CN108893520A - A kind of method of Chinese rose downy mildew resistance identification - Google Patents
A kind of method of Chinese rose downy mildew resistance identification Download PDFInfo
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Abstract
The present disclosure provides a kind of methods of Chinese rose downy mildew resistance identification, include the following steps：(1) typical downy mildew sample is acquired；(2) downy mildew pathogen is expanded numerous；(3) downy mildew pathogen is inoculated with；(4) Chinese rose downy mildew disease resistance evaluation：Chinese rose downy mildew incidence and morbidity severity are counted, disease index is calculated, Chinese rose downy mildew disease resistance is evaluated by disease index average in the statistics phase；Chinese rose downy mildew disease resistance grade scale is as follows：It is immune：Average disease index=0.00；Highly resistance：0.01≤be averaged disease index≤5.00；It is disease-resistant：5.01≤be averaged disease index≤10.00；In resist/middle sense：10.01≤be averaged disease index≤20.00；It is susceptible：20.01≤be averaged disease index≤40.00；Height sense：Average disease index >=40.01 or more.Identification method of the present invention is easy to operate, can be used for the high frequency zone modern rose cultivars strong to Chinese rose downy mildew resistance.
Description
Technical field
The present invention relates to plant disease Resistance Identification technical field, more particularly to a kind of Chinese rose downy mildew resistance
The method of identification.
Background technique
Chinese rose is rosaceae rose, originating in China, is current state away from modern existing more than 2000 years cultivation histories
The maximum cut-flower of border Market clearing quantity.Chinese rose downy mildew is a kind of important disease and a kind of destructive disease of greenhouse Chinese rose,
Caused by rose downy mildew is infected.The egg spore or mycelium of the pathogen remain on it is overwintering in diseased tissues, it is low, opposite in temperature
In the case that humidity is higher, there is water droplet on plant surface, disease easily occurs and spreads.Therefore, greenhouse ventilation is bad, plant mistake
It is close, when humidity is high and nitrogenous fertilizer is excessive, disease is just more serious.Outdoor cropping mostly occurs mainly to be sent out in rainy season, greenhouse or greenhouse
Life is in the 3-4 month and the 9-10 month.
Chinese rose downy mildew main harm blade, tender tip, bennet and flower.There is irregular light green speckle in initial stage, blade face, after
Expanding is in yellowish-brown and purple, is afterwards taupe, edge color is deeper, with health tissues without obvious boundary, finally blade is caused to be turned round
Bent, deformity.When wet weather, the visible sparse canescence downy mildew layer at blade back scab.Leaflet often turns yellow, some scabs
For aubergine, center is canescence, similar to pesticide injury and chemical fertilizer shape of burning.When young sprout and flower infection, scab and blade phase
Seemingly, but in tender tip scab shows slightly recess.When serious, lobar atrophy falls off, and young sprout is withered.
Compared with other diseases, Chinese rose downy mildew disease symptoms are unobvious, but when waiting discoveries morbidity more when having already passed by prevention and control
Phase, larger to Chinese rose production harm, after Chinese rose is susceptible, treatment will cause the total crop failure of current year cut-flower not in time, seriously affect its yield
And quality, largely hinder industry development.
According to existing report, Chinese rose downy mildew mainly passes through selection disease-resistant variety, rational close planting, avoid high temperature and humidity and
Application medicament is prevented and treated；Therefore, disease resistance of the research different cultivars Chinese rose to downy mildew, the high kind plantation of screening resistance,
The generation that downy mildew can be reduced promotes the development of the industry of flowers and plants.However, there is no the Resistance Identification side for Chinese rose downy mildew at present
Method and technology, most of Chinese rose of field planting has downy mildew, therefore carries out the identification of different cultivars downy mildew resistance
Technical research filters out the stronger modern rose cultivars of disease resistance and is of great significance.
Summary of the invention
In view of this, the present invention provides a kind of methods of Chinese rose downy mildew resistance identification, to filter out downy mildew resistance
Stronger modern rose cultivars.
