CN108277165A - Wheat scab grain spawn makes and application method - Google Patents

Abstract

The present invention relates to fungal diseases of plants Bacteria culturings, and specially wheat scab grain spawn makes and application method, the existing dientification of bacteria method that connects of solution need to cultivate bacterium solution, method is complicated, bacterium solution cannot be stored for a long time, connect the low success rate of problem of bacterium, prepared by grain spawn：Band shell grain steeps 5% mung bean water and keeps 60% specific retention, is distributed into bag and gently compresses close, sealing bacterium cylinder processed, autoclave sterilization dries in the air to room temperature, two termination bacterium dark constant temperature ventilation high humility cultures, are transferred to ventilative paper bag, press scattered shady and cool ventilation to dry in sealed bag and keeps in dark place at low temperature.Application method：First detection activity, basal spikelet side little Hua glumelles connect bacterium before flowering after heading, and package moisturizing to lemma browning removes wrapping layer, high humility atomizing moisturizing 28 days.Advantage：1, strain can long-term preservation；2, it produces once, is used for multiple times, dosage is few, is readily transported；3, it connects bacterium method to be simple and efficient, incidence is high, and Disease Resistance Identification result is accurate.

Description

Wheat scab grain spawn makes and application method

Technical field

The present invention relates to fungal diseases of plants Bacteria culturings, the specially making of wheat scab grain spawn and user Method.

Background technology

Wheat scab（Fusarium head blight, FHB）It is by Fusarium graminearum（Fusarium graminearum）The caused a kind of destructive disease for seriously endangering wheat fringe portion and seed, under taking place mostly in the Changjiang river The humidity such as trip, Basin of Huaihe River Winter Wheat Area and Northeasten Spring Wheat Area of China and semi-humid region.The plant disease epidemic time can cause wheat 70%- 100% production loss, the wheat infected seed infected through gibberella, toxin, especially DON toxin containing there are many are equal to humans and animals There is very strong toxicity, humans and animals vomiting, diarrhea, skin irritatin, food refusal, neurological disorders, miscarriage, stillborn foetus etc. can be caused.Work as disease When wheat rate reaches 4% or more, i.e., it cannot eat, lose commodity value.

In recent years, as global warming aggravation and the variation of cropping system, EI Nino and Ramsey numbers are handed over For appearance, extreme weather takes place frequently, and wheat scab gradually rises to commonly-occurring disease evil by accidental disease, and occurring area also constantly expands Greatly, head blight expands to Yellow River-Huai River region and the North China area of wheat by the middle and lower reach of Yangtze River area of wheat often sent out, the Basin of Huaihe River area of wheat at For one of the area for the most serious that caused harm by head blight.But the effective ways that there is no prevention head blight in the world, also do not develop energy The chemical agent of enough thoroughly prevention wheat scabs, therefore, selection and breeding with promote anti gibberellic disease kind just and become control at present with it is pre- Anti- wheat scab approach the most economic and environment-friendly.

The important means that the dientification of bacteria is selection and breeding wheat anti gibberellic disease kind is connect, common method has single flower inoculation method, bacterium solution Spray-on process, native table bacterination process etc., these types of method respectively has advantage and disadvantage.It is accurate that single flower inoculation method connects bacterium, can ensure to connect bacterium success Rate can carry out qualitative, quantitative identification；But it needs to carry out bacterium solution culture before connecing bacterium, required equipment is complex, to environment and training The condition of supporting requires strictly, to need to detect viable bacteria number under the microscope before connecing bacterium every time, and cultured bacterium solution cannot deposit for a long time It puts, easily loss of activity, therefore extremely inconvenient for the identification of head blight.Bacterium solution spray-on process connects bacterium conveniently, is conducive to carry out big face Product connects the dientification of bacteria on a large scale；But it also needs to cultivate bacterium solution before connecing bacterium every time, and viable count mesh, bacterium solution is detected in microscope It preserves inconvenience and the holding time is short, it is relatively low to connect bacterium success rate.Native table bacterination process uses cereals seed culture strain, spills in native table Spore is relied on to launch natural occurrence afterwards, it is higher to environmental requirement, it is relatively low to connect bacterium success rate, it is difficult to which it is qualitative fixed to be carried out to wheat breed Amount identification.

