CN101750478B - In-vitro verification method for apple rot disease resistance - Google Patents
In-vitro verification method for apple rot disease resistance Download PDFInfo
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Abstract
The invention discloses an in-vitro verification method for apple rot disease resistance. The method comprises the following steps: (1) culturing conidiophores of Valsa mali Miyabe et Yamada at 20-30 DEG C for 6-24 hours to obtain the bourgeoned conidiophores; (2) inoculating the isolated branch with the bourgeoned conidiophores in step (1), and culturing at 20-30 DEG C under the relative humidity of 60-100%; and after culturing for 5-16 days, taking out the branches, and identifying the resistance. The verification method for apple rot disease resistance uses the cultured spores in the bourgeoning state to infect the branches according to the characteristics of plant disease resistance research development and rot disease occurrence. The invention conforms to the law of infection of Valsa mali Miyabe et Yamada, has the advantages of moderate pathogenicity and uniform disease occurrence. The method can verify the resistance of apple varieties to the rot disease, and has the advantages of convenience, high speed, simple and easy realization, reliable result and the like.
Description
Technical field
The present invention relates to a kind of in-vitro verification method of apple rot disease resistance.
Background technology
Canker of apple fruit is commonly called as bark rot, is one of important disease of northern China apple tree, and cause of disease Valsa maliMiyabe et Yamada claim apple black skin shell bacterium, belongs to the Ascomycotina fungi.Asexual generation is that Cytosporamandshurica Miura claims the dried apple slices shell capsule spore bacterium of rotting, and belongs to the Deuteromycotina fungi.This disease has latent infection and continuous characteristics such as outbreak, in case fall ill, pasts medical help, and causes that tree vigo(u)r is weak, limb is withered, dead tree, even ruins the garden.Therefore, identifying the apple rot disease resistance germplasm, cultivate resistant variety, is the Important Action of preventing and treating rot.The multiplex mycelia of apple rot disease resistance evaluation is at present cooked source of infection.Chen Ce (Chen Ce. the separation of pathogen, manually cultivate and inoculate [J]. fruit tree 1978, (5)) propose, with the isolated shoot of inoculated by hypha block through scalding, identify apple rot disease resistance, but this method has no and is applied to concrete practice.That Liu defends is medium (Liu Hanzhong, Ren Qingmian, Liu Linian. the anti-canker of apple fruit proterties investigation of the main germ plasm resource of apple tree [J]. Shanxi fruit tree, 1990, (02)) with the wild seeds of apple tree that inoculated by hypha block is being grown, identify 12 parts of resistance resources.Japanese scholars (Abe, K.; Kotoda, N.; Kato, H.; Soejima, (2007) Resistance sources to Valsacanker (Valsa ceratosperma) in a germplasm collection of diverse Malus species[J] PlantBreeding, 126, p449-453) mycelia that does with the system of collection, be made into mycelia suspending liquid, the inoculation isolated shoot, identify the disease resistance of the cultivars such as the wild seeds of apple tree and Fuji, gold hat, carbuncle, obtained four wild resources that resistance is relatively strong, and between cultivar resistance without obvious difference.Contain the enzyme of more toxin and degraded bark cell membrane due to mycelia, this inoculation method intoxicating power is large, and morbidity is difficult to differentiate the resistance of apple variety rapidly.By the practice of scab of apple resistance breeding and molecular labeling, everybody recognizes, identifies resistance resource and the resistant gene of cultivar, by breeding, gene is gathered, and is the important directions of resistance breeding from now on.Therefore, developing a kind of suitable Resistance Identification method, identify the resistance of different cultivars, cultivate resistant variety, is very necessary.Studies show that by model plant, disease resistance mechanism is the interactional result of disease fungus and plant host; A series of inducible factors that conidia germination, intrusion produce are to affect key factor (the JanA.L.van Kan that plant produces resistance, (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogenTRENDS in Plant Science.11,247-253).
Summary of the invention
The in-vitro verification method that the purpose of this invention is to provide a kind of apple rot disease resistance.
The in-vitro verification method of apple rot disease resistance provided by the present invention comprises the following steps:
(1) conidium of Valsa mali (Valsa mali Miyabe et Yamada) was cultivated 6-24 hour the conidium that obtains sprouting at 20-30 ℃;
The branch that the conidium inoculation of (2) sprouting with step (1) is exsomatized, at 20-30 ℃, relative humidity is to cultivate under the condition of 60-100%, cultivates and takes out branch after 5-16 days, carries out Resistance Identification.