To achieve the goals above, the present invention adopts the following technical scheme that：
A kind of method of Chinese rose downy mildew resistance identification, which is characterized in that include the following steps：
(1) typical downy mildew sample is acquired：In field, acquisition has the Chinese rose blade of downy mildew disease symptom；
(2) downy mildew pathogen is expanded numerous：Typical downy mildew sample collected is placed in moisturizing culture in culturing room, makes disease
Downy mildew pathogen on spot expands numerous growth；
(3) downy mildew pathogen is inoculated with：Visible mycelia and spore are covered with to downy mildew disease leaf blade back, is made on superclean bench
Standby spore suspension or clip scab, are inoculated with plant to be identified, isolated shoot or excised leaf；
(4) Chinese rose downy mildew disease resistance evaluation：Investigation statistics Chinese rose downy mildew incidence and serious journey of falling ill after inoculation
Degree calculates disease index, evaluates Chinese rose downy mildew disease resistance by disease index average in the statistics phase；
Chinese rose downy mildew disease resistance grade scale is as follows：
It is immune：Average disease index=0.00；
Highly resistance：0.01≤be averaged disease index≤5.00；
It is disease-resistant：5.01≤be averaged disease index≤10.00；
In resist/middle sense：10.01≤be averaged disease index≤20.00；
It is susceptible：20.01≤be averaged disease index≤40.00；
Height sense：Average disease index >=40.01 or more.
Preferably, blade is installed using freshness protection package in the step (1), and the sterile filter of 1-2 piece aqua sterilisa wetting is put in bag
Paper 1-2 piece, sealing of blowing are taken back, and then promote the subsequent numerous culture of expansion.
Preferably, in the step (2)：
On superclean bench, the blade back of blade is put in upward in the sterile tray for being lined with double-layer sterile blotting paper, and tray is deep
5-8 centimetres of degree, blotting paper is soaked with sterile water；In spraying misty sterile water on blade, guarantee that blotting paper is wet, slightly inclination plate
When water will not be under plume, but there is a small amount of ponding at plate edge；Using sealed membrane by disk seal mouth, it is placed in culturing room and cultivates；Training
Support room condition of culture：Intensity of illumination 1800-3200lx, photophase photoperiod 12-16 hour, 8-12 hours dark phases, air is relatively wet
75-90% is spent, is cultivated 5-10 days under the conditions of 15-22 DEG C of room temperature.
Preferably, expanding numerous temperature is 15-20 DEG C；Fast-propagation is completed by living body moisturizing culture, scab mycelia extends leaf
Piece rate reaches 32-56%.
Preferably, the preparation method of the spore suspension is：On superclean bench, sterile water is dipped in using sterile brush,
The spore of downy mildew disease leaf blade back is gently washed down, being configured to concentration is 1 × 104-1×106The spore suspension of a/mL, 4-10
DEG C place 2h it is spare.
Preferably, plant and isolated shoot are spraying using spore suspension brushing or spore suspension in the step (3)
Mode be inoculated with；Excised leaf is amplexiformed using scab, is inoculated with by the way of spore suspension brushing or spore suspension drop transposing.
Preferably, in vitro tender leaf uses concentration for 1 × 104The spore suspension of a/mL is inoculated with, it is in vitro in old leaf, in vitro
Branch and plant use 1 × 105The spore suspension of a/mL is inoculated with.
Preferably, it carries out needle thorn in the step (3) before inoculation around the vein of blade to be seeded, but blade is not pierced
It wears.Needle thorn can promote downy mildew pathogen and quickly infect blade.
Preferably, investigation statistics Chinese rose downy mildew incidence, incidence are indicated with disease incidence D after inoculation, by following
Formula calculates：
D (%)=(N/T) × 100
In formula：D-disease incidence, unit are percentage (%)；
N-diseased plant number or the sick number of sheets, unit are strain or leaf；
T-investigation total strain number or total number of sheets, unit are strain or leaf；
Morbidity severity is indicated with disease index, is calculated according to the following formula：
ID=[∑ (N × R)/(M × T)] × 100
In formula：ID-disease index；
The summation of Σ-investigation disease relative disease value of series and its strain number or number of sheets product；
The plant number or the number of sheets of a certain rank of N-disease, unit are strain or leaf；
R-disease relative disease value of series；
M-disease highest disease value of series；
T-investigation total strain number or total number of sheets, unit are strain or leaf.
Preferably, excised leaf incidence survey grade scale：
0 grade-blade disease-free spot；
1 grade-lesion area accounts for leaf area 1-10%；
3 grades-lesion area accounts for leaf area 11-25%；
5 grades-lesion area accounts for leaf area 26-40%；
7 grades-lesion area accounts for leaf area 41-60%；
9 grades-lesion area accounts for 61% or more leaf area or sick leaf is withered；
Field or plant incidence survey grade scale：
0 grade-the whole normal blade disease-free spot of strain；
1 grade-disease leaf accounts for complete stool expansion leaf 1-10%, and downy mildew is unobvious；
3 grades-disease leaf accounts for complete stool expansion leaf 11-25%, and downy mildew is unobvious；
5 grades-disease leaf accounts for complete stool expansion leaf 26-50%, and downy mildew is obvious；
7 grades-disease leaf accounts for complete stool expansion leaf 51-75%, and sick leaf portion point is withered；
75% or more or the most of jaundice that 9 grades-disease leaf accounts for complete stool blade are withered.