Therefore, a kind of making of research, application method are simple, connect bacterium success rate height, wheat scab paddy with long preservation period Grain and application method are necessary.

Invention content

The present invention, which solves the dientification of bacteria method that connects existing at present, need to cultivate bacterium solution, and preparation method is complicated, cultured bacterium Liquid cannot be stored for a long time, connect the low success rate of problem of bacterium, provide a kind of preparation method it is simple, without cultivating bacterium solution, prepare Grain can connect the high wheat scab grain spawn making of bacterium success rate and application method with long-term preservation without loss of activity.

The present invention is realized by following operating procedure：Wheat scab grain spawn production method, including following behaviour Make step：

A, prepare fresh full, free from insect pests and mouldy mung bean 50g, pure water 1200ml is added, is placed in boiled on fire, boiling water After boil 15-20min, when mung bean is just bloomed close fire, with crocus cloth filter off mung bean residue, only retain mung bean water, mend Pure water is filled to 1000ml, is configured to 5% mung bean water, it is spare；

B, prepare fresh band shell grain 1kg dry, without go mouldy, remove outer skin, disintegrating slag, be placed in clean container, warm is added 5% mung bean water, being sufficiently stirred makes grain moisten completely, and capping moisturizing 4 hours makes grain fully absorb water；

C, excessive moisture is filtered off, soaked grain is spread out at shady and cool ventilation and dries 30min, grain specific retention is made to reach about 60% or so, it is pinched with hand not agglomerating；

D, the grain handled well is dispensed into high temperature high voltage resistant polypropylene sterilizing bag, every bag of packing 300-500g, light pressure makes it Keep more close state；

E, it is sealed at high temperature high voltage resistant polypropylene sterilizing bag both ends with ventilative strain bag sponge sealing lantern ring, is fabricated to bacterium Cylinder, prevents living contaminants；

F, the bacterium cylinder made is placed in high-pressure sterilizing pot, and 125 DEG C of autoclave sterilizations 2 hours are dried in the air after deflation to room temperature；

G, the sponge lid that the sponge sealing lantern ring of bacterium cylinder one end is opened on superclean bench, soya bean is gripped with sterilized tweezers A big fritter gibberella kind, is put on the grain of bacterium cylinder one end, covers ventilative sponge lid；The other end is followed the prescribed rules, and bacterium cylinder two is made End all connects strain；

H, connect bacterium bacterium cylinder be placed between the incubator or culture of complete darkness in 25 DEG C of constant temperature incubations, keep ventilation, incubator Or 60 % of relative humidity or so is kept between culture, it is spaced the bacterium cylinder of overturning in 1-2 days during Spawn incubation, ensures mycelia growth one It causes, about 7-10 days or so, after mycelia covers with bacterium cylinder, bacterium cylinder culture terminated；

I, cultured bacterium cylinder one end is opened on superclean bench, strain is transferred in sterile ventilative paper bag, by band The grain pressure of bacterium dissipates, and is placed in hang airing at shady and cool ventilation；

J, the strain dried completely can be fitted into sterile Self-enclosing polybag, stick strain label, indicate strain name and date, It is kept in dark place under the conditions of 4 DEG C；Also it can be fitted into vacuum packaging bag, after vacuumizing, long-term preservation under the conditions of being placed in -20 DEG C.

