CN108048601A - A kind of capsicum Cucumber Mosaic Virus Seedling Inoculation method and its application - Google Patents
A kind of capsicum Cucumber Mosaic Virus Seedling Inoculation method and its application Download PDFInfo
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Abstract
The present invention relates to a kind of capsicum Cucumber Mosaic Virus Seedling Inoculation method and its application.For having the germ plasm resource of Cucumber Mosaic Virus resistance in Rapid identification and screening China's pepper resource.The manual injection on samsun NN of cucumber mosaic virus poison source after purification is taken to be inoculated with, the sick leaf of harvesting in 12~15 days after inoculation, sick leaf adds in the homogenate of phosphate buffer ice bath, double gauze filtering or centrifugation, and filtrate or supernatant are inoculation liquid.Shoot Organogenesis from Cotyledon of Pepper phase to the 56 leaf phases can be inoculated with.The wherein 12 leaf phases are optimal inoculation seedling age, 1mL inoculation liquids are drawn with disposable syringe (1mL), 2 pin holes is pricked out on cotyledon with syringe needle, removes syringe needle, inoculation liquid is slowly injected at the pin hole of the cotyledon back side, two panels cotyledon is made to be full of virus inoculation liquid.Inoculation carries out Disease investigation after 14 days.Advantage of this approach is that easily controllable virus inoculation amount, when inoculation, is directly injected into mesophyll cell, is easy to observe, reduction is missed.
Description
Technical field
The invention belongs to plant protection arts, are related to a kind of capsicum Cucumber Mosaic Virus Seedling Inoculation method and its answer
With.
Background technology
Temperate zone is the main Distribution Area of Cucumber Mosaic Virus (CMV), and the trend aggravated year by year is presented in torrid areas.Mesh
Before until, the host range of CMV is more than 100 sections, 1200 kinds of plants, to the harm of various vegetables crop and ornamental plant particularly
Seriously.CMV is mainly propagated by a variety of aphids in a manner of non-continuous, and Activities of Some Plants can seed biography poison.Extensive host range,
The reasons such as aphid transmission and crop varieties replacement frequency are small cause CMV that can survive in the anniversary, it is caused to be difficult to prevent.China capsicum master
Producing region CMV verification and measurement ratios are very high, and Beijing, Shaanxi, the recall rate in the province of the Yuan Dynasty and Jiangsu and Zhejiang Provinces area are more than 50%.
CMV shows as progressive pathogenic process after infecting capsicum.Morbidity early stage tender blade base yellow, then yellow
Symptom gradually expands to entire blade；Then new life blade shows apparent floral leaf；With plant development, blade occurs serious
Shrinkage, plant are downgraded, and leaf color shows as lacklustre bottle green.When the severity of capsicum infection CMV symptoms depends on infection
Strain age, general Seedling Stage infection CMV can cause it is bald, downgrade and the underproduction, can cause to have no harvest when serious；And strain infection CMV
Symptom afterwards is relatively light.
Disease Resistance Identification is the basis of disease-resistant material screening, genetics of resistance law study and disease-resistant variety selection and breeding, crucial
It is the resistance level that can accurately reflect material.The method that the anti-CMV identifications of capsicum use artificial infection idenfication mostly.At present
CMV Inoculation Methods include finger friction inoculation, the inoculation of frosted glass spoon, spray gun inoculation and viral juice dipping inoculation.First 3 kinds
Method need to spray the diamond dust of 600 mesh of a thin layer (diameter is about 23 μm) before inoculation in leaf table, will be extra after inoculation 30min
Inoculum wash.Finger friction is inoculated with and the inoculation of frosted glass spoon is easy to operate, but is inoculated with uneven.Used in spray gun inoculation
Spray gun for paint sprayer, for spray gun away from seedling 2cm or so, the operating pressure of air compressor is 2.1~2.5kg during inoculation
Cm, spray gun inoculation have that inoculation is uneven and misses suitable for the Resistance Identification of lot of materials.In this kind of China method
There are certain applications in the anti-CMV materials screening of capsicum.Viral juice leaching root method refers to clean in the soil of seedling root, and immersion connects
In kind liquid, the effect of inoculation of this method is best, but heavy workload, not easy to operate, using few.For in more than inoculation method
There are the problem of, establish it is a kind of it is new, the more efficiently anti-CMV inoculation methods of capsicum are imperative.
The content of the invention
It is an object of the invention to：For problem present in the anti-CMV sprout period authenticating methods of current China capsicum, one is provided
New, the more effective and perfect Seedling Inoculation method and its application of kind.