By lesion area and sick number of sheets amount to severity Scaling, facilitate the calculating of disease index；Calculate simple, method section
It learns, as a result accurately.
It can be seen via above technical scheme that compared with prior art, the invention discloses a kind of Chinese rose downy mildew resistances
The method of identification carries out fast-propagation to collected downy mildew sample, and scab mycelia extended blades rate is higher, and pathogen is connect
It is cultivated in kind to excised leaf to be identified, branch or plant, statistics Chinese rose downy mildew incidence and morbidity severity,
Disease index is calculated, the i.e. evaluable Chinese rose downy mildew disease resistance of disease index average in the statistics phase is passed through；Evaluation result it is accurate and
Intuitively, by above-mentioned identification method to different cultivars Chinese rose carry out downy mildew resistance identification, can Effective selection go out resistant variety.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical solution of the present invention is clearly and completely described, it is clear that retouched
The embodiment stated is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, originally
Field those of ordinary skill every other embodiment obtained without making creative work, belongs to the present invention
The range of protection.
Embodiment 1 acquires typical downy mildew sample
According to field different onset situation, the serious plant that falls ill can acquire branch, insert in band in the plastic barrel equipped with water
It returns；The plant sporadicly to fall ill acquires typical incidence of leaf, and the aseptic filter paper of 1-2 piece sterile water wetting is installed and be put into freshness protection package
Sealing of blowing is taken back.
Downy mildew disease symptom is as follows：There is irregular light green speckle in early stage, blade face, it is rear expand in yellowish-brown and
Purple is afterwards taupe, and edge color is deeper, with health tissues without obvious boundary, finally causes leaf curling, deformity.Weather tide
When wet, the visible sparse canescence downy mildew layer at blade back scab.Leaflet often turns yellow, and some scabs are aubergine, center
It is similar to pesticide injury and chemical fertilizer shape of burning for canescence.When young sprout and flower infection, scab is similar to blade, but sick in tender tip
Spot shows slightly recess.When serious, lobar atrophy falls off, and young sprout is withered.
2 downy mildew pathogen of embodiment is expanded numerous
Moisturizing culture is carried out to typical downy mildew sample collected, the downy mildew pathogen on scab is made to expand numerous growth,
Incubation time is 5-10 days：
Branch is in intensity of illumination 2000lx, photophase 14 hour photoperiod, 10 hours dark phases, relative air humidity 75-80%,
Clear water bottle is inserted in culturing room under the conditions of 15-22 DEG C of room temperature, the wet preservative film package moisturizing of branch middle and lower part use, outside branch top
Dew, bottle are inserted water and are changed daily once；2/3 test branch is withered after 5-8 days, and blade is withered, and 1/3 test branch Spot expansion is unobvious,
It gradually withers after continuing observation 10-15 days, downy mildew expands numerous failure.
On superclean bench, the blade back of sick leaf blade is put in upward in the sterile tray for being lined with double-layer sterile blotting paper, shallowly
5 centimetres of disk depth, blotting paper is soaked with sterile water；In spraying misty sterile water on blade, guarantee that blotting paper is wet, slightly pitch
Period of the day from 11 p.m. to 1 a.m water will not be under plume, but there is a small amount of ponding at plate edge；Using sealed membrane by disk seal mouth, it is placed in culturing room and cultivates.
Culturing room's condition of culture：Intensity of illumination 3200lx, photophase 16 hour photoperiod, 8 hours dark phases, relative air humidity 80-85%,
15-22 DEG C of room temperature, 5-10 days rear blade scabs of culture successfully extend, and mycelia and Spores successfully expand numerous.
Further, to study the most suitable numerous temperature of expansion, in intensity of illumination 3200lx, photophase 16 hour photoperiod, 8 hours are dark
Phase under the conditions of relative air humidity 80-85%, tests numerous to the expansion of 7 days scab mycelia of above-mentioned leaf culture in 12-28 DEG C
Effect；Wherein, scab mycelia extended blades rate is 23-28% within the scope of 12-14 DEG C, scab mycelia extension within the scope of 15-20 DEG C
Blade rate is 32-56%, and scab mycelia extended blades rate is 29-51%, scab bacterium within the scope of 23-28 DEG C within the scope of 21-22 DEG C
Silk extended blades rate is 4-21%；As it can be seen that it is 15-20 DEG C that Chinese rose downy mildew, which is suitable for the numerous temperature of expansion,.
