CN108486033A - A kind of in-vitro verification method of coffee germplasm rust resistance - Google Patents
A kind of in-vitro verification method of coffee germplasm rust resistance Download PDFInfo
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Abstract
The invention discloses a kind of in-vitro verification method of coffee germplasm rust resistance, including the preparation of rest fungus uredospore, the preparation of excised leaf and inoculated identification；The preparation of wherein rest fungus uredospore includes the rest fungus uredospore on the coffee incidence of leaf of the preparation of inoculated and cultured box, acquisition with typical coffee rest fungus scab, and rest fungus uredospore is coated in the vacuum side of blade of susceptible variety, and repeated inoculation obtains purifying rest fungus strain.The in-vitro verification method of coffee germplasm rust resistance in the present invention, it may insure that inoculation condition, inoculation pressure and the onset condition of every part of germplasm materials to be identified are highly consistent, the experimental error caused by inoculation environment and onset environment are inconsistent is reduced to greatest extent, improve the accuracy of qualification result, in addition Vitro Inoculation Technique for Identification has broken away from dependence of the crop field identification to season, make Resistant Gene To Rust identification that can carry out at any time, greatly improves work efficiency.
Description
Technical field
The present invention relates to breeding for disease resistance technical field, more particularly to a kind of coffee germplasm rust resistance it is in vitro
Identification method.
Background technology
Coffee rust is coffee (Coffea caused by coffee rest fungus (Hemileia vastatrix Berk.＆Br.)
Arabica a kind of disease) generally occurred in production, seriously threatens the yield and quality of China's coffee, causes coffee every year
Production loss is between 15%~80%.The disease has generation, main harm coffee blade to cause in whole world Coffee Production area
Leaf abscission, plant are weak, even dead.Since coffee rust is aeroborne disease, it is difficult to chemical prevention and biological control etc.
Means are eradicated, and breeding resistant variety is the prevention most economical effective method of coffee rust.
But the method that Natural infection and inoculated identification are mainly used to the identification of coffee rust at present, pass through field
Between natural occurrence or artificial infection rust uredospore, after the onset investigate leaf spot lesion symptom and production spore situation, be classified accordingly,
Finally become by field weather although this method is simple, quick according to the ability of each its Resistant Gene To Rust of germplasm state of an illness level evaluation
That changes is affected, and inoculation success or not is difficult to control, and investigates time stage difficulty, and the accuracy of qualification result can not also protect
Card.It obtains Resistant Gene To Rust germ plasm resource, excavate the premise and basis that disease-resistant gene is rust-proofing breeding, and reliable rust-proofing dientification of bacteria skill
Art is the key that realize this target.In order to improve the accuracy of Resistant Gene To Rust identification, established in succession on wheat, soybean in vitro
The method of blade identification, but on coffee, there is no the method and technology of rust Detached-leaf test.
Therefore, it is those skilled in the art's urgent need to resolve to establish a kind of in-vitro verification method of coffee germplasm rust resistance
Problem.
Invention content
In view of this, the present invention provides a kind of in-vitro verification method of coffee germplasm rust resistance, this method is using warp
It is inoculated with the single biological strain of rest fungus of purifying in vitro coffee tender leaf, the growth of rest fungus uredospore is observed under controllable environment
Situation evaluates the Resistant Gene To Rust ability of germplasm according to the upgrowth situation of spore.The technical operation is simple, is easy to apply, and connects in vitro
Dependence of the crop field identification to season has been broken away from kind identification, makes Resistant Gene To Rust identification that can need to carry out at any time according to researcher,
It greatly improves work efficiency.
To achieve the goals above, the present invention adopts the following technical scheme that：
1, a kind of rest fungus uredospore for coffee rust resistance Detached-leaf test, which is characterized in that the rest fungus uredospore
Preparation method it is as follows：
1) preparation of inoculated and cultured box：Impregnate sponge 3~5 minutes with a concentration of 70~75% alcohol, after dry, be layered on
Sponge is soaked with sterile water in plastic culture cassette bottom portion；
2) acquisition has the coffee incidence of leaf of typical coffee rest fungus scab, scrapes rest fungus uredospore, and by the rest fungus
Uredospore is coated in the vacuum side of blade of susceptible variety Caturra or Matari, so that the rest fungus uredospore is uniformly distributed, and spray water
Mist is to there is moisture film generation；Then vacuum side of blade will be inoculated with upward, be placed on the sponge in the inoculated and cultured box, 20~25 DEG C dark
40-50h is cultivated, later light and shade alternate culture 35~40 days；
3) the single rest fungus uredium that will occur on the inoculation blade, is seeded in new Caturra or Matari leaves
On piece repeats step 2) and step 3) 2~3 times, obtains the rest fungus strain of purifying；
4) production spore blade is taken out, capsule is put into after the rest fungus uredospore is collected, is the dry of 45-55% in relative humidity
It is dry in dry device, rear 5 DEG C of preservations.
Further, the blade of susceptible variety Caturra described in step 2) or Matari are just to be unfolded, the tender softness of children
Blade.Just it is unfolded, the leaf growth of the tender softness of children is not healthy and strong and more delicate enough, resists force difference, is easy to infect rest fungus,
Success ratio of inoculation is increased, and shorten compared to ripe healthy and strong blade using the blade of the tender softness of children as infection matrix
Infection time is conducive to the screening for accelerating purifying rest fungus strain.
Further, the rest fungus uredospore is made to be evenly distributed on susceptible variety Caturra or Matari leaf in step 2)
The method at the piece back side is gently to be brushed using writing brush.Brush writing skill is soft and disperses, not easy damaged rest fungus when being swiped on blade
Uredospore, and can be quickly so that rest fungus uredospore is evenly distributed on susceptible variety Caturra or Matari vacuum side of blade.
Further, in step 2) during the light and shade alternate culture, sponge moistening is kept, is inhaled within 3-4 days after inoculation
The water of the dry inoculation leaf back, is used in combination absorbent cotton to wipe the inoculation leaf back.
The coffee rest fungus incubation period takes 35 days or more, under proper culture conditions, will generate a large amount of coffees within 35 days~40 days
Rest fungus uredospore；Coffee rest fungus uredospore starts to sprout for 2 hours in chance water, largely completes to infect host in 48 hours,
Alternate culture is to ensure that coffee blade keeps fresh and alive later, and coffee blade can survive in crisper in the case of moisture abundance
45-50 days or so.Sprouting is completed in the case where there is sufficient moisture condition and successfully invades for coffee rest fungus uredospore in 2 days after inoculation
Dye, 3-4 days suck dry moistures infect rest fungus uredospore and do not influence after inoculation, and at this moment suck dry moisture, and energy is wiped with absorbent cotton
Except the parasitic category Hyperparasites such as (Darluca) and Verticillium (Verticillium spp.) that derust, and these hyperparasites are true
Bacterium cause damages to rest fungus uredospore if there is meeting.
Preferably, rest fungus uredospore collection method is in step 4), using scalpel by rest fungus uredospore from production spore blade
On scrape on template, capsule is placed into after collection.
The rest fungus uredospore is used for the in-vitro verification method of coffee germplasm rust resistance, includes the following steps：
1) preparation of excised leaf：The blade for picking germplasm to be identified, cutting off petiole keeps blade back face-up, is put into and prepares
Inoculated and cultured box as described in claim 1 in；
2) inoculated identification：The rest fungus uredospore as described in claim 1 prepared is coated on excised leaf, is made described
Rest fungus uredospore is uniformly distributed, and sprays water mist to there is moisture film generation, in artificial incubator, 20~25 DEG C, and light culture 40-
50h is cultivated 35~40 days in the environment of light and shade alternating later, and rear expert evidence scab type, size, the scab investigated produces spore feelings
Condition.
Further, the blade of germplasm to be identified described in step 1) is the blade of the just tender softness of expansion children.Just it is unfolded,
The leaf growth of the tender softness of children is not healthy and strong and more delicate enough, resists force difference, is easy to infect rest fungus, utilizes the leaf of the tender softness of children
Piece increases success ratio of inoculation, and shorten infection time, favorably as infection matrix compared to ripe healthy and strong blade
In the screening for accelerating purifying rest fungus strain.
Further, it is gently to be brushed with writing brush so that rest fungus uredospore is evenly distributed on the method on excised leaf in step 2).
Brush writing skill is soft and disperses, not easy damaged rest fungus uredospore when being swiped on blade, and can be quickly so that the rest fungus summer
Spore is evenly distributed on susceptible variety Caturra or Matari vacuum side of blade.
Further, in step 2) during the light and shade alternate culture, sponge moistening, and 3-4 days after inoculation are kept
The water for blotting inoculation leaf back is used in combination absorbent cotton to wipe the inoculation leaf back.
Further, rust resistance is divided into 0-4 grades, 0 grade of the resistance does not generate any scab, resistance for blade surface
Type is immune (I)；1 grade of the resistance has colour fading spot or tumor scab to occur for blade surface, and resistance type is disease-resistant (R)；It is described
2 grades of resistance is that the production spore area of leaf spot lesion accounts for the 50% of leaf spot lesion area hereinafter, resistance type is moderate resistance (MR)；It is described anti-
Property 3 grades of production spore areas for leaf spot lesion be more than 50% but be less than 95%, resistance type is middle sense (MS)；4 grades of the resistance is
The production spore area of scab is more than 95%, and resistance type is susceptible (S).
The present disclosure provides a kind of in-vitro verification methods of coffee germplasm rust resistance, at least have the advantage that：
(1) use the Resistant Gene To Rust of Vitro Inoculation Technique for Identification evaluation coffee germplasm horizontal, it can be ensured that every part of to be identified kind of material
The inoculation condition of material, inoculation pressure and onset condition it is highly consistent, reduced to greatest extent because of inoculation environment and onset environment
Experimental error caused by inconsistent improves the accuracy of qualification result.
(2) Vitro Inoculation Technique for Identification has broken away from dependence of the crop field identification to season, makes Resistant Gene To Rust identification can be according to researcher
Need carry out at any time, greatly improve work efficiency.
(3) compared with greenhouse inoculated identification and crop field inoculated identification, space needed for Vitro Inoculation Technique for Identification is small, is suitable for advising greatly
Mould identifies that management cost and labor intensity are greatly reduced.
(4) Vitro Inoculation Technique for Identification is easy to operate, easy to spread universal.
Specific implementation mode
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment
Only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
The every other embodiment that art personnel are obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1：
A kind of preparation method of rest fungus uredospore for coffee rust resistance Detached-leaf test, includes the following steps：
1) prepared by inoculated and cultured box：Sponge is impregnated with a concentration of 72% alcohol 4 minutes, wherein sponge thickness 1cm, length
15cm, width 10cm, after dry, be layered on plastic culture cassette bottom portion, soaked sponge with sterile water；
2) out of, Yunnan Province Yingjiang County coffee main producing region crop field, the coffee morbidity with typical coffee rest fungus scab is acquired
Blade gently scrapes rest fungus uredospore with scalpel, and rest fungus uredospore is coated in susceptible variety Matari and has just been unfolded, and children is tender
Soft vacuum side of blade, is gently brushed with writing brush, so that rest fungus uredospore is uniformly distributed, and gently spray water mist, until there is moisture film to be produced as
Only, it then will be inoculated with vacuum side of blade upward, and be placed on the sponge in culture box, cover lid, it is dark to train in 22 DEG C of artificial incubator
48h is supported, later light and shade alternate culture 37 days, during which keeps sponge moistening, and blot inoculation blade back with blotting paper within 3 days after inoculation
The water in face is used in combination absorbent cotton gently to wipe inoculation leaf back；
Rest fungus is obligate parasite, and this method can keep coffee blade to survive 45-50 days or so, and rest fungus breeding is most
When thermophilic is spent, the production spore time is within 35 days；If temperature is not optimum temperature, the production spore time can be elongated；
3) it will be inoculated with the single rest fungus uredium that occurs on blade, be seeded in rest fungus uredospore with scalpel new
On Matari blades, step 2) and step 3) 3 times are repeated, the rest fungus strain of purifying is obtained；
4) production spore blade is taken out, gently rest fungus uredospore is scraped with pocket knife and is put into capsule after collection, is in relative humidity
It is dry in 50% drier, it is placed in 5 DEG C of refrigerators and stores for future use.
The blade picked Matari, Catimor7963 respectively, the tender softness of children has S.795 just been unfolded with HDT germplasm, cuts off leaf
Handle blade back is face-up, is put into the inoculated and cultured box prepared；The rest fungus uredospore prepared is coated in scalpel and is prepared
Excised leaf on, gently being brushed with writing brush makes rest fungus uredospore be uniformly distributed on excised leaf, and light atomized water spray is to there is moisture film production
It is raw, the lid of culture box is covered, 22 DEG C of light culture 48h in artificial incubator are placed on, is cultivated in the environment of light and shade alternating later
37 days, sponge moistening is during which kept, and blot within 3 days the water of inoculation leaf back after inoculation, absorbent cotton wiping inoculation blade back is used in combination
Face.It is recorded in table 1 by Matari, Catimor7963, S.795 with HDT germplasm scabs type, size, scab production spore situation.
Identification method is as follows to be identified using greenhouse artificial inoculation method to same a collection of germplasm materials Resistance Identification simultaneously：
Choose Matari, Catimor7963, S.795 with HDT germplasm coffee species Potted orchards, 5 plants of each kind, with solution
The above-mentioned rest fungus uredospore prepared is coated in above-mentioned germplasm and has just been unfolded by cut open cutter, the vacuum side of blade of the tender softness of children, light with writing brush
Brush, makes rest fungus uredospore be uniformly distributed, and gently spray water mist, until having moisture film generation, then puts on polybag, wet shading
48h is handled, is cultivated in the greenhouse.The rust of each germplasm is investigated when control group susceptible variety Matari shows serious rust symptom
A situation arises for disease, and remembers by Matari, Catimor7963, S.795 with HDT germplasm scabs type, size, scab production spore situation
Record is in table 1.
Table 1：Different germplasm coffee Detached-leaf tests and the artificial qualification result of room temperature
By the experimental result in table 1 it is found that the comparison of Detached-leaf test and greenhouse artificial infection idenfication, the result of Detached-leaf test
It is believed that and is consistent with the result of greenhouse artificial infection idenfication.
Embodiment 2：
A kind of rest fungus uredospore for coffee rust resistance Detached-leaf test, the preparation method of the rest fungus uredospore is such as
Under：
1) prepared by inoculated and cultured box：Sponge is impregnated with a concentration of 75% alcohol 5 minutes, wherein sponge thickness 1cm, length
15cm, width 10cm, after dry, be layered on plastic culture cassette bottom portion, soaked sponge with sterile water；
2) out of, Yunnan Province Yingjiang County coffee main producing region crop field, the coffee morbidity with typical coffee rest fungus scab is acquired
Blade gently scrapes uredospore with scalpel, and rest fungus uredospore is coated in susceptible variety Caturra and has just been unfolded, and children is tender soft
Soft vacuum side of blade is gently brushed with writing brush, so that rest fungus uredospore is uniformly distributed, and gently spray water mist, until having moisture film generation,
Then vacuum side of blade will be inoculated with upward, and will be placed on the sponge in culture box, lid is covered, in 25 DEG C of artificial incubator, light culture
45h, light and shade alternate culture 35 days, during which keep sponge moistening, and blot inoculation leaf back with blotting paper within 4 days after inoculation later
Water, be used in combination absorbent cotton gently wipe inoculation leaf back；
3) it will be inoculated with the single rest fungus uredium that occurs on blade, be seeded in rest fungus uredospore with scalpel new
On Caturra blades, step 2) and step 3) 2 times are repeated, the rest fungus strain of purifying is obtained；
4) production spore blade is taken out, gently rest fungus uredospore is scraped with pocket knife and is put into capsule after collection, is in relative humidity
It is dry in 53% drier, it is placed in 5 DEG C of refrigerators and stores for future use.
It is picked respectively in Dehong Tropical Agriculture Institute of Yunnan, Ministry of Agriculture's Ruili coffee Germplasm Resources
The blade of the tender softness of children has just been unfolded in Caturra, Matari, Catimor7963, Typica, PT and HDT germplasm, cuts off petiole leaf
The back side upward, is put into the inoculated and cultured box prepared；The rest fungus uredospore prepared is coated in scalpel prepare from
On body blade, gently being brushed with writing brush makes rest fungus uredospore be uniformly distributed on excised leaf, and light atomized water spray is covered to there is moisture film generation
The lid for cultivating box well, is placed on 25 DEG C of light culture 45h in artificial incubator, is cultivated 35 days in the environment of light and shade alternating later,
Period keeps sponge moistening, and blots within 4 days the water of inoculation leaf back after inoculation, and absorbent cotton wiping inoculation leaf back is used in combination.It will
Caturra, Matari, Catimor7963, Typica, PT and HDT germplasm scab type, size, scab production spore situation are recorded in
In table 2.
Identification method is as follows to be identified using Natural infection method to same a collection of germplasm materials Resistance Identification simultaneously：
When susceptible variety Matari shows serious rust symptom, in Dehong Tropical Agriculture Institute of Yunnan's coffee
The rust that coffee Germplasm Resources investigate each coffee germplasm of Caturra, Matari, Catimor7963, Typica, PT and HDT occurs
Situation, and Caturra, Matari, Catimor7963, Typica, PT and HDT germplasm scab type, size, scab are produced into spore
Situation is recorded in table 2.
Table 2：Different germplasm coffee Detached-leaf tests and natural appraisal result
By the experimental result in table 2 it is found that Detached-leaf test is compared with natural appraisal, the result and natural appraisal of Detached-leaf test
Result be substantially consistent.
In conclusion the identification method in the present invention can be compared with greenhouse inoculated identification and crop field inoculated identification, in vitro
Space needed for inoculated identification is small, and the time is short, is suitable for Large scale identification, and management cost and labor intensity are greatly reduced；And it is in vitro
Inoculated identification is easy to operate, easy to spread universal；In addition Vitro Inoculation Technique for Identification has broken away from dependence of the crop field identification to season, makes to resist
Rust identification can be carried out at any time, greatly improve work efficiency.
Each embodiment is described by the way of progressive in this specification, the highlights of each of the examples are with other
The difference of embodiment, just to refer each other for identical similar portion between each embodiment.For device disclosed in embodiment
For, since it is corresponded to the methods disclosed in the examples, so description is fairly 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 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 range caused.
