 0.45m, in field corn plant 7-8 leaf phase, the Semen Maydiss that step d is uprooped
Helminthosporium maydis diseased plant, takes 4 plants of tilings respectively and is disposed across on the moisture in the soil face in the middle of milpa, combine field conditions normal management afterwards,
Normal pouring, keeps ground moistening.
Then, repeat step a-d, sweet No. 1 to top grade, banyan respectively, forever precious No. 7 this other 3 kinds of different corn germplasms enter
The natural induction in fields of row corn southern leaf blight, investigates the incidence of each corn germplasm in Semen Maydiss milk stage.
In order to the corn southern leaf blight natural induction in fields technology for verifying the present invention resists to corn southern leaf blight in corn germplasm
Superiority in characteristic of disease identification application, inventor has also carried out field natural occurrence as a comparison, and result of the test is shown in Table 2：
As a result with analysis：
For try corn variety under the conditions of different tests, with different morbidity effects.Little using the Semen Maydiss of the present invention
Pinta natural induction in fields technology, can significantly improve the morbidity effect of plant, and 4 corn variety state of an illness difference for trying are obvious,
Between kind, the effect of anti-sense trend and field natural occurrence in table 22 (morbidity is preferable) is consistent, all with banyan sweet No. 1, forever precious No. 7 anti-
Property is better than top grade, Guangdong sweet 22.Show the corn southern leaf blight natural induction in fields technology using the present invention, can react interracial anti-
Gender gap, and in the poor field of field natural occurrence, 4 corn variety disease indexs for trying are all low, it is impossible to participating in the experiment
The resistance of kind is rationally screened and is evaluated.
The Disease investigation of different corn variety helminthosporium maydis under the conditions of 2 different tests of table
Embodiment eight：Corn southern leaf blight natural induction in fields technology is in medicament and the research of screening control of leaf spot of corn
Application in the application method of control of leaf spot of corn medicament
A. materials and methods
There is Semen Maydiss susceptible variety Guangdong sweet 22 for trying corn variety, in the Semen Maydiss 7-8 leaf phase, carried out using the provocation method of the present invention
Corn southern leaf blight natural induction in fields (i.e. sweet 22 culturing and transplanting seedlings in Guangdong, 4 leaf of covering with ground sheeting duplicate rows single-strain planting, 1 heart maize seedling, OK
Spacing 0.45m, in the field corn plant 7-8 leaf phase, takes 4 plants of tiling traverses of fresh corn helminthosporium maydis diseased plant of warm canopy artificial vaccination
On the moisture in the soil face in the middle of milpa, normally pour in conjunction with field, moisten Soil conservation), while (8-9 after diseased plant 5d is applied
Ye Qi, sick leaf rate reaches 20%) and 15d after (take out the male initial stage, sick leaf rate reaches 60%) chemical control for carrying out corn southern leaf blight, for examination
Medicament has 30% mixture medicine BY, 30% love Seedling EC, and its concentration, times for spraying and insecticide spraying time are referring to table 3, per plant of spray
Amount about 30mL (8-9 leaf phase) and 50mL (taking out the male initial stage).In the incidence that Semen Maydiss milk stage investigation medicament is respectively processed, test knot
Fruit is shown in Table 3.Wherein described mixture medicine BY is the medicament with propiconazole and RP-26019 as active component, in patent CN
Disclose in 105638713A.
B. result and analysis
Using the corn southern leaf blight natural induction in fields technology of the present invention, the morbidity effect of milpa can be significantly improved,
Under identical use condition, the mixture medicine BY prevention effect for trying is significantly better than love Seedling, wherein mixture medicine BY medicament medication 2 times
Preventive effect be better than the preventive effect of medication 1 time, and the prevention effect in Semen Maydiss 8-9 leaf their early stage medication 1 time is slightly better than Semen Maydiss and takes out hero
Initial stage disease leaf rate reaches 60% preventive effect.Using the corn southern leaf blight natural induction in fields technology of the present invention, can be Chemicals and
Medicament provides good, stable experimental condition using technical research.