Scab mycelia extended blades rate=when lesion area-sample (lesion area) after sample culturing/sample
Lesion area * 100% when product sample.
Further, on superclean bench, it is right with four kinds of culture mediums (PDA, oat, rye, Chinese rose fresh leaf juice) to attempt
Downy mildew pathogen is cultivated, and processing method includes that Chinese rose downy mildew scab directly amplexiforms, chooses that mould layer connects, healthy leaves amplexiform
It is amplexiformed afterwards plus scab and full wafer disease leaf.Each processing, which is put into 22 DEG C of ± 1 DEG C of incubators, cultivates, and sees within culture the 5th day, 10 days
It examines, the results are shown in Table 1.Not long downy mildew pathogen bacterium colony or mould layer on four kinds of culture mediums are not detected on culture medium
Downy mildew spore only sees mould layer on original scab, detects downy mildew spore, it can be seen that, expanding propagation method of the present invention
It is simple and practical, and it is good to expand numerous effect.
1 four kinds of culture medium Fiber differentiation downy mildew pathogen results of table
The test of 3 pre-vaccination of embodiment
Visible mycelia and spore are covered with to downy mildew disease leaf blade back, dip in sterile water using sterile brush on super workbench,
The spore of downy mildew disease leaf blade back is gently washed down, obtained spore suspension spore concentration is big, without in diluted situation
Inoculation, inoculation method is spraying.
The consistent fresh and healthy Chinese rose branch of same collecting location, growth period is selected, it is outstanding using above-mentioned not diluted spore
Supernatant liquid is sprayed leave dual sides；Then branch clear water bottle is inserted, wet preservative film is wrapped up outside branch middle and lower part and is carried out
Moisturizing is put into intensity of illumination 1800lx, photophase 12 hour photoperiod, 12 hours dark phases, relative air humidity 85-90%, temperature
It is cultivated under conditions of being 15~22 DEG C, observation is primary every two days and records incidence, takes out after preservative film moisturizing to third day.
The test is repeated 3 times, every time 5 branch, every 20 leaf of branches and leaves the piece number.
Test result：The slight disease symptom of 3 leaves is observed within the 4th day without obvious disease symptom within inoculation 2 days, the 6th day accumulative
There is the morbidity of 5 leaves, gradually yellow is withered for the 8th day branch blade, and incidence of leaf there are 7 leaves.The result shows that spore suspension connects by spraying
Moisturizing 72 hours after kind, downy mildew pathogen infection Chinese rose blade can lead to morbidity.
4 spore suspension concentration tests of embodiment
Expand numerous by the sick leaf that embodiment 1,2 acquires stone forest base, chooses the scab more than mould layer, dipped in hairbrush sterile
Mould layer is washed, then spore suspension is placed under microscope by water, observes quantity with blood counting chamber, uses sterile water tune
Whole is 1 × 103、104、105、106、107A/ml spore or 1 × 103、104、105、106A/ml spore is put into 4 DEG C of refrigerators and puts
Set 2 hours it is spare.
Same collecting location, from Chinese rose ' movie star ' shoot apex downwards number be fully deployed it is strong containing 5 or 7 leaves
The one 5 leaflet of health or the one 7 leaflet complete leaf are used as to test material material, are cleaned and are dried.On superclean bench, blade back is put in pad upward
Have in the sterile tray of double-layer sterile blotting paper, tray is 5 centimetres deep, and blotting paper is soaked with sterile water.To part health before inoculation
Blade is concentrated around vein under slight needle thorn 2-4, blade is not exposed, another part healthy leaves do not do needling treatment.It will
The spore suspension of each concentration rocks mixing, draws 20ul with liquid-transfering gun, drop is pierced around position or vein in blade needle, each
1~2 drop of blade drop.After inoculation, in spraying misty sterile water on blade, guarantee that blotting paper is wet, slightly pitch period of the day from 11 p.m. to 1 a.m water will not
At under plume, but there is a small amount of ponding at plate edge, covers sealed membrane moisturizing, and being placed in intensity of illumination is 1800lx, photoperiod 16
Hour photophase, 8 hours dark phases, relative air humidity 75-80%, temperature is cultivated under the conditions of being 15-20 DEG C, every 2- after inoculation
The incidence of observation in 3 days records disease incidence and disease index, and carries out parallel test, and the results are shown in Table 2.