Described branch also need be handled as follows before inoculation: branch is cleaned dried, punch on the branch that dries, hole depth is removed bark to xylem; The diameter in described hole is 4-10mm.
Wherein, described in step (1), the Valsa mali conidium is cultivated in the PDA fluid nutrient medium, and the initial concentration of described Valsa mali conidium in the PDA fluid nutrient medium can be 1 * 10
4-1 * 10
8The CFU/ milliliter.
Cultivate described in step (1) as concussion and cultivate, the rotating speed of concussion can be 100--300 rev/min.
The conidium that sprouts described in step (2) is mixed with suspending liquid and inoculates, and conidial concentration of sprouting in described suspending liquid is 1 * 10
4-1 * 10
8The CFU/ milliliter.
Canker of apple fruit of the present invention can betide Malus (Malus) fruit tree, as apple, Chinese pear-leaved crabapple, Malus spectabilis, fragrant fruit etc.
Described branch specifically can be apple branch; The diameter of described branch can be 0.1-4.0 centimetre.
The multiplex mycelia of apple rot disease resistance evaluation is at present cooked source of infection, but on the one hand because the mycelial growth state is inconsistent, causes the qualification result reliability not high; Be on the other hand have to infect method intoxicating power large, morbidity is difficult to differentiate the resistance of apple variety rapidly.Although doing source of infection with the canker of apple fruit conidium is more satisfactory vaccination ways, because conidia germination needs nutrition, and is difficult to support spore to infect morbidity only according to the nutrition that wound provides, become the restraining factors of using conidium to do source of infection.The present invention identifies that the method for apple rot disease resistance is the spore that is in germinating that adopts through cultivating, infect branch, not only meet the rule that rotten pathogenic bacteria infects, and pathogenicity is moderate, morbidity evenly can identify preferably that apple variety is to the resistance of rot.
The advantage of the inventive method is: according to disease resistance of plant progress and rot characteristics of incidence, with treated rotten pathogenic bacteria conidium, inoculate treated stripped branch, carry out apple rot disease resistance and identify, have easily and fast, the advantage such as simple, reliable results.Do source of infection with the conidium that sprouts, meet the rule that rotten pathogenic bacteria infects morbidity; Do the examination material with isolated shoot, effectively avoided doing with the growth fruit tree loss and the inconvenience that the examination material brings; The conidium quantity of inoculation is controlled, more can show the difference of apple variety resistance.
Description of drawings
Fig. 1 is the pycnidial picture of Valsa mali.
Fig. 2 is conidial picture that Valsa mali is sprouted.
Fig. 3 is the picture of morbidity and morbidity branch in embodiment 1, and figure left side be the picture of the individual 26-88 of high resistance, be the picture of Resistant Individuals 22-98 in figure, and scheming the right side is the picture that height is felt individual 22-117.
Embodiment
The rot disease resistance of embodiment 1, gold hat and carbuncle filial generation is identified and screening
1, the configuration of nutrient culture media
(1) preparation of PDA nutrient culture media
Formula: peeling potato: 200g
Sucrose (C
12H
22O
11, CHG/T3462-1999, Beijing Chemical Plant): 20g
Agar (DH0110-1.1, Beijing ancient cooking vessel state biotechnology Ltd): 15-20g
Distilled water (GB50172-92): 1000ml
Operation steps:
1) will remove the peel potato and weigh 200g, be cut into small pieces, put into beaker, add water 800ml, boil 30min, with gauze elimination potato and residue thereof, collect filtrate;
2) add respectively agar and sucrose heating and melting in filtrate, be settled to 1000ml with distilled water;
3) solution is divided install in Erlenmeyer flask, sealing, numbering;
4) will divide the solution that installs to be put in high-pressure steam sterilizing pan, 121 ℃ of high pressure steam sterilization 20min;
5) under aseptic condition, the nutrient culture media that 25ml melts to be poured in double dish, cooled and solidified is numbered stand-by.