Wheat scab grain spawn application method, including following operating procedure：

One, detection activity：Filter paper is opened in clean culture dish middle berth one, a small amount of sterile purified water is added, filter paper is made to moisten completely, 50 grain spawns are put into each culture dish, so that strain is scattered completely, strain label is sticked after capping, is placed in complete darkness 25 DEG C of constant temperature incubations in incubator are observed mycelia growing state and are shown if covering with white hypha around grain spawn after 36 hours Strain is active, if grain spawn does not grow mycelia, shows that strain loses activity, and arranges to repeat for 2-3 times, records and counts The ratio of active strain can be used for field and connect bacterium if active strain accounts for 90% or more；

Two, it carries out connecing bacterium using single flower bacterination process, it be after wheat heading to bacterium position before flowering, is connect is wheat base portion to connect bacterium period The right side little Hua of bottom small ear side up the 6th small ear of number chooses the tassel eared 2-3 days, with tweezers one paddy of gripping Grain strain is put on the right side of target small ear in the glumelle of little Hua, and pure water under bacterium tassel spray 2-3 is connect with the alignment of hand-held spraying kettle, Tassel moistening is kept, the wheat head moisturizing, hang tag, record are entangled by tassel packet layer of transparent preservative film, or with PE polybags The bacterium time is connect, often row material connects bacterium at least ten tassel；

Three, it connects and notices that observation connects bacterium position after bacterium, moisturizing about 3-5 days after bacterium small ear lemma browning waiting, removes preservative film or modeling Material bag；

Four, install atomizing moisture-keeping system additional, daily water spray 5-10 time makes growth environment keep humidity more than 50%, humidity it is more big more Be conducive to gibberella extension, it is appropriate to increase water spray number if dry；

Five, 21 days and 28 days investigation head blight incidences, investigation method are investigated using conventional method after connecing bacterium.

Wheat scab grain spawn of the present invention compared with prior art, has the following advantages：1, with single flower inoculation method Compare：Single flower inoculation method need to use constant-temperature table culture bacterium solution, need to detect spore number under the microscope before inoculation every time, train The bacterium solution supported cannot be stored for a long time（It is preserved no more than 1 month under the conditions of 4 DEG C）, the easy loss of activity of spore, strange land identification It is inconvenient to carry；After the grain spawn that the present invention makes impregnates grain using 5% mung bean water, contained nutritional ingredient compared with horn of plenty, The growth for not only improving gibberella silk trophosome, is also beneficial to the formation of gibberella spore, and do not need constant-temperature table, only needs It to be cultivated 7-10 days under 25 DEG C or so of constant temperature（Because breeding bacterium amount is big, so incubation time is longer）, cultivate item Part is more prone to realize compared to mung bean water Liquid Culture, it is dry after the grain that carries disease germs, gibberella spore in a dormant state, favorably In the long-term preservation of strain, it is also convenient for being carried to strange land progress inoculated identification, and grain spawn is being placed in suitable humiture Under the conditions of, spore is sprouted quickly, restores nutrient growth, and experiment shows the strain preserved under the conditions of 4 DEG C and preserved under the conditions of -20 DEG C Strain, preserve 6 months after, active strain all reaches 95% or more.2, compared with bacterium solution spray-on process：Bacterium solution is sprayed Method largely connects the dientification of bacteria suitable for large area, but affected by environment big, connects that bacterium success rate is low, and it is accurately fixed to be carried out to identification target Property quantitative analysis, be suitable only for the anti-disease enzyme of improved variety, be not suitable for genetic group single plant（Or single fringe）Identification；The present invention makes Grain spawn can carry out single plant inoculation, can ensure effect of inoculation with morbidity success rate, and suitable for carry out genetic group Single plant（Or single fringe）Identification.3, compared with native table bacterination process：Native table bacterination process is frequently with cereals seed（Such as wheat, corn, water Grain of rice etc.）Strain is cultivated, spills and launches natural occurrence by spore after Yu Tubiao, higher to environmental requirement, it is relatively low to connect bacterium success rate, It is difficult to carry out qualitative, quantitative identification to wheat breed；Grain spawn simple grain prepared by the present invention is small, because of millet band shell, strain Adhesion is not susceptible in manufacturing process, the adhesion grown by mycelia is easily dispersed into single grain, and soil can be spread on after dry Earth surface carries out native table inoculation, it is possible to use tweezers grip single grain and are placed in wheat head little Hua glumelles, reach and single flower inoculation method Identical effect of inoculation can carry out genetic group single plant（Or single fringe）Identification, therefore, scope of application ratio using wheat, corn, The strain of the making such as rice grain is wider.4, it in addition, grain spawn prepared by the present invention is produced once, being used for multiple times, grain is smaller, Mass of 1000 kernel is only 2.2-4.0g or so, and produce once 1kg, can get strain 22-40 ten thousand, you can ten thousand small to meet bacterium 22-40 Wheat head, dosage is few, is readily transported, and strain of making, which can be supplied, mostly to be used for multiple times；5, it connects bacterium method to be simple and efficient, send out Sick rate is high, and Disease Resistance Identification result is accurate, and completely alternative traditional single flower inoculation method identifies scab resistance.