Disease incidence D is calculated according to the following formula：
D (%)=(N/T) × 100
In formula：D-disease incidence, unit are percentage (%)；
N-disease the number of sheets, unit are leaf；
T-investigation total number of sheets, unit are leaf；
Disease index is calculated according to the following formula：
ID=[∑ (N × R)/(M × T)] × 100
In formula：ID-disease index；
The summation of Σ-investigation disease relative disease value of series and its number of sheets product；
The number of sheets of a certain rank of N-disease, unit are leaf；
R-disease relative disease value of series；
M-disease highest disease value of series；
T-investigation total number of sheets, unit are leaf.
Wherein, excised leaf incidence survey grade scale is as follows：
0 grade-blade disease-free spot；
1 grade-lesion area accounts for leaf area 1-10%；
3 grades-lesion area accounts for leaf area 11-25%；
5 grades-lesion area accounts for leaf area 26-40%；
7 grades-lesion area accounts for leaf area 41-60%；
9 grades-lesion area accounts for 61% or more leaf area or sick leaf is withered.
The different downy mildew bacteria concentrations of table 2 handle Chinese rose ' movie star ' tender leaf stab inoculation incidence
Data are the average value of test result twice in table 2.By above-mentioned data it is found that downy mildew pathogen spore suspension
Liquid concentration is 1 × 103、104、105、106、107A/ml can cause a disease, and pathogenicity difference is obvious between various concentration.Low concentration 1
×103、104A/ml spore suspension morbidity is obvious, wherein 1 × 104The morbidity of a/ml spore suspension is prominent；High concentration 1 ×
105、106、107A/ml spore suspension morbidity is lighter；It is more serious that occurring degree extends morbidity at any time.
Further, expand numerous by the sick leaf that embodiment 1,2 acquires the peace base Feng Ying, spore suspension is made, according to
The above method is 1 × 10 using concentration3、104、105、106A/ml spore suspension to ' movie star ' kind in vitro in old leaf in August
It is inoculated within 16th, August progress first time statistics on the 23rd, experimental result is as shown in table 3.
The in vitro old leaf stab inoculation incidence of 3 modern rose cultivars of table ' movie star '
Data are the average value of test result twice in table 3, and 4 various concentrations have morbidity, wherein 1 × 105A/ml spore
Sub- suspension morbidity protrudes；The state of an illness does not extend after morbidity five days.
In conclusion Chinese rose downy mildew pathogen inoculum density is 1 × 104With 1 × 105The intercurrent disease of a/ml is preferable, from
The tender leaf of body blade uses 1 × 104A/ml spore suspension inoculation, in vitro middle old leaf and plant use 1 × 105A/ml spore
Inoculation of suspension liquid can obtain better result.
5 different vaccination method comparative test of embodiment
Prepare spore suspension：On superclean bench, selection downy mildew expands the sick leaf more than numerous good, visible mould layer, with nothing
Pili brush dips in sterile water, and mould layer is washed, is placed under microscope, observes quantity with blood counting chamber, is adjusted to 1 × 104A/
Ml spore places promotion in 2 hours in 4 DEG C of refrigerators and produces spore.
Make scab：On superclean bench, chooses downy mildew and expand numerous good, visible sick leaf selected more than mould layer, cut scab portion
Position (avoiding clip is in the scab position of withered and yellow state), it is spare.
Balance：It takes and picks up from maritime and growth period consistent ' Monroe ', ' healthy leaves each 48 of fruit bubble ' 2 kind
Leaf is cleaned and is dried；On superclean bench, blade back swings in sterile tray upwards, and each kind fills a disk, every 6 row of disk, every 8 leaf of row,
Every 4 leaf of row concentration around vein is pierced with the slight needle of aseptic inoculation needle, and 4 leaves do not pierce, and wet sterile blotting paper is lined in disk.
Inoculation：On superclean bench, each kind blade, the 1st, 2 rows do scab and amplexiform processing, one piece of scab of every leaf, disease
The spot back side is affixed on around needle thorn position or vein；3rd, 4 rows do sterile brush brushing spore suspension and handle, and the one of brush blade
Side；5th, 6 rows do spore suspension drop transposing 20ul processing, and every leaf one drips, drop needle thorn position or vein week on blade
It encloses.