Claims (9)
1. a kind of preparation method of rest fungus uredospore for coffee rust resistance Detached-leaf test, which is characterized in that including as follows
Step：
1) preparation of inoculated and cultured box：Impregnate sponge 3~5 minutes with a concentration of 70~75% alcohol, after dry, be layered on plastics
Cassette bottom portion is cultivated, is soaked sponge with sterile water；
2) acquisition has the coffee incidence of leaf of typical coffee rest fungus scab, scrapes rest fungus uredospore, and by the rest fungus summer spore
Son is coated in the vacuum side of blade of susceptible variety Caturra or Matari, so that the rest fungus uredospore is uniformly distributed, and spray water mist extremely
There is moisture film generation；Then vacuum side of blade will be inoculated with upward, be placed on the sponge in the inoculated and cultured box, 20~25 DEG C of light cultures
40-50h, later light and shade alternate culture 35~40 days；
3) the single rest fungus uredium that will occur on the inoculation blade, is seeded on new Caturra or Matari blades,
Step 2) and step 3) 2~3 times are repeated, the rest fungus strain of purifying is obtained；
4) production spore blade is taken out, capsule is put into after the rest fungus uredospore is collected, in the drier that relative humidity is 45-55%
In 5 DEG C preservation.
2. a kind of rest fungus uredospore for coffee rust resistance Detached-leaf test according to claim 1, which is characterized in that
The blade of susceptible variety Caturra described in step 2) or Matari are just to be unfolded, the blade of the tender softness of children.
3. a kind of rest fungus uredospore for coffee rust resistance Detached-leaf test according to claim 1, which is characterized in that
It is to utilize to make the method that the rest fungus uredospore is evenly distributed on susceptible variety Caturra or Matari vacuum side of blade in step 2)
Writing brush is gently brushed.
4. a kind of rest fungus uredospore for coffee rust resistance Detached-leaf test according to claim 1, which is characterized in that
In step 2) during the light and shade alternate culture, sponge moistening is kept；The inoculation leaf back is blotted within 3-4 days after inoculation
Water is used in combination absorbent cotton to wipe the inoculation leaf back.
5. a kind of rest fungus uredospore for coffee rust resistance Detached-leaf test according to claim 1 is used for coffee germplasm
The in-vitro verification method of rust resistance, which is characterized in that include the following steps：
1) preparation of excised leaf：The blade for picking germplasm to be identified, cutting off petiole keeps blade back face-up, be put into prepare as
In inoculated and cultured box described in claim 1；
2) inoculated identification：The rest fungus uredospore as described in claim 1 prepared is coated on excised leaf, the rest fungus is made
Uredospore is uniformly distributed, and sprays water mist to there is moisture film generation, in artificial incubator, 20~25 DEG C, light culture 40-50h, it
It is cultivated 35~40 days in the environment of light and shade alternating afterwards, rear expert evidence scab type, size, the scab investigated produces spore situation.
6. a kind of in-vitro verification method of coffee germplasm rust resistance according to claim 5, which is characterized in that step 1)
Described in germplasm to be identified blade be the just tender softness of expansion children blade.
7. a kind of in-vitro verification method of coffee germplasm rust resistance according to claim 5, which is characterized in that step 2)
In make rest fungus uredospore be evenly distributed on the method on excised leaf be gently brushed with writing brush.
8. a kind of in-vitro verification method of coffee germplasm rust resistance according to claim 5, which is characterized in that step 2)
During the light and shade alternate culture, keep sponge moistening, and after inoculation blot within 3-4 days be inoculated with leaf back water, be used in combination
Absorbent cotton wipes the inoculation leaf back.
9. a kind of in-vitro verification method of coffee germplasm rust resistance according to claim 5, which is characterized in that by rust
Resistance is divided into 0-4 grades, and 0 grade of the resistance does not generate any scab for blade surface, and resistance type is immune；1 grade of the resistance
There are colour fading spot or tumor scab to occur for blade surface, resistance type is disease-resistant；2 grades of the resistance accounts for for the production spore area of leaf spot lesion
The 50% of leaf spot lesion area is hereinafter, resistance type is moderate resistance；3 grades of the resistance is more than 50% for the production spore area of leaf spot lesion
But it is less than 95%, resistance type is middle sense；4 grades of the resistance is more than 95% for the production spore area of scab, and resistance type is susceptible.
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