The purpose of the present invention can be achieved through the following technical solutions：
Capsicum Cucumber Mosaic Virus Seedling Inoculation method, includes the following steps：
(1) Cucumber Mosaic Virus bacterial strain after purification is taken, in samsun NN (Nicotiana tabaccum CVS
Samun NN) on manual injection's inoculation, 12~15 days sick leaves of harvesting after inoculation；Sick leaf is added in into the homogenate of phosphate buffer ice bath,
Filtering or centrifugation, filtrate or supernatant are inoculation liquid；
(2) manually being cultivated to 2 true leaves of seedling after pepper seed disinfection can be inoculated with when being fully deployed；
(3) 1mL bacterium solutions are drawn with 1mL asepsis injectors, pricks out 2 pin holes on cotyledon with syringe needle, remove syringe needle, in son
Inoculation liquid is slowly injected at leaf back pin hole, making two panels cotyledon, after inoculation, diurnal temperature is maintained at 25 full of virus inoculation liquid
DEG C, seedling normal growth management.
Wherein, the phosphate buffer is preferably the 0.02MolL that pH value is 7-1Phosphate buffer；Every gram of sick leaf
It adds in the phosphate buffer described in 5mL and carries out ice bath homogenate.
The preferred double gauze filtering of the filter method；The condition of the centrifugation is 3500 turns points-115 points of centrifugation
Clock.
Pepper seed sterilizes after twenty minutes in 5% sodium hypochlorite, with aseptic water washing 3-4 times.
Manually the method for culture pepper seed is preferably：After pepper seed disinfection in culture dish under 25 DEG C of dark conditions
It sprouts, seed is seeded in 8 × 8 nutritive cube and grows after sprouting, and growth period temperature is 20 DEG C of 25 degrees Celsius/night of daytime, seedling 2
True leaf can be inoculated with when being fully deployed.
Method of the present invention has the kind of Cucumber Mosaic Virus resistance in Rapid identification and screening pepper resource
Application in matter resource.
The application is preferably inoculated with pepper seedling according to the capsicum Cucumber Mosaic Virus Seedling Inoculation method, connects
Kind carries out Disease investigation after 14 days to pepper seedling.
The application is further preferred, the classification mark preferably formulated according to China " eight or five " pepper disease resistance breeding project group
Standard is classified the state of an illness, 0 grade-without any symptom；The bright arteries and veins of 1 grade-lobus cardiacus or the inoculation acute small withered spot of leaf；3 grades-system floral leaf or
Necrotic spot is generated on stem；Downright bad streak is generated on 5 grades-system weight floral leaf, deformity or stem；7 grades-most leaf malformation, fern leaf,
Plant downgrades or stem, branch and the necrosis of vein system；9 grades-plant is seriously downgraded, and stops growing or serious systemic is downright bad, until complete stool
It is dead；Disease index=∑ (state of an illness rank × this grade of strain number)/(investigation total strain number × 9)] × 100.Material or group's disease resistance
The criteria for classifying is：Immune (I)：DI=0；Highly resistance (HR)：0 ﹤ DI≤5；Disease-resistant (R)：5 ﹤ DI≤20；Moderate resistance (MR)：20 ﹤ DI≤
40；Susceptible (S)：DI ﹥ 40.
A kind of anti-CMV Seedling Inoculations identification method of capsicum, comprises the following steps：
(1) Cucumber Mosaic Virus bacterial strain after purification is taken, in samsun NN (Nicotiana tabaccum CVS
Samun NN) on manual injection's inoculation, 12~15 days sick leaves of harvesting after inoculation, it is 7 that 5ml Ph values are added in per 1g diseases leaf
0.02Mol·L-1Phosphate buffer ice bath homogenate, double gauze filtering or 3500 turns points-1Centrifugation 15 minutes, filtrate or
Supernatant is inoculation liquid；
(2) pepper seed sterilizes after twenty minutes in 5% sodium hypochlorite, with aseptic water washing 3-4 times；
(3) sprouted after pepper seed disinfection in culture dish under 25 DEG C of dark conditions, seed is seeded in 8 × 8 after sprouting
Nutritive cube in grow, growth period temperature is 20 DEG C of 25 DEG C/night in daytime, and 2 true leaves of seedling can be inoculated with when being fully deployed；
(4) 1mL bacterium solutions are drawn with disposable syringe, pricks out 2 pin holes on cotyledon with syringe needle, remove syringe needle, in son
Inoculation liquid is slowly injected at leaf back pin hole, two panels cotyledon is made to be full of virus inoculation liquid.After inoculation, diurnal temperature is maintained at 25
DEG C, seedling normal growth management；
(5) Disease investigation is carried out to pepper seedling after being inoculated with 14 days, according to China " eight or five " pepper disease resistance breeding project group
The grade scale of formulation is classified, and calculates disease index；Grade scale is：0 grade-without any symptom；The bright arteries and veins of 1 grade-lobus cardiacus or
It is inoculated with the acute small withered spot of leaf；Necrotic spot is generated on 3 grades-system floral leaf or stem；It is generated on 5 grades-system weight floral leaf, deformity or stem
Downright bad streak；7 grades-majority leaf malformation, fern leaf, plant downgrades or stem, branch and the necrosis of vein system；9 grades-plant is seriously short
Change, stop growing or serious systemic is downright bad, until complete stool is dead.