(2) preparation of liquid PDA nutrient culture media
Formula: peeling potato: 200g
Glucose (HG/T3475-1999, Beijing chemical reagents corporation): 20g
Distilled water (GB50172-92): 1000ml
Operation steps:
1) will remove the peel potato and weigh 200g, be cut into small pieces, put into beaker, add water 800ml, boil 30min, with gauze elimination potato and residue thereof, collect filtrate;
2) add glucose in filtrate, heating and melting is settled to 1000ml with distilled water;
3) solution is divided install in Erlenmeyer flask, sealing, numbering;
4) will divide the solution that installs to be put in high-pressure steam sterilizing pan (YXQ-LS-18SIO, the sincere laboratory equipment of Southeast Region of Beijing instrument company limited), 121 ℃ of high pressure steam sterilization 20min, stand-by.
2, the conidial cultivation of Valsa mali (Valsa mali Miyabe et Yamada) ACCC 30052 is sprouted
1) Valsa mali (Valsa mali Miyabe et Yamada) ACCC 30052 is inoculated on the PDA nutrient culture media, put constant temperature illumination box (HPG-280BX, Harbin Donglian Electronic ﹠ Technology Development Co., Ltd.) in, 26 ℃ of illumination constant temperature culture 30 days;
2) pycnidia (seeing Fig. 1) with blackening takes off, and under aseptic condition, puts in the mortar of sterilization and grinds, and adds liquid PDA nutrient culture media;
3) liquid PDA nutrient culture media is changed in the 45ml centrifuge tube over to the rotating speed of 1500-3000 rev/min centrifugal 5 minutes;
4) get supernatant, change in the Erlenmeyer flask of sterilization, the suspending liquid that takes a morsel is counted conidium concentration with blood counting chamber (36XL Shanghai cosmos mountain precision optical instrument company limited) under 400 times, then use liquid PDA nutrient culture media with conidial concentration adjustment to 1 * 10 in suspending liquid
6The CFU/ milliliter;
5) suspending liquid is put in 26 ℃ of constant-temperature shaking incubators (BS-1EA Jintan City Jie Ruier Electrical Appliances Co., Ltd), cultivated 12 hours with the rotating speed of 100-300 rev/min of concussion, make conidia germination (seeing Fig. 2);
6) will through the suspending liquid cultivation with 1500-3000 rev/min centrifugal 10 minutes, remove supernatant, with the resuspended conidium of distilled water, spore concentration is adjusted to 1 * 10
6The CFU/ milliliter.
3, inoculation, cultivation and Resistance Identification
1) gold hat (Unified number: PGB0196) with carbuncle (Unified number: PGB0057) in hybridization in 2003, be colonizated in breeding nursery after the seed germination that obtains.Winter in 2008, choose 68 strain seedling trees of gold hat and carbuncle hybridization, every strain tree is got three of the dormancy apple branch of 1 centimetre of diameter, be cut into the length of 20 centimetres, wash and dry, make a call to three holes with the distance that card punch (Jiangdu three glass apparatus factory) interval equates, the diameter in hole is 10mm, hole depth is removed bark to xylem, and three repetitions are established in test;
2) be 1 * 10 with 40 μ l concentration
6The spore suspension of CFU/ milliliter adds each to set in the hole with pipettor, after solution absorbs fully, branch is wrapped up moisturizing (relative humidity is greater than 60%) with wet towel, put into porcelain dish, 26 ℃ of constant temperature culture in incubator (HPG-280BX Harbin Donglian Electronic ﹠ Technology Development Co., Ltd.);
3) cultivation was taken out branch after seven days, measured the scab extension length, divided the resistance grade;
Disease resistance classification: divide by vaccination scab spread scenarios
High resistance is not fallen ill;
Anti-morbidity, but not expansion;
In anti-spreading coefficient less than 0.25;
Middle sense spreading coefficient is less than 0.5;
The sense spreading coefficient is less than 0.75;
High sense spreading coefficient is more than or equal to 0.75 or equal 1;
The individual average extension length of sense of spreading coefficient=average extension length/;
The resistance classification results:
According to the disease resistance classification, test in 68 selected strain seedling trees to obtain individual 4 strains of high resistance, anti-individual 6 strains, in anti-individual 18 strains, susceptible individual 12 strains, individual 17 strains of middle sense, high sense individual 11 strains (seeing Table 1).