3 medicament variety classes of table and different dispenser periods compare to the prevention effect of corn southern leaf blight
Claims (6)
1. a kind of corn southern leaf blight natural induction in fields technology, it is characterised in that：It comprises the following steps：
A. the separation of corn southern leaf blight pathogen；
B. the activation of corn southern leaf blight pathogenic bacteria；
C. corn southern leaf blight pathogenic bacteria spore suspension is prepared:After corn southern leaf blight pathogenic bacteria activates, with the card punch of a diameter of 5mm
Truffle is cut in colony edge, and with Inoculating needle, truffle is moved to PDA culture medium surface, after 25-28 DEG C of dark culturing 7-10d,
Obtain conidium；In PDA culture medium surface injected clear water, and the spore on PDA culture medium surface is scraped, dilute with clear water after filtration
Spore suspension is released, makes conidium final concentration be about 1-10 × 105Individual/mL；
D. the artificial spray inoculation of maize seedling:Maize planting susceptible variety in warm canopy, treats that maize seedling length, to 5-7 leaf, sprays Semen Maydiss
Helminthosporium maydis pathogenic bacteria spore suspension, per plant of milpa sprays spore suspension 15-25mL, the milpa modeling after spray inoculation
Material thin film covering moisturizing process 24-48h, onset environment temperature 22-32 DEG C, after inoculation 7d, maize leaf is covered with scab, and diseased plant is even
Having eradicated, bacterium source has been induced as field；
E. field corn helminthosporium maydis spontaneous induction：Semen Maydiss susceptible variety culturing and transplanting seedlings, covering with ground sheeting, duplicate rows single-strain planting 3-4 leaf 1
Heart maize seedling, line space 0.4-0.5m, in field corn plant 7-8 leaf phase, the corn southern leaf blight disease that step d is uprooped
Strain, 4-6 strain tiling is disposed across on the moisture in the soil face in the middle of milpa, is combined field conditions normal management afterwards, is normally poured, keeps
Ground moistening.
2. corn southern leaf blight natural induction in fields technology according to claim 1, it is characterised in that：In step a, Semen Maydiss are little
The separation method of pinta pathogen is：Corn southern leaf blight disease leaf standard specimen of the collection with classical symptom, with shears by maize leaf
Single scab blade is cut into, with sterilizing tweezers, the blade for shearing is put into leaching 25-30s in the ethanol that concentration is 75%, then proceeds to
2-3min is processed in 0.1% mercuric chloride solution, with aseptic water washing 3-5 time, with aseptic nipper by blade, proceeds to PDA culture dish
In, per 4 blades are put in ware, after 25-28 DEG C of culture 4-6d, picking scab blade edge mycelia in new PDA culture dish,
4-6d is cultivated in 25-28 DEG C, through single spore separation method after purification, single bacterium colony is proceeded on PDA culture medium inclined-plane 5-7d is cultivated, 4 DEG C
Save backup.
3. corn southern leaf blight natural induction in fields technology according to claim 1, it is characterised in that：In step b, Semen Maydiss are little
The activation method of pinta bacterium is：The southern corn leaf blight of step a gained is proceeded in PDA culture medium, 7- is cultivated in 25-28 DEG C
10d.
4. corn southern leaf blight natural induction in fields technology according to claim 1, it is characterised in that：Described Semen Maydiss are susceptible
Kind is that Guangdong is sweet 22, the one kind in top grade, carbuncle 2 and agriculture section jade 368.
5. the application of the corn southern leaf blight natural induction in fields technology according to claim 1-4 any one：Its feature exists
In：Application in Disease Resistance Identification research of the corn germplasm to corn southern leaf blight.
6. the application of the corn southern leaf blight natural induction in fields technology according to claim 1-4 any one：Its feature exists
In：Application in the medicament of screening control of leaf spot of corn and the application method of research control of leaf spot of corn medicament.
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