The disease index computational methods are preferably：Disease index=∑ (state of an illness rank × this grade of strain number)/(investigation is total
Strain number × 9)] × 100.Material or group's disease resistance criteria for classifying are：Immune (I)：DI=0；Highly resistance (HR)：0 ﹤ DI≤5；It is disease-resistant
(R)：5 ﹤ DI≤20；Moderate resistance (MR)：20 ﹤ DI≤40；Susceptible (S)：DI ﹥ 40.
Advantageous effect the advantage of the invention is that：Compared with current common inoculation method, this method need not spray golden steel
Sand, reduces operating procedure, direct needle injection, easily controllable virus inoculation amount and smaller to blade injury, and inoculation liquid can be straight
It connects and is injected into mesophyll cell, be easy to observe, reduction is missed.The present invention reflects to the existing part hot pepper germ plasm resource in research department
Surely satisfactory result is achieved.Show that this method can be used in quickly screening China's pepper resource the capsicum germplasm with anti-CMV
Resource is identified, widens the utilization of resources of the anti-CMV of capsicum.
Specific embodiment
Embodiment 1
(1) Cucumber Mosaic Virus bacterial strain after purification is taken, in samsun NN (Nicotiana tabaccum CVS
Samun NN) on breed, manual injection's inoculation, 12~15 days sick leaves of harvesting after inoculation.It is 7 that 5ml Ph values are added in per 1g diseases leaf
0.02Mol·L-1Phosphate buffer ice bath homogenate, double gauze filtering or 3500 turns points-1Centrifugation 15 minutes, filtrate or
Supernatant is inoculation liquid；
(2) pepper seed sterilizes after twenty minutes in 5% sodium hypochlorite, with aseptic water washing 3-4 times；
(3) sprouted after pepper seed disinfection in culture dish under 25 DEG C of dark conditions, seed is seeded in 8 × 8 after sprouting
Nutritive cube in grow, growth period temperature is 20 DEG C of 25 degrees Celsius/night in daytime, and 2 true leaves of seedling can be inoculated with when being fully deployed.
(4) 1mL bacterium solutions are drawn with disposable syringe (1mL), removes syringe needle, bacterium solution is slow at the back side of Shoot Organogenesis from Cotyledon of Pepper
Injection makes two panels cotyledon be full of virus inoculation liquid.After inoculation, diurnal temperature is maintained at 25 DEG C, seedling normal growth management.
(5) Disease investigation is carried out to pepper seedling after being inoculated with 14 days.Mainly according to China " eight or five " pepper disease resistance breeding
The grade scale that tackling key problem group is formulated, 0 grade-without any symptom；The bright arteries and veins of 1 grade-lobus cardiacus or the inoculation acute small withered spot of leaf；3 grades-system is spent
Necrotic spot is generated on leaf or stem；Downright bad streak is generated on 5 grades-system weight floral leaf, deformity or stem；7 grades-majority leaf malformation,
Fern leaf, plant downgrades or stem, branch and the necrosis of vein system；9 grades-plant is seriously downgraded, and stops growing or serious systemic is downright bad, until
Complete stool is dead.Disease index=∑ (state of an illness rank × this grade of strain number)/(investigation total strain number × 9)] × 100.Varietal population is disease-resistant
The property criteria for classifying is：Immune (I)：DI=0；Highly resistance (HR)：0 ﹤ DI≤5；Disease-resistant (R)：5 ﹤ DI≤20；Moderate resistance (MR)：20 ﹤ DI≤
40；Susceptible (S)：DI ﹥ 40.Qualification test is set to be repeated three times, often handles 10 plants.Test material is with easy susceptible material C .annumn
G29 and disease-resistant material C .frutescens PBC688 (G.J.Guo, S.B.Wang, J.B.Liu et al, Rapid
identification and characterization of QTLs Underlying Resistance to Cucumber
Mosaic Virus in Pepper (Capsicum frutescens) .Theor Appl Genet, 2016,130,1:41-
52.) it is used as check variety.