Specific implementation mode

Wheat scab grain spawn production method, including following operating procedure：

A, prepare fresh full, free from insect pests and mouldy mung bean 50g, pure water 1200ml is added, is placed in boiled on fire, boiling water After boil 15-20min, when mung bean is just bloomed close fire, with crocus cloth filter off mung bean residue, only retain mung bean water, mend Pure water is filled to 1000ml, is configured to 5% mung bean water, it is spare；

B, prepare fresh band shell grain 1kg dry, without go mouldy, remove outer skin, disintegrating slag, be placed in clean container, warm is added 5% mung bean water, being sufficiently stirred makes grain moisten completely, and capping moisturizing 4 hours makes grain fully absorb water；

C, excessive moisture is filtered off, soaked grain is spread out at shady and cool ventilation and dries 30min, grain specific retention is made to reach about 60% or so, it is pinched with hand not agglomerating；

D, the grain handled well is dispensed into high temperature high voltage resistant polypropylene sterilizing bag, every bag of packing 300-500g, light pressure makes it Keep more close state；

E, it is sealed at high temperature high voltage resistant polypropylene sterilizing bag both ends with ventilative strain bag sponge sealing lantern ring, is fabricated to bacterium Cylinder, prevents living contaminants；

F, the bacterium cylinder made is placed in high-pressure sterilizing pot, and 125 DEG C of autoclave sterilizations 2 hours are dried in the air after deflation to room temperature；

G, the sponge lid that the sponge sealing lantern ring of bacterium cylinder one end is opened on superclean bench, soya bean is gripped with sterilized tweezers A big fritter gibberella kind, is put on the grain of bacterium cylinder one end, covers ventilative sponge lid；The other end is followed the prescribed rules, and bacterium cylinder two is made End all connects strain；

H, connect bacterium bacterium cylinder be placed between the incubator or culture of complete darkness in 25 DEG C of constant temperature incubations, keep ventilation, incubator Or 60 % of relative humidity or so is kept between culture, it is spaced the bacterium cylinder of overturning in 1-2 days during Spawn incubation, ensures mycelia growth one It causes, about 7-10 days or so, after mycelia covers with bacterium cylinder, bacterium cylinder culture terminated；

I, cultured bacterium cylinder one end is opened on superclean bench, strain is transferred in sterile ventilative paper bag, by band The grain pressure of bacterium dissipates, and is placed in hang airing at shady and cool ventilation；

J, the strain dried completely can be fitted into sterile Self-enclosing polybag, stick strain label, indicate strain name and date, It is kept in dark place under the conditions of 4 DEG C；Also it can be fitted into vacuum packaging bag, after vacuumizing, long-term preservation under the conditions of being placed in -20 DEG C.

Wheat scab grain spawn application method, including following operating procedure：

One, detection activity：Filter paper is opened in clean culture dish middle berth one, a small amount of sterile purified water is added, filter paper is made to moisten completely, 50 grain spawns are put into each culture dish, so that strain is scattered completely, strain label is sticked after capping, is placed in complete darkness 25 DEG C of constant temperature incubations in incubator are observed mycelia growing state and are shown if covering with white hypha around grain spawn after 36 hours Strain is active, if grain spawn does not grow mycelia, shows that strain loses activity, and arranges to repeat for 2-3 times, records and counts The ratio of active strain can be used for field and connect bacterium if active strain accounts for 90% or more；

Two, it carries out connecing bacterium using single flower bacterination process, it be after wheat heading to bacterium position before flowering, is connect is wheat base portion to connect bacterium period The right side little Hua of bottom small ear side up the 6th small ear of number chooses the tassel eared 2-3 days, with tweezers one paddy of gripping Grain strain is put on the right side of target small ear in the glumelle of little Hua, and pure water under bacterium tassel spray 2-3 is connect with the alignment of hand-held spraying kettle, Tassel moistening is kept, the wheat head moisturizing, hang tag, record are entangled by tassel packet layer of transparent preservative film, or with PE polybags The bacterium time is connect, often row material connects bacterium at least ten tassel；

Three, it connects and notices that observation connects bacterium position after bacterium, moisturizing about 3-5 days after bacterium small ear lemma browning waiting, removes preservative film or modeling Material bag；

Four, install atomizing moisture-keeping system additional, daily water spray 5-10 time makes growth environment keep humidity more than 50%, humidity it is more big more Be conducive to gibberella extension, it is appropriate to increase water spray number if dry；

Five, 21 days and 28 days investigation head blight incidences, investigation method are investigated using conventional method after connecing bacterium.

Embodiment one：In April, 2017 is in certain northern indoor wheat of Academy of Agricultural Sciences of provinces and cities science and technology innovation bases sunlight In experimental plot, the gibberella saubinetii grain spawn made using the present invention connects the dientification of bacteria to 695 tassels of 139 row wheats.Its In 12 tassels fracture because of artificial origin's tassel, identification is invalid.In remaining 683 tassel, fringe of effectively falling ill is 671, connects bacterium Incidence is 98.24%.Highly resistance is identified altogether to the new strain of wheat 35 of immune head blight, wherein 6 strain disease small ear rates are 5.48%, it is the kind that resistance is best in 139 strains.The sick small ear rate in susceptible check variety Mianyang 8545 is 55.56%, disease-resistant The sick small ear rate for compareing Sumai 3 is 10.01%.

Embodiment two：April in the same year, in certain Academy of Agricultural Sciences of provinces and cities modern agriculture scientific and technical innovation Demonstration Garden wheat experiment of south Tanaka, the gibberella saubinetii grain spawn made using the present invention connect the dientification of bacteria to 1200 tassels of 120 row wheats.Wherein 39 For a tassel because reasons, the moisture film such as blow, rain fall off, identification is invalid.Remaining 1161 tassel, fringe of effectively falling ill is 1093 A, it is 94.14% to connect bacterium incidence.Highly resistance is identified altogether to the new strain of wheat 27 of immune head blight, wherein 8 strain diseases Small ear rate be 6% hereinafter, and full line all inoculation fringe resistances it is consistent.The sick small ear rate in susceptible check variety Mianyang 8545 is 48.22%, the sick small ear rate of susceptible control Alondra is 57.64%, and the sick small ear rate of disease-resistant control Sumai 3 is 9.26%, is resisted The sick small ear rate of disease control Wangshuibai is 11.38%.

Claims (2)

1. a kind of wheat scab grain spawn production method, including following operating procedure：

A, prepare fresh full, free from insect pests and mouldy mung bean 50g, pure water 1200ml is added, is placed in boiled on fire, boiling water After boil 15-20min, when mung bean is just bloomed close fire, with crocus cloth filter off mung bean residue, only retain mung bean water, mend Pure water is filled to 1000ml, is configured to 5% mung bean water, it is spare；

B, prepare fresh band shell grain 1kg dry, without go mouldy, remove outer skin, disintegrating slag, be placed in clean container, warm is added 5% mung bean water, being sufficiently stirred makes grain moisten completely, and capping moisturizing 4 hours makes grain fully absorb water；

C, excessive moisture is filtered off, soaked grain is spread out at shady and cool ventilation and dries 30min, grain specific retention is made to reach about 60% or so, it is pinched with hand not agglomerating；

D, the grain handled well is dispensed into high temperature high voltage resistant polypropylene sterilizing bag, every bag of packing 300-500g, light pressure makes it Keep more close state；

E, it is sealed at high temperature high voltage resistant polypropylene sterilizing bag both ends with ventilative strain bag sponge sealing lantern ring, is fabricated to bacterium Cylinder, prevents living contaminants；

F, the bacterium cylinder made is placed in high-pressure sterilizing pot, and 125 DEG C of autoclave sterilizations 2 hours are dried in the air after deflation to room temperature；

G, the sponge lid that the sponge sealing lantern ring of bacterium cylinder one end is opened on superclean bench, soya bean is gripped with sterilized tweezers A big fritter gibberella kind, is put on the grain of bacterium cylinder one end, covers ventilative sponge lid；The other end is followed the prescribed rules, and bacterium cylinder two is made End all connects strain；

H, connect bacterium bacterium cylinder be placed between the incubator or culture of complete darkness in 25 DEG C of constant temperature incubations, keep ventilation, incubator Or 60 % of relative humidity or so is kept between culture, it is spaced the bacterium cylinder of overturning in 1-2 days during Spawn incubation, ensures mycelia growth one It causes, about 7-10 days or so, after mycelia covers with bacterium cylinder, bacterium cylinder culture terminated；

I, cultured bacterium cylinder one end is opened on superclean bench, strain is transferred in sterile ventilative paper bag, by band The grain pressure of bacterium dissipates, and is placed in hang airing at shady and cool ventilation；

J, the strain dried completely can be fitted into sterile Self-enclosing polybag, stick strain label, indicate strain name and date, It is kept in dark place under the conditions of 4 DEG C；Also it can be fitted into vacuum packaging bag, after vacuumizing, long-term preservation under the conditions of being placed in -20 DEG C.

2. wheat scab grain spawn application method, including following operating procedure：

One, detection activity：Filter paper is opened in clean culture dish middle berth one, a small amount of sterile purified water is added, filter paper is made to moisten completely, 50 grain spawns are put into each culture dish, so that strain is scattered completely, strain label is sticked after capping, is placed in complete darkness 25 DEG C of constant temperature incubations in incubator are observed mycelia growing state and are shown if covering with white hypha around grain spawn after 36 hours Strain is active, if grain spawn does not grow mycelia, shows that strain loses activity, and arranges to repeat for 2-3 times, records and counts The ratio of active strain can be used for field and connect bacterium if active strain accounts for 90% or more；

Two, it carries out connecing bacterium using single flower bacterination process, it be after wheat heading to bacterium position before flowering, is connect is wheat base portion to connect bacterium period The right side little Hua of bottom small ear side up the 6th small ear of number chooses the tassel eared 2-3 days, with tweezers one paddy of gripping Grain strain is put on the right side of target small ear in the glumelle of little Hua, and pure water under bacterium tassel spray 2-3 is connect with the alignment of hand-held spraying kettle, Tassel moistening is kept, the wheat head moisturizing, hang tag, record are entangled by tassel packet layer of transparent preservative film, or with PE polybags The bacterium time is connect, often row material connects bacterium at least ten tassel；

Three, it connects and notices that observation connects bacterium position after bacterium, moisturizing about 3-5 days after bacterium small ear lemma browning waiting, removes preservative film or modeling Material bag；

Four, install atomizing moisture-keeping system additional, daily water spray 5-10 time makes growth environment keep humidity more than 50%, humidity it is more big more Be conducive to gibberella extension, it is appropriate to increase water spray number if dry；

Five, 21 days and 28 days investigation head blight incidences, investigation method are investigated using conventional method after connecing bacterium.