The data of table 1 show, after a certain amount of treated conidium of inoculation, through 7 days, gold hat and carbuncle filial generation rot disease resistance difference were obvious, can distinguish preferably anti-, sense is individual, for Resistant Individuals screens, molecular labeling is laid a good foundation.This experiment triplicate, favorable reproducibility as a result.
Fig. 3 is the picture of morbidity and morbidity branch in embodiment, and wherein, the individual 26-88 branch of high resistance downright bad phenomenon do not occur around the individual inoculation of high resistance hole as seen from the figure, and grows callus as shown in Fig. 3 left side; The branch of Resistant Individuals 22-98 as shown in Figure 3, Resistant Individuals scab expansion rate is slower as seen from the figure, occurs slough around the hole in inoculation, bark rots, slough and the difference of morbidity part are obvious; The individual 22-117 branch of high sense as shown in Fig. 3 right side, as seen from the figure the individual morbidity of high sense rapidly, slough namely extended to full branch in 7 days, bark is rotten.And the individual 22-117 branch of high sense has obvious vinasse flavor.
Table 1 scab extension length and resistance classification
| Individual number | Total extension length (cm) | Average extension length (cm) | Spreading coefficient | The resistance classification |
| 26--88 | 0 | 0 | 0 | High resistance |
| 25--15 | 0 | 0 | 0 | High resistance |
| 37--56 | 0 | 0 | 0 | High resistance |
| 22--98 | 0 | 0 | 0 | High resistance |
| 42--114 | 0.5 | 0.17 | 0.01 | Anti- |
| 45--109 | 0.6 | 0.2 | 0.01 | Anti- |
| 40--123 | 0.8 | 0.27 | 0.02 | Anti- |
| 22--33 | 0.9 | 0.3 | 0.02 | Anti- |
| 38--102 | 4 | 1.33 | 0.08 | Anti- |
| 44--115 | 4.2 | 1.4 | 0.08 | Anti- |
| 40--99 | 4.9 | 1.6 | 0.1 | In anti- |
| 43--99 | 5 | 1.7 | 0.11 | In anti- |
| 41--94 | 5.8 | 1.9 | 0.12 | In anti- |
| 39--81 | 6 | 2 | 0.13 | In anti- |
| 40--89 | 6.9 | 2.3 | 0.15 | In anti- |
| 22--28 | 7 | 2.3 | 0.15 | In anti- |
| 22--61 | 7.8 | 2.6 | 0.17 | In anti- |
| 44--119 | 7.9 | 2.6 | 0.17 | In anti- |
| 37--88 | 8 | 2.7 | 0.17 | In anti- |
| 40--124 | 8.6 | 2.9 | 0.18 | In anti- |
| 39-102 | 9 | 3 | 0.19 | In anti- |
| 40--91 | 9.2 | 3.1 | 0.2 | In anti- |
| 38--83 | 9.9 | 3.3 | 0.21 | In anti- |
| 43--98 | 10 | 3.3 | 0.21 | In anti- |
| 40--87 | 10.2 | 3.4 | 0.22 | In anti- |
| 40--88 | 11 | 3.7 | 0.24 | In anti- |
| 42--81 | 11.2 | 3.7 | 0.24 | In anti- |
| 39--75 | 12 | 4 | 0.25 | In anti- |
| 22--47 | 12.2 | 4.1 | 0.26 | Sense |
| 39-121 | 13.3 | 4.4 | 0.28 | Sense |
| 22--40 | 14.5 | 4.8 | 0.31 | Sense |
| 38--73 | 15 | 5 | 0.32 | Sense |
| 42--84 | 15.8 | 5.3 | 0.34 | Sense |
| 44--132 | 16 | 5.3 | 0.34 | Sense |
| 45--90 | 16.6 | 5.5 | 0.35 | Sense |
| 40--116 | 17.6 | 5.9 | 0.38 | Sense |
| 43--100 | 18.9 | 6.3 | 0.4 | Sense |
| 38--76 | 19 | 6.3 | 0.4 | Sense |
| 38--88 | 20.3 | 6.8 | 0.43 | Sense |
| 22--95 | 20.5 | 6.8 | 0.43 | Sense |
| 45--110 | 24 | 8 | 0.51 | Middle sense |
| 22--87 | 24.2 | 8 | 0.51 | Middle sense |
| 38--109 | 25 | 8.3 | 0.53 | Middle sense |
| 43--105 | 26 | 8.7 | 0.55 | Middle sense |
| 39--130 | 27.4 | 9.1 | 0.58 | Middle sense |
| 39--71 | 27.8 | 9.3 | 0.59 | Middle sense |
| 43--102 | 28.1 | 9.4 | 0.6 | Middle sense |
| 43--83 | 29.4 | 9.8 | 0.62 | Middle sense |
| 46--123 | 29.8 | 9.93 | 0.63 | Middle sense |
| 43--88 | 30 | 10 | 0.64 | Middle sense |
| 37--83 | 30.5 | 10.2 | 0.65 | Middle sense |
| 45--99 | 31 | 10.3 | 0.66 | Middle sense |
| 40--85 | 31.1 | 10.4 | 0.66 | Middle sense |
| 37--117 | 32.8 | 10.9 | 0.69 | Middle sense |
| 37--75 | 33.6 | 11.2 | 0.72 | Middle sense |
| 37--136 | 34 | 11.3 | 0.72 | Middle sense |
| 22--93 | 34 | 11.3 | 0.72 | Middle sense |
| 22-96 | 36.3 | 12.1 | 0.77 | High sense |
| 41--81 | 37.1 | 12.4 | 0.8 | High sense |
| 39--80 | 37.5 | 12.5 | 0.8 | High sense |
| 38--122 | 38 | 12.7 | 0.81 | High sense |
| 37--134 | 38.6 | 12.9 | 0.82 | High sense |
| 38--105 | 39 | 13 | 0.83 | High sense |
| 22--97 | 39.8 | 13.3 | 0.85 | High sense |
| 22--67 | 40.7 | 13.6 | 0.87 | High sense |
| 37--66 | 41.8 | 13.9 | 0.89 | High sense |
| 41--115 | 42.1 | 14.3 | 0.91 | High sense |
| 22--117 | 47.2 | 15.7 | 1 | High sense |
Claims (8)
1. the in-vitro verification method of apple rot disease resistance comprises the following steps:
(1) conidium of Valsa mali (Valsa mali Miyabe et Yamada) was cultivated 6-24 hour the conidium that obtains sprouting at 20-30 ℃;
The branch that the conidium inoculation of (2) sprouting with step (1) is exsomatized is to cultivate under the condition of 60-100% at 20-30 ℃, relative humidity, cultivates and takes out branch after 5-16 days, carries out Resistance Identification;
The conidium that sprouts described in step (2) is mixed with suspending liquid and inoculates, and conidial concentration of sprouting in described suspending liquid is 1 * 10
4-1 * 10
8The CFU/ milliliter.
2. method according to claim 1, it is characterized in that: described branch is handled as follows before inoculation: branch is cleaned dried, punch on the branch that dries, hole depth is removed bark to xylem; The diameter in described hole is 4-10mm.
3. method according to claim 1, it is characterized in that: described in step (1), the conidium of Valsa mali is cultivated in liquid PDA nutrient culture media, and the initial concentration of the conidium of described Valsa mali in liquid PDA nutrient culture media is 1 * 10
4-1 * 10
8The CFU/ milliliter.
4. method according to claim 2, it is characterized in that: described in step (1), the conidium of Valsa mali is cultivated in liquid PDA nutrient culture media, and the initial concentration of the conidium of described Valsa mali in liquid PDA nutrient culture media is 1 * 10
4-1 * 10
8The CFU/ milliliter.
5. method according to claim 3 is characterized in that: cultivate described in step (1) as concussion and cultivate, the rotating speed of concussion is 100-300 rev/min.
6. method according to claim 4 is characterized in that: cultivate described in step (1) as concussion and cultivate, the rotating speed of concussion is 100-300 rev/min.
7. arbitrary described method according to claim 1-6, it is characterized in that: described branch is apple branch.
8. method according to claim 7, it is characterized in that: the diameter of described branch is 0.1-4.0 centimetre.
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| CN107517726A (en) * | 2017-10-14 | 2017-12-29 | 中国科学院新疆生态与地理研究所 | A kind of fast quick access bacterium of canker of apple fruit pathogen and the method for pathogenicity Rapid identification |
| CN109988808A (en) * | 2019-03-20 | 2019-07-09 | 新疆农业大学 | A kind of method of the in vitro water culture measurement erwinia amylovora pathogenicity of bergamot pear branch |
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