According to degree of disease investigation result, pepper resource is evaluated according to disease scale and disease index.
As in this experiment, portion of material qualification result such as table 1, in 140 parts of capsicum materials of identification, 15 parts of material state of an illness
Index belongs to resistant material between 5-20；The disease index of 47 parts of materials belongs to moderate resistance material between 20-40；78 parts of materials
Disease index is more than 40, belongs to susceptible material；It does not find that CMV is immunized and the pepper resource of highly resistance, illustrates to grind in the anti-CMV of capsicum
Study carefully middle needs and further expand Screening of Germplasm scope, material foundation is provided for the anti-CMV researchs of capsicum.
The anti-CMV qualification results of 1 hot pepper germ plasm resource of table
Claims (10)
1. capsicum Cucumber Mosaic Virus Seedling Inoculation method, it is characterised in that include the following steps：
(1) Cucumber Mosaic Virus bacterial strain after purification is taken, in samsun NN (Nicotiana tabaccum CVS Samun
NN manual injection is inoculated on), the sick leaf of harvesting in 12~15 days after inoculation；Sick leaf is added in into the homogenate of phosphate buffer ice bath, filtering or
Centrifugation, filtrate or supernatant are inoculation liquid；
(2) manually being cultivated to 2 true leaves of seedling after pepper seed disinfection can be inoculated with when being fully deployed；
(3) 1mL bacterium solutions are drawn with 1mL asepsis injectors, pricks out 2 pin holes on cotyledon with syringe needle, remove syringe needle, carried on the back in cotyledon
Inoculation liquid is slowly injected at the pin hole of face, two panels cotyledon is made to be full of virus inoculation liquid, after inoculation, diurnal temperature is maintained at 25 DEG C,
Seedling normal growth management.
2. the according to the method described in claim 1, it is characterized in that 0.02MolL that it is 7 that the phosphate buffer, which is pH value,-1Phosphate buffer；The phosphate buffer that every gram of sick leaf is added in described in 5mL carries out ice bath homogenate.
3. according to the method described in claim 1, it is characterized in that the filter method filters for double gauze；It is described from
The condition of the heart is 3500 turns points-1Centrifugation 15 minutes.
4. according to the method described in claim 1, it is characterized in that pepper seed sterilizes 20 minutes in 5% sodium hypochlorite
Afterwards, with aseptic water washing 3-4 times.
5. according to the method described in claim 1, it is characterized in that manually the method for culture pepper seed is：Pepper seed sterilizes
It is sprouted afterwards in culture dish under 25 DEG C of dark conditions, seed, which is seeded in after sprouting in 8 × 8 nutritive cube, to be grown, growth period temperature
It spends for 20 DEG C of 25 degrees Celsius/night of daytime, 2 true leaves of seedling can be inoculated with when being fully deployed.
6. method described in claim 1 has the kind of Cucumber Mosaic Virus resistance in Rapid identification and screening pepper resource
Application in matter resource.
7. application according to claim 1, it is characterised in that according to the capsicum cucumber any one of claim 1-5
Mosaic virus Seedling Inoculation method is inoculated with pepper seedling, and inoculation carries out Disease investigation after 14 days to pepper seedling.
8. application according to claim 7, it is characterised in that formulated according to China " eight or five " pepper disease resistance breeding project group
Grade scale the state of an illness is classified, 0 grade-without any symptom；The bright arteries and veins of 1 grade-lobus cardiacus or the inoculation acute small withered spot of leaf；3 grades-be
Necrotic spot is generated on system floral leaf or stem；Downright bad streak is generated on 5 grades-system weight floral leaf, deformity or stem；7 grades-most blades are abnormal
Shape, fern leaf, plant downgrades or stem, branch and the necrosis of vein system；9 grades-plant is seriously downgraded, and stops growing or serious systemic is bad
Extremely, until complete stool is dead；Disease index=∑ (state of an illness rank × this grade of strain number)/(investigation total strain number × 9)] × 100；Varietal population
The disease resistance criteria for classifying is：Immune (I)：DI=0；Highly resistance (HR)：0 ﹤ DI≤5；Disease-resistant (R)：5 ﹤ DI≤20；Moderate resistance (MR)：20 ﹤
DI≦40；Susceptible (S)：DI ﹥ 40.
9. a kind of anti-CMV Seedling Inoculations identification method of capsicum, it is characterised in that comprise the following steps：
(1) Cucumber Mosaic Virus bacterial strain after purification is taken, in samsun NN (Nicotiana tabaccum CVS Samun
NN manual injection is inoculated on), and the sick leaf of harvesting in 12~15 days after inoculation adds in the 0.02MolL that 5ml Ph values are 7 per 1g diseases leaf-1
Phosphate buffer ice bath homogenate, double gauze filtering or 3500 turns points-1Centrifugation 15 minutes, filtrate or supernatant are to connect
Kind liquid；
(2) pepper seed sterilizes after twenty minutes in 5% sodium hypochlorite, with aseptic water washing 3-4 times；
(3) sprouted after pepper seed disinfection in culture dish under 25 DEG C of dark conditions, seed is seeded in 8 × 8 battalion after sprouting
It supports and is grown in alms bowl, growth period temperature is 20 DEG C of 25 DEG C/night of daytime, and 2 true leaves of seedling can be inoculated with when being fully deployed；
(4) 1mL bacterium solutions are drawn with disposable syringe, pricks out 2 pin holes on cotyledon with syringe needle, remove syringe needle, carried on the back in cotyledon
Inoculation liquid is slowly injected at the pin hole of face, two panels cotyledon is made to be full of virus inoculation liquid, after inoculation, diurnal temperature is maintained at 25 DEG C,
Seedling normal growth management；
(5) Disease investigation is carried out to pepper seedling after being inoculated with 14 days, is formulated according to China " eight or five " pepper disease resistance breeding project group
Grade scale be classified, calculate disease index；Grade scale is：0 grade-without any symptom；The bright arteries and veins of 1 grade-lobus cardiacus or inoculation
The acute small withered spot of leaf；Necrotic spot is generated on 3 grades-system floral leaf or stem；Necrosis is generated on 5 grades-system weight floral leaf, deformity or stem
Streak；7 grades-majority leaf malformation, fern leaf, plant downgrades or stem, branch and the necrosis of vein system；9 grades-plant is seriously downgraded, and stops
Only growth or serious systemic necrosis, until complete stool is dead.
10. the anti-CMV Seedling Inoculations identification method of capsicum according to claim 9, it is characterised in that disease index calculating side
Method is：Disease index=∑ (state of an illness rank × this grade of strain number)/(investigation total strain number × 9)] × 100；Varietal population's disease resistance is drawn
Minute mark standard is：Immune (I)：DI=0；Highly resistance (HR)：0 ﹤ DI≤5；Disease-resistant (R)：5 ﹤ DI≤20；Moderate resistance (MR)：20 ﹤ DI≤40；
Susceptible (S)：DI ﹥ 40.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108990586A (en) * | 2018-06-15 | 2018-12-14 | 云南农业大学 | The method that injection inoculation obtains infection 4 kinds of pathogen diseased plants of Yunnan tobacco clump top sickness |
| CN109937736A (en) * | 2019-04-11 | 2019-06-28 | 江苏省农业科学院 | A kind of sponge gourd cucumber mosaic virus Seedling Inoculation, method of resistance identification and application |
| CN112538516A (en) * | 2020-12-18 | 2021-03-23 | 山东省华盛农业股份有限公司 | Seedling-stage injection inoculation method for cucurbita pepo mosaic virus |
| CN113125643A (en) * | 2021-04-02 | 2021-07-16 | 宁波市农业科学研究院 | Method for rapidly identifying tomato bacterial wilt seedling stage resistance by injection inoculation method |
| CN114561424A (en) * | 2022-03-24 | 2022-05-31 | 浙江中医药大学 | Method for seedling injection inoculation of salvia miltiorrhiza and cucumber mosaic virus |
| CN114561424B (en) * | 2022-03-24 | 2024-01-26 | 浙江中医药大学 | Method for injection inoculation of red sage root cucumber mosaic virus at seedling stage |
| CN116076252A (en) * | 2022-11-28 | 2023-05-09 | 华中农业大学 | Precise application method of exogenous substances for strong seedling cultivation of grafted seedlings |
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