CN105766573A - Method for inoculating wound tobacco root system with phytophthora nicotianae - Google Patents
Method for inoculating wound tobacco root system with phytophthora nicotianae Download PDFInfo
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Abstract
The invention discloses a method for inoculating a wound tobacco root system with phytophthora nicotianae. The method comprises the following steps of 1, first-stage seedling culture, 2, second-stage seedling culture, 3, preparation of a phytophthora nicotianae zoospore suspension solution, 4, root wound soaking inoculation, 5, disease resistance identification. According to the method, disease resistance identification can be performed on a large number of tobacco cultivars, the disease resistance identification efficiency can be effectively improved, and the method is particularly suitable for screening out disease resistance target varieties from a huge number of genetic resources; seedling transplanting and inoculation are convenient, personal errors among different operators are reduced, meanwhile, the identification time is shortened, labor cost is saved, the occupied space is small, and management is easy; meanwhile, the tobacco seedling disease resistance identification result can accurately reflect the disease resistance of tobacco seedlings in a field.
Description
Technical field
The invention belongs to tobacco disease resistance breeding technical field, the method being specifically related to a kind of Phytophthora nicotianae inoculation wound tobacco root system.
Background technology
Black shank be by Phytophthora nicotianae (Phytophthoranicotianae) infect that Tobacco Root stem causes a kind of widely distributed, endanger serious tobacco diseases, all having in each main Chan Yan district of China and occur in various degree, controlling the most economical effective method of this disease is select disease-resistant variety.
The Disease Resistance Identification carrying out kind is typically required for finding disease-resistant variety.Generally, the Disease Resistance Identification of tobacco bred is many inoculates Phytophthora nicotianae with tobacco seedlings root or rhizome portion for object, then the incidence adding up tobacco seedlings draws tobacco seedlings disease resistance, such as apply more indoor bacterium paddy and inoculate disease-resistant evaluation (GB/T23224), but this method is transplanting tobacco seedlings, after cultivation survives, need every strain tobacco seedlings is quantitatively inoculated, moisturizing is cultivated again, especially tobacco seedlings is various in style, can cause when quantity is big that workload is very big, and take a lot of space just can complete, therefore this method is only applicable to novel wheat cultivars or the Disease Resistance Identification of a small amount of kind；Also have is exactly wash root leaching root inocalation method, first seedling is washed root, again seedling is fixed on floating disc, then the inoculation of leaching root carries out Disease Resistance Identification again, but it is too big and be difficult to control to wash injury that tobacco root system causes by root in this method, during inoculation, the surface area of tobacco seedlings root contact zoospore is big, causes that morbidity is often laid particular stress on, can not reflect tobacco seedlings field resistance.
For this, research and development one can identify a large amount of tobacco seedlings kind simultaneously, and workload is little, efficiency is high, and the method that can accurately reflect the wound tobacco root system inoculation Phytophthora nicotianae of tobacco seedlings field resistance is the key solving the problems referred to above.
Summary of the invention
The method that it is an object of the invention to provide a kind of Phytophthora nicotianae inoculation wound tobacco root system.