After inoculation, spray fumulus shape sterile water on blade, and guarantee on blotting paper moisten, pitch period of the day from 11 p.m. to 1 a.m water not at
Under plume, but there is a small amount of ponding at plate edge, then covers sealed membrane moisturizing, is 3200lx in intensity of illumination, the photoperiod is 14 small
When photophase, 10 hours dark phases, relative air humidity 75-80%, room temperature be 15-20 DEG C under the conditions of moisturizing culture, every 2-4 days
It observes and records once, the results are shown in Table 4.
4 Chinese rose Pseudoperonospora cubensis different vaccination method comparative test result of table
As seen from the above table, Monroe's kind either scab amplexiforms, hairbrush brushing spore suspension, spore suspension drop move
It connects, after stabbing at blade inoculation and not piercing processing, shows disease-resistant characteristic；Heart chief minister's seal kind is handled using the above method,
Show susceptible after middle hairbrush brushing, other methods show high sense.
By the above test data it is found that stab inoculation incidence probability is generally higher than not piercing processing；Excised leaf passes through scab
Amplexiform, the method for hairbrush brushing spore suspension, 20 μ l of spore suspension drop transposing inoculation, after 25 days scab amplexiform morbidity compared with
It is good, it is more mainly due to Pseudoperonospora cubensis spore amount on scab；2 kinds have different degrees of downy mildew, disease hair in table 4
Raw trend shows as aggravating day by day.
The test of 6 plant Resistance Identification of embodiment
Natural occurrence record is carried out to 18 modern rose cultivars seedlings, 18 modern rose cultivars seedlings come from maritime same enterprise
Industry, 15-25 centimetres of seedling height of seedling；
Disease incidence D is calculated according to the following formula：
D (%)=(N/T) × 100
In formula：D-disease incidence, unit are percentage (%)；
N-diseased plant number, unit are strain；
T-investigation total strain number, unit are strain；
Disease index is calculated according to the following formula：
ID=[∑ (N × R)/(M × T)] × 100
In formula：ID-disease index；
The summation of Σ-investigation disease relative disease value of series and its strain number product；
The plant number of a certain rank of N-disease, unit are strain；
R-disease relative disease value of series；
M-disease highest disease value of series；
T-investigation total strain number, unit are strain.
Plant incidence survey grade scale：
0 grade-the whole normal blade disease-free spot of strain；
1 grade-disease leaf accounts for complete stool expansion leaf 1-10%, and downy mildew is unobvious；
3 grades-disease leaf accounts for complete stool expansion leaf 11-25%, and downy mildew is unobvious；
5 grades-disease leaf accounts for complete stool expansion leaf 26-50%, and downy mildew is obvious；
7 grades-disease leaf accounts for complete stool expansion leaf 51-75%, and sick leaf portion point is withered；
75% or more or the most of jaundice that 9 grades-disease leaf accounts for complete stool blade are withered.
Chinese rose downy mildew disease resistance grade scale is as follows：
It is immune：Average disease index=0.00；
Highly resistance：0.01≤be averaged disease index≤5.00；
It is disease-resistant：5.01≤be averaged disease index≤10.00；
In resist/middle sense：10.01≤be averaged disease index≤20.00；
It is susceptible：20.01≤be averaged disease index≤40.00；
Height sense：Average disease index >=40.01 or more.
The results are shown in Table 5 for record.
5 18, table common modern rose cultivars seedling natural occurrence situations
As can be seen from the table, under natural occurrence situation, to downy mildew resistance, there are bright between Chinese rose different cultivars resource
Significant difference is different, in 18 parts of variety sources：7 variety sources (red lip, Jin Hui, revival, more Trottas, fruit bubble, revelry bubbles, purple
Rosy clouds fairy maiden) immune, better resistance is showed downy mildew；Other kinds are horizontal in disease-resistant and highly resistance.It should be noted that 7
Kind (red lip, Jin Hui, revival, more Trottas, the mystery of ocean, Jin Yan, orange Bobby) resource September 6 days by powdery mildew etc. other
Disease infection, in 18 parts of modern rose cultivars resources, 3 variety sources (fruit bubble, revelry bubble, purple light fairy maiden) are anti-to downy mildew
Property is preferable, while also resisting other diseases, can be used as preferred Resistance resource.
