CN101750478A - In-vitro verification method for apple rot disease resistance - Google Patents

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

A kind of in-vitro verification method of apple rot disease resistance

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 claims the dried apple slices shell capsule spore bacterium of rotting for Cytosporamandshurica Miura, 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 behave that prevents and treats rot.Apple rot disease resistance identifies how to do source of infection with mycelia at present.Chen Ce (Chen Ce. the separation of pathogen, ARTIFICIAL CULTURE and inoculation [J]. Chinese fruit tree 1978, (5)) propose, with the isolated shoot of inoculated by hypha block, identify apple rot disease resistance, but this method is not seen and is applied to concrete practice through scalding.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.Japan scholar (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 cultivars such as wild seeds of apple tree and Fuji, gold hat, carbuncle, obtained four resistances stronger wild resource relatively, and resistance there is not evident difference between the cultivar.Because mycelia contains the enzyme of more toxin and degraded bark cell membrane, this inoculation method intoxicating power is big, 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 authentication method, identify the resistance of different cultivars, cultivate resistant variety, is very necessary.Studies show that by model plant disease resistance mechanism is disease fungus and plant host results of interaction; A series of inducible factors that conidia germination, intrusion are produced are to influence 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 may further comprise the steps:

(1) conidium of Valsa mali (Valsa mali Miyabe et Yamada) was cultivated the conidium that obtains sprouting 6-24 hour at 20-30 ℃;

(2) branch that exsomatizes of the conidium inoculation of sprouting with step (1), 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 the resistance evaluation.

Described branch also need carry out following processing 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, the Valsa mali conidium is cultivated in the PDA fluid nutrient medium described in the step (1), 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 the step (1) to concussion and cultivate, the rotating speed of concussion can be 100--300 rev/min.

The conidium that sprouts described in the step (2) is mixed with suspending liquid and inoculates, and conidial concentration of sprouting in the 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.

Apple rot disease resistance identifies how to do source of infection with mycelia at present, 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 big, morbidity is difficult to differentiate the resistance of apple variety rapidly.Though doing source of infection with the canker of apple fruit conidium is more satisfactory vaccination ways, because conidia germination needs nutrition, and be 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 be identified the resistance of apple variety to rot preferably.

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, 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 loss and the inconvenience that the examination material brings with the growth fruit tree; The conidium controllable number of inoculation more can show the difference of apple variety resistance.

Description of drawings

Fig. 1 is the pycnidial picture of Valsa mali.

Conidial picture that Fig. 2 sprouts for Valsa mali.

Fig. 3 is the picture of morbidity and morbidity branch among the embodiment 1, and a figure left side be the picture of high anti-individual 26-88, be the picture of the individual 22-98 of resistance among the 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) PDA culture medium preparation

Prescription: 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,, collect filtrate with gauze elimination potato and residue thereof;

2) in filtrate, add agar and sucrose heating and melting respectively, be settled to 1000ml with distilled water;

3) the solution branch is installed in the Erlenmeyer flask sealing, numbering;

4) solution that branch is installed is put in the 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 the double dish, cooled and solidified is numbered stand-by.

(2) liquid PDA culture medium preparation

Prescription: 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,, collect filtrate with gauze elimination potato and residue thereof;

2) add glucose in filtrate, heating and melting is settled to 1000ml with distilled water;

3) the solution branch is installed in the Erlenmeyer flask sealing, numbering;

4) solution that branch is installed is put in the high-pressure steam sterilizing pan (YXQ-LS-18SI0, the southeast, Beijing sincere laboratory equipment of instrument company limited), and 121 ℃ of high pressure steam sterilization 20min are stand-by.

2, Valsa mali (Valsa mali Miyabe et Yamada) ACCC 30052 conidial cultivations are 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) the pycnidia (see figure 1) of blackening is taken off, under aseptic condition, put in the mortar of sterilization and grind, add liquid PDA nutrient culture media;

3) liquid PDA nutrient culture media is changed in the 45ml centrifuge tube over to centrifugal 5 minutes with 1500-3000 rev/min rotating speed;

4) get supernatant, change in the Erlenmeyer flask of sterilization, the suspending liquid that takes a morsel 400 times of counting conidium concentration down, uses liquid PDA nutrient culture media with conidial concentration adjustment to 1 * 10 in the suspending liquid with blood counting chamber (cosmos mountain, 36XL Shanghai precision optical instrument company limited) then ⁶The CFU/ milliliter;

5) suspending liquid is put in 26 ℃ of constant-temperature shaking culture casees (BS-1EA Jintan City Jie Ruier Electrical Appliances Co., Ltd), cultivated 12 hours, make the conidia germination (see figure 2) with the rotating speed of 100-300 rev/min of concussion;

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 are identified

1) gold hat (unified numbering: PGB0196) with carbuncle (unified numbering: PGB0057), be colonizated in breeding nursery behind the seed germination that obtains in hybridization in 2003.Winter in 2008, choose 68 strain seedling trees of gold hat and carbuncle hybridization, three of the about 1 centimetre dormancy apple branch in cut-off footpath are set in every strain, be cut into 20 centimetres length, wash and dry, make a call to three holes with the distance that card punch (Jiangdu three glass apparatus factories) equates at interval, 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 in each tree hole with pipettor, after treating that solution absorbs fully, branch is preserved moisture (relative humidity is greater than 60%) with the wet towel parcel, 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 inoculation point scab spread scenarios

High anti-not morbidity;

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 the 68 selected strain seedling trees to obtain high anti-individual 4 strains, 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 that behind 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 anti-, sense individuality preferably, for the individual screening of resistance, molecular labeling are laid a good foundation.This experiment triplicate, favorable reproducibility as a result.

Fig. 3 is the picture of morbidity and morbidity branch among the embodiment, and wherein, high anti-individual 26-88 branch is shown in Fig. 3 left side, and high as seen from the figure anti-individual inoculation downright bad phenomenon do not occur around the hole, and grows callus; The branch of the individual 22-98 of resistance as shown in Figure 3, the individual scab expansion rate of resistance is slower as seen from the figure, slough occurs around the inoculation hole, bark rots, slough and the difference of morbidity part are obvious; The individual 22-117 branch of high sense is shown in Fig. 3 right side, and the individual morbidity of high as seen from the figure sense is rapid, and slough promptly extended to full branch in 7 days, and bark rots.And the individual 22-117 branch of high sense has tangible 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 anti-
??25--15	??0	??0	??0	High anti-
					??37--56	??0	??0	??0	High anti-
??22--98	??0	??0	??0	High anti-
					??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-

Individual number	Total extension length (cm)	Average extension length (cm)	Spreading coefficient	The resistance classification
					??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-

Individual number	Total extension length (cm)	Average extension length (cm)	Spreading coefficient	The resistance classification
					??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

Individual number	Total extension length (cm)	Average extension length (cm)	Spreading coefficient	The resistance classification
					??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

Individual number	Total extension length (cm)	Average extension length (cm)	Spreading coefficient	The resistance classification
					??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 may further comprise the steps:

(1) conidium of Valsa mali (Valsa mali Miyabe et Yamada) was cultivated the conidium that obtains sprouting 6-24 hour at 20-30 ℃;

(2) branch that exsomatizes of the conidium inoculation of sprouting with step (1) 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 the resistance evaluation.

2. method according to claim 1 is characterized in that: described branch carries out following processing 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 and 2, it is characterized in that: the conidium of Valsa mali is cultivated in liquid PDA nutrient culture media described in the step (1), 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 3 is characterized in that: cultivate described in the step (1) to concussion and cultivate, the rotating speed of concussion is 100-300 rev/min.

5. method according to claim 1 and 2 is characterized in that: the conidium that sprouts described in the step (2) is mixed with suspending liquid and inoculates, and conidial concentration of sprouting in the described suspending liquid is 1 * 10 ⁴-1 * 10 ⁸The CFU/ milliliter.

6. method according to claim 3 is characterized in that: the conidium that sprouts described in the step (2) is mixed with suspending liquid and inoculates, and conidial concentration of sprouting in the described suspending liquid is 1 * 10 ⁴-1 * 10 ⁸The CFU/ milliliter.

7. according to arbitrary described method among the claim 1-6, it is characterized in that: described branch is an apple branch.

8. method according to claim 7 is characterized in that: the diameter of described branch is 0.1-4.0 centimetre.

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