The object of the present invention is achieved like this, including (1) first stage nursery, (2) second stage nursery, the preparation of (3) Phytophthora nicotianae zoospore suspension, the leaching root inoculation of (4) root wound, (5) Disease Resistance Identification, specifically comprises the following steps that
(1) first stage nursery: tobacco seed is sowed in the first floating seedling tray of carrying substrates nursery 30 ~ 40 days, then forge Seedling 2 ~ 3 days, then pick out the seedling of neat and consistent；
(2) second stage nursery: seedling step (1) picked out is transplanted to without in the second floating seedling tray of substrate together with substrate, nursery 10 ~ 20 days, seedling culture to fibrous root is entered floating pool through the second floating disc, then weak Seedling is rejected, the tobacco seedlings that screening fibrous root growing way is consistent, carry out leaf-cutting again, obtain fibrous root tobacco seedlings, in order to inoculation；
(3) preparation of Phytophthora nicotianae zoospore suspension: Phytophthora nicotianae is seeded to OA culture medium flat plate, at 25 ~ 28 DEG C, dark culturing covers with whole flat board to mycelia in 7 ~ 10 days, take mycelia to be cut into small pieces and obtain mycelia block, mycelia block produces zoosporangium through liquid culture induction, then promote zoosporangium release zoospore through K cryogenic treatment, then regulate zoospore concentration to 10 with sterilized water4~6Individual/ml, obtains Phytophthora nicotianae zoospore suspension；
(4) root wound leaching root inoculation: the fibrous root tobacco seedlings that step (2) obtains is taken out dewatering 1 ~ 2 day together with the second floating seedling tray, strike off the fibrous root that the second floating seedlings tray bottom exposes again, the Phytophthora nicotianae zoospore suspension that step (3) obtains is inserted in tray, then the second seedlings nursing plate is dipped in tray, under 12h illumination 12h dark alternation condition, in tray, tobacco seedlings is maintained inoculation 24 ~ 36 hours；
(5) Disease Resistance Identification: postvaccinal tobacco seedlings continues floating cultivation, the sickness rate a of tobacco seedlings balck shank of investigation in every 7 days, investigate 3 ~ 4 times altogether, carry out statistics using the highest morbidity survey result as the state of an illness and divide anti-perception, sickness rate a=0 is immunity or high resistance kind, sickness rate 0 < a≤25% is disease-resistant variety, sickness rate 25% < a≤45% be in anti-kind, sickness rate 45% < a≤65% is middle sense kind, sickness rate 65% < a≤80% is susceptible variety, sickness rate 80% < a≤100% is high sense kind, wherein immunity or high resistance, disease-resistant, in anti-kind be resistant material.
Compared with prior art, the method have the advantages that
1, the floating seedlings of the present invention multiple tobacco seedlings kind of nursery simultaneously, inoculation is immersed after wound tobacco root system, carry out again identifying after cultivation is observed, the tobacco bred Disease Resistance Identification of extensive quantity can be realized, it is effectively improved Disease Resistance Identification efficiency, is particularly suitable for from the germ plasm resource of substantial amounts, screen disease-resistant target kind；
2, the present invention adopts two-part floating seedlings and tray inoculation, transplants seedlings, inoculates conveniently, decrease the personal error between different operating personnel, save qualification time simultaneously, save manual labor's cost, and it is little to take up room, it is easy to management；
3, the wound amount that tobacco seedlings root is caused by the technology scraping root process in the present invention is stable, and during inoculation, the surface area of tobacco seedlings root contact zoospore is stable and consistent, and qualification result can accurately reflect the disease resistance in tobacco seedlings field；
4, tobacco seedlings is being soaked root and is quantitatively being inoculated by the tray of the present invention, and infection processs is closer to field natural infection, and disease resistance selection pressure is suitable, and Disease Resistance Identification result can accurately reflect the resistance against diseases in tobacco seedlings field.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further illustrated, but never in any form the present invention is any limitation as, and based on present invention teach that any conversion or replacement made, belongs to protection scope of the present invention.
A kind of method of Phytophthora nicotianae inoculation wound tobacco root system, including (1) first stage nursery, (2) second stage nursery, the preparation of (3) Phytophthora nicotianae zoospore suspension, the leaching root inoculation of (4) root wound, (5) Disease Resistance Identification, specifically comprise the following steps that
(1) first stage nursery: tobacco seed is sowed in the first floating seedling tray of carrying substrates nursery 30 ~ 40 days, then forge Seedling 2 ~ 3 days, then pick out the seedling of neat and consistent；
(2) second stage nursery: seedling step (1) picked out is transplanted to without in the second floating seedling tray of substrate together with substrate, nursery 10 ~ 20 days, seedling culture to fibrous root is entered floating pool through the second floating disc, then reject weak Seedling, screen the tobacco seedlings that fibrous root growing way is consistent, carry out leaf-cutting again, obtain fibrous root tobacco seedlings, in order to inoculation；
(3) preparation of Phytophthora nicotianae zoospore suspension: Phytophthora nicotianae is seeded to OA culture medium flat plate, at 25 ~ 28 DEG C, dark culturing covers with whole flat board to mycelia in 7 ~ 10 days, take mycelia to be cut into small pieces and obtain mycelia block, mycelia block produces zoosporangium through liquid culture induction, then promote zoosporangium release zoospore through K cryogenic treatment, then regulate zoospore concentration to 10 with sterilized water4~6Individual/ml, obtains Phytophthora nicotianae zoospore suspension；
(4) root wound leaching root inoculation: the fibrous root tobacco seedlings that step (2) obtains is taken out dewatering 1 ~ 2 day together with the second floating seedling tray, strike off the fibrous root that the second floating seedlings tray bottom exposes again, the Phytophthora nicotianae zoospore suspension that step (3) obtains is inserted in tray, then the second seedlings nursing plate is dipped in tray, under 12h illumination 12h dark alternation condition, in tray, tobacco seedlings is maintained inoculation 24 ~ 36 hours；
(5) Disease Resistance Identification: postvaccinal tobacco seedlings continues floating cultivation, the sickness rate a of tobacco seedlings balck shank of investigation in every 7 days, investigate 3 ~ 4 times altogether, carry out statistics using the highest morbidity survey result as the state of an illness and divide anti-perception, sickness rate a=0 is immunity or high resistance kind, sickness rate 0 < a≤25% is disease-resistant variety, sickness rate 25% < a≤45% be in anti-kind, sickness rate 45% < a≤65% is middle sense kind, sickness rate 65% < a≤80% is susceptible variety, sickness rate 80% < a≤100% is high sense kind, wherein immunity or high resistance, disease-resistant, in anti-kind be resistant material.