Further, the brushing of spore suspension is carried out to the blade of each modern rose cultivars health seedling plant, bagging moisturizing,
And pay attention to adding water moisturizing by spraying, inoculation institutes an inquiry incidence in 4-6 days.The rational application of fertilizer after plant inoculation, plant moisturizing light transmission
(reaching medium temperature high humidity).Powdery mildew such as occurs, gray mold, the non-purpose disease such as red spider, disease generation is lighter, sprays to plant
Pesticide grown processing, or non-purpose disease blade is cut；It falls ill heavier, plant is removed into healthy plant management region, no
It is re-used as test and uses plant, prevent non-purpose disease spread；Other diseases occur, unsuitable spraying pesticide processing, in order to avoid inhibit frost
Mildew occurs, and should wipe out disease leaf or remove healthy plant management region.It keeps blade face wet when watering, insect pest occurs and locates in time
Reason, is administered or is rinsed with water.
First inoculation test：Spore suspension concentration is 1 × 104A/ml, 10 groups of tests of each kind progress, every group 3
Strain, the results are shown in Table 6, and data are 10 groups of test mean values in table.
6 18 modern rose cultivars healthy leaves of table are inoculated with downy mildew 1 × 104A/ml spore suspension resistance situation
18 modern rose cultivars resource downy mildew disease indexs difference, 5 variety sources (red lips, more Trottas, Ma Li
Sub-, basin show 6) show as immune and highly resistance；Revival, dreamlike Bobby, orange Bobby, purple light fairy maiden, revelry bubble, ocean mystery,
The Performance of cultivar such as basin show 1, basin show 2, basin show 3, basin show 4 are flat for middle water resistant；Fruit bubble and basin show 5 show disease-resistant；Jin Hui, Jin Yan
It shows as susceptible.
Second batch inoculation test：Spore suspension concentration is 1 × 105A/ml, every group 3 plants, the results are shown in Table 7.
712 modern rose cultivars healthy leaves of table are inoculated with downy mildew 1 × 105A/ml spore suspension resistance situation
12 modern rose cultivars resource downy mildew disease indexs difference.Wherein red lip, Jin Hui, Jin Yan show as high sense；
Revival, more Trottas, AVE MAROA, dreamlike Bobby, purple light fairy maiden show as susceptible；Orange Bobby, fruit bubble, revelry bubble, sea
The mystery in ocean shows as middle sense；12 modern rose cultivars resources reach onset peak in -30 days 20 days after artificial infection.In summary
Test, revelry bubble downy mildew resistance is preferably and performance is stablized, the resistant variety as filtered out.
All in all, the downy mildew incidence difference of natural occurrence and spore suspension inoculation plant leaf, people
Work inoculation morbidity is even more serious；Mainly due to natually morbid pathogen limited amount, and pathogen is generally hidden in soil,
It is easy to be influenced by various factors such as soil environment, humidity, temperature；And artificial infection pathogen quantity is sufficient, blade humidity is high,
Be conducive to infecting for pathogen；In practical qualification process, natural occurrence and artificial infection test can be carried out respectively, in conjunction with the two
Different modern rose cultivars can be obtained to the relevant antagonism of downy mildew in test result, and then filter out the higher kind of relevant antagonism.
Each embodiment in this specification is described in a progressive manner, the highlights of each of the examples are with other
The difference of embodiment, the same or similar parts in each embodiment may refer to each other.For device disclosed in embodiment
For, since it is corresponded to the methods disclosed in the examples, so being described relatively simple, related place is said referring to method part
It is bright.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Claims (9)
1. a kind of method of Chinese rose downy mildew resistance identification, which is characterized in that include the following steps：
(1) typical downy mildew sample is acquired：In field, acquisition has the Chinese rose blade of downy mildew disease symptom；
(2) downy mildew pathogen is expanded numerous：Typical downy mildew sample collected is set into moisturizing culture in culturing room, is made on scab
Downy mildew pathogen expands numerous growth；
(3) downy mildew pathogen is inoculated with：Visible mycelia and spore are covered with to downy mildew disease leaf blade back, spore suspension is prepared or cuts
Scab is taken, plant to be identified, isolated shoot or excised leaf are inoculated with；
(4) Chinese rose downy mildew disease resistance evaluation：Investigation statistics Chinese rose downy mildew incidence and morbidity severity, meter after inoculation
Disease index is calculated, Chinese rose downy mildew disease resistance is evaluated by disease index average in the statistics phase；
Chinese rose downy mildew disease resistance grade scale is as follows：
It is immune：Average disease index=0.00；
Highly resistance：0.01≤be averaged disease index≤5.00；
It is disease-resistant：5.01≤be averaged disease index≤10.00；
In resist/middle sense：10.01≤be averaged disease index≤20.00；
It is susceptible：20.01≤be averaged disease index≤40.00；
Height sense：Average disease index >=40.01 or more.
2. a kind of method of Chinese rose downy mildew resistance identification according to claim 1, which is characterized in that the step (1)
Middle blade is installed using freshness protection package, and the aseptic filter paper 1-2 piece of 1-2 piece aqua sterilisa wetting is put in bag, and sealing of blowing is taken back.
3. a kind of method of Chinese rose downy mildew resistance identification according to claim 1, which is characterized in that the step (2)
In：
On superclean bench, the blade back of blade is put in upward in the sterile tray for being lined with double-layer sterile blotting paper, shallow pan depth
5-8 centimetres, blotting paper is soaked with sterile water；In spraying misty sterile water on blade, guarantee that blotting paper is wet, slightly the pitch period of the day from 11 p.m. to 1 a.m
Water will not be under plume, but there is ponding at plate edge；Using sealed membrane by disk seal mouth, it is placed in culturing room and cultivates；Culturing room's training
The condition of supporting：Intensity of illumination 1800-3200lx, photophase photoperiod 12-16 hour, 8-12 hours dark phases, relative air humidity 75-
90%, it cultivates 5-10 days under the conditions of 15-22 DEG C of room temperature.
4. a kind of method of Chinese rose downy mildew resistance identification according to claim 1, which is characterized in that the step (3)
The preparation method of miospore suspension is：On superclean bench, sterile water is dipped in using sterile brush, by downy mildew disease leaf blade back
Spore gently wash down, be configured to concentration be 1 × 104-1×106The spore suspension of a/mL, 4-10 DEG C of placement 2h are spare.
5. a kind of method of Chinese rose downy mildew resistance identification according to claim 4, which is characterized in that the step (3)
Middle plant and isolated shoot are inoculated in such a way that spore suspension brushing or spore suspension are spraying；Excised leaf uses scab
It amplexiforms, the mode of spore suspension brushing or spore suspension drop transposing is inoculated with.
6. a kind of method of Chinese rose downy mildew resistance identification according to claim 5, which is characterized in that in vitro tender leaf uses
Concentration is 1 × 104The spore suspension of a/mL is inoculated with, and in vitro middle old leaf, isolated shoot and plant use 1 × 105The spore of a/mL
Sub- inoculation of suspension liquid.
7. a kind of method of Chinese rose downy mildew resistance identification according to claim 1, which is characterized in that the step (3)
Needle thorn is carried out before middle inoculation around the vein of blade to be seeded, but blade is not pierced through.
8. a kind of method of Chinese rose downy mildew resistance identification according to claim 1, which is characterized in that investigate system after inoculation
Incidence is counted, incidence is indicated with disease incidence D, is calculated according to the following formula：
D (%)=(N/T) × 100
In formula：D-disease incidence, unit are percentage (%)；
N-diseased plant number or the sick number of sheets, unit are strain or leaf；
T-investigation total strain number or total number of sheets, unit are strain or leaf；
Morbidity severity is indicated with disease index, is calculated according to the following formula：
ID=[∑ (N × R)/(M × T)] × 100
In formula：ID-disease index；
The summation of Σ-investigation disease relative disease value of series and its strain number or number of sheets product；
The plant number or the number of sheets of a certain rank of N-disease, unit are strain or leaf；
R-disease relative disease value of series；
M-disease highest disease value of series；
T-investigation total strain number or total number of sheets, unit are strain or leaf.
9. a kind of method of Chinese rose downy mildew resistance identification according to claim 8, which is characterized in that
Excised leaf incidence survey grade scale：
0 grade-blade disease-free spot；
1 grade-lesion area accounts for leaf area 1-10%；
3 grades-lesion area accounts for leaf area 11-25%；
5 grades-lesion area accounts for leaf area 26-40%；
7 grades-lesion area accounts for leaf area 41-60%；
9 grades-lesion area accounts for 61% or more leaf area or sick leaf is withered；
Field or plant incidence survey grade scale：
0 grade-the whole normal blade disease-free spot of strain；
1 grade-disease leaf accounts for complete stool expansion leaf 1-10%, and downy mildew is unobvious；
3 grades-disease leaf accounts for complete stool expansion leaf 11-25%, and downy mildew is unobvious；
5 grades-disease leaf accounts for complete stool expansion leaf 26-50%, and downy mildew is obvious；
7 grades-disease leaf accounts for complete stool expansion leaf 51-75%, and sick leaf portion point is withered；
75% or more or the most of jaundice that 9 grades-disease leaf accounts for complete stool blade are withered.
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