The tobacco seedlings that screening fibrous root growing way is consistent in described step (2) is have 8 ~ 16 through the fibrous root quantity of floating disc, fibrous root length 4 ~ 5 centimetres, and in pure white.
The first described floating seedling tray is 300 ~ 400 hole floating seedling trays, and the second floating seedling tray is 100 ~ 200 hole floating seedling trays, and the area equation of the area of the first floating seedling tray and the second floating seedling tray.
After the tobacco seedlings that in described step (2), screening fibrous root growing way is consistent, quantity at least 12 strain of each kind tobacco seedlings in second floating disc, and the quantity of the second floating disc is no less than 3, namely the total quantity of each kind tobacco seedlings is no less than 36 strains.
In described step (4), the water level of tray is 1 ~ 2cm, and temperature is 25 ~ 28 DEG C, relative humidity more than 75%.
In described step (2), leaf-cutting is to wipe out along tobacco seedlings blade tip to launch the 1/3 ~ 1/2 of blade, then cuts off the 2-4 sheet Huang leaf of cigarette strain bottom and whole leaves of Lao Ye and petiole.
In described step (3), liquid culture is to be joined by mycelia block in the culture dish of 10%V8 juice fluid medium, every ware adds mycelia block 20 ~ 40, continue at dark culturing 3 ~ 4 days at 25 ~ 28 DEG C, incline culture fluid, rinse 3 ~ 5 times with aquesterilisa again, then aquesterilisa is changed every day 1 ~ 3 time, the generation of continuous 2 ~ 3 days induction zoosporangiums.
In described step (3), K cryogenic treatment is when mycelia block zoosporangium is 104/cm2Time more than individual, in 5 ~ 8 DEG C of K cryogenic treatment 20 ~ 30min, take out and be placed in room temperature 15 ~ 30min induction zoosporangium release zoospore.
In described step (5), the temperature of floating cultivation is 22 ~ 30 DEG C.
In described step (1), forging Seedling is to be taken out from floating pool by the first floating disc, ventilation dewatering 2 ~ 3 days.
Below in conjunction with specific embodiment, the invention will be further described.
In embodiment 1 ~ 5, Phytophthora nicotianae is No. 0 biological strain (People's Republic of China (PRC) tobacco business standard YC/T39-1996).
Investigation is to investigate all inoculation tobacco seedlings in units of strain, and wherein rhizome portion blackening, tobacco seedlings be wilting is denoted as morbidity, and the invariant color of rhizome portion, tobacco seedlings are upright, growth is normally denoted as and does not fall ill.
Embodiment 1
(1) first stage nursery: tobacco seed is sowed in the first floating seedling tray of carrying substrates nursery 30 days, then forge Seedling 2 days, then pick out the seedling of neat and consistent；
(2) second stage nursery: seedling step (1) picked out is transplanted to without in the second floating seedling tray of substrate together with substrate, nursery 10 days, seedling culture to fibrous root is entered floating pool through the second floating disc, then reject weak Seedling, screen the tobacco seedlings that fibrous root growing way is consistent, carry out leaf-cutting again, obtain fibrous root tobacco seedlings, in order to inoculation；
(3) preparation of Phytophthora nicotianae zoospore suspension: Phytophthora nicotianae is seeded to OA culture medium flat plate, at 25 DEG C, dark culturing covers with whole flat board to mycelia in 7 days, take mycelia to be cut into small pieces and obtain mycelia block, mycelia block produces zoosporangium through liquid culture induction, then promote zoosporangium release zoospore through K cryogenic treatment, then regulate zoospore concentration to 10 with sterilized water4~6Individual/ml, obtains Phytophthora nicotianae zoospore suspension；
(4) root wound leaching root inoculation: the fibrous root tobacco seedlings that step (2) obtains is taken out dewatering 1 day together with the second floating seedling tray, strike off the fibrous root that the second floating seedlings tray bottom exposes again, the Phytophthora nicotianae zoospore suspension that step (3) obtains is inserted in tray, then the second seedlings nursing plate is dipped in tray, under 12h illumination 12h dark alternation condition, in tray, tobacco seedlings is maintained inoculation 24 hours；
(5) Disease Resistance Identification: postvaccinal tobacco seedlings continues floating cultivation, the sickness rate a of tobacco seedlings balck shank of investigation in every 7 days, investigation is to investigate all inoculation tobacco seedlings in units of strain, investigation 4 times altogether, carry out statistics using the highest morbidity survey result as the state of an illness and divide anti-perception, sickness rate a=0 is immunity or high resistance kind, sickness rate 0 < a≤25% is disease-resistant variety, sickness rate 25% < a≤45% be in anti-kind, sickness rate 45% < a≤65% is middle sense kind, sickness rate 65% < a≤80% is susceptible variety, sickness rate 80% < a≤100% is high sense kind, wherein immunity or high resistance, disease-resistant, in anti-kind be resistant material.
Embodiment 2
(1) first stage nursery: tobacco seed is sowed in the first floating seedling tray of carrying substrates nursery 40 days, then forge Seedling 3 days, then pick out the seedling of neat and consistent；
(2) second stage nursery: seedling step (1) picked out is transplanted to without in the second floating seedling tray of substrate together with substrate, nursery 20 days, seedling culture to fibrous root is entered floating pool through the second floating disc, then reject weak Seedling, screen the tobacco seedlings that fibrous root growing way is consistent, carry out leaf-cutting again, obtain fibrous root tobacco seedlings, in order to inoculation；
(3) preparation of Phytophthora nicotianae zoospore suspension: Phytophthora nicotianae is seeded to OA culture medium flat plate, at 28 DEG C, dark culturing covers with whole flat board to mycelia in 10 days, take mycelia to be cut into small pieces and obtain mycelia block, mycelia block produces zoosporangium through liquid culture induction, then promote zoosporangium release zoospore through K cryogenic treatment, then regulate zoospore concentration to 10 with sterilized water4~6Individual/ml, obtains Phytophthora nicotianae zoospore suspension；
(4) root wound leaching root inoculation: the fibrous root tobacco seedlings that step (2) obtains is taken out dewatering 2 days together with the second floating seedling tray, strike off the fibrous root that the second floating seedlings tray bottom exposes again, the Phytophthora nicotianae zoospore suspension that step (3) obtains is inserted in tray, then the second seedlings nursing plate is dipped in tray, under 12h illumination 12h dark alternation condition, in tray, tobacco seedlings is maintained inoculation 36 hours；
(5) Disease Resistance Identification: postvaccinal tobacco seedlings continues floating cultivation, the sickness rate a of tobacco seedlings balck shank of investigation in every 7 days, investigation is to investigate all inoculation tobacco seedlings in units of strain, investigation 4 times altogether, carry out statistics using the highest morbidity survey result as the state of an illness and divide anti-perception, sickness rate a=0 is immunity or high resistance kind, sickness rate 0 < a≤25% is disease-resistant variety, sickness rate 25% < a≤45% be in anti-kind, sickness rate 45% < a≤65% is middle sense kind, sickness rate 65% < a≤80% is susceptible variety, sickness rate 80% < a≤100% is high sense kind, wherein immunity or high resistance, disease-resistant, in anti-kind be resistant material.
Embodiment 3
(1) first stage nursery: tobacco seed is sowed in the first floating seedling tray of carrying substrates nursery 35 days, then forge Seedling 2.5 days, then pick out the seedling of neat and consistent；
(2) second stage nursery: seedling step (1) picked out is transplanted to without in the second floating seedling tray of substrate together with substrate, nursery 15 days, seedling culture to fibrous root is entered floating pool through the second floating disc, then reject weak Seedling, screen the tobacco seedlings that fibrous root growing way is consistent, carry out leaf-cutting again, obtain fibrous root tobacco seedlings, in order to inoculation；
(3) preparation of Phytophthora nicotianae zoospore suspension: Phytophthora nicotianae is seeded to OA culture medium flat plate, at 26 DEG C, dark culturing covers with whole flat board to mycelia in 8 days, take mycelia to be cut into small pieces and obtain mycelia block, mycelia block produces zoosporangium through liquid culture induction, then promote zoosporangium release zoospore through K cryogenic treatment, then regulate zoospore concentration to 10 with sterilized water4~6Individual/ml, obtains Phytophthora nicotianae zoospore suspension；
(4) root wound leaching root inoculation: the fibrous root tobacco seedlings that step (2) obtains is taken out dewatering 1.5 days together with the second floating seedling tray, strike off the fibrous root that the second floating seedlings tray bottom exposes again, the Phytophthora nicotianae zoospore suspension that step (3) obtains is inserted in tray, then the second seedlings nursing plate is dipped in tray, under 12h illumination 12h dark alternation condition, in tray, tobacco seedlings is maintained inoculation 30 hours；
(5) Disease Resistance Identification: postvaccinal tobacco seedlings continues floating cultivation, the sickness rate a of tobacco seedlings balck shank of investigation in every 7 days, investigation is to investigate all inoculation tobacco seedlings in units of strain, investigation 3 times altogether, carry out statistics using the highest morbidity survey result as the state of an illness and divide anti-perception, sickness rate a=0 is immunity or high resistance kind, sickness rate 0 < a≤25% is disease-resistant variety, sickness rate 25% < a≤45% be in anti-kind, sickness rate 45% < a≤65% is middle sense kind, sickness rate 65% < a≤80% is susceptible variety, sickness rate 80% < a≤100% is high sense kind, wherein immunity or high resistance, disease-resistant, in anti-kind be resistant material.
Embodiment 4
(1) first stage nursery: the tobacco seed of big for Flos Carthami gold dollar, NC71, K326, Beinhart1000-1 kind is sowed in order the first of 360 holes and floats nursery 30 days in seedlings nursing plate, then forge Seedling 2 days, then pick out the seedling of neat and consistent；
(2) second stage nursery: seedling step (1) picked out is transplanted to without in 122 hole the second floating seedling trays of substrate together with substrate, nursery 15 days, seedling culture to fibrous root is entered floating pool through the second floating disc, then reject weak Seedling, screen the tobacco seedlings that fibrous root growing way is consistent, carry out leaf-cutting again, obtain fibrous root tobacco seedlings, in order to inoculation；
(3) preparation of Phytophthora nicotianae zoospore suspension: Phytophthora nicotianae is seeded to OA culture medium flat plate, at 27 DEG C, dark culturing covers with whole flat board to mycelia in 8 days, take mycelia to be cut into small pieces and obtain mycelia block, mycelia block produces zoosporangium through liquid culture induction, then promote zoosporangium release zoospore through K cryogenic treatment, then regulate zoospore concentration to 10 with sterilized water4~6Individual/ml, obtains Phytophthora nicotianae zoospore suspension；
(4) root wound leaching root inoculation: the fibrous root tobacco seedlings that step (2) obtains is taken out dewatering 1 day together with the second floating seedling tray, strike off the fibrous root that the second floating seedlings tray bottom exposes again, the Phytophthora nicotianae zoospore suspension that step (3) obtains is inserted in tray, then the second seedlings nursing plate is dipped in tray, then in tray, tobacco seedlings is inoculated, wherein tray water level 1 centimetre, temperature 27 DEG C, relative humidity more than 75%, under 12h illumination 12h dark alternation condition, in tray, tobacco seedlings is maintained inoculation 33 hours；
(5) Disease Resistance Identification: postvaccinal tobacco seedlings continues floating cultivation, the sickness rate a of tobacco seedlings balck shank of investigation in every 7 days, investigation 3 times altogether, carry out statistics using the highest morbidity survey result as the state of an illness and divide anti-perception, sickness rate a=0 is immunity or high resistance kind, sickness rate 0 < a≤25% is disease-resistant variety, sickness rate 25% < a≤45% be in anti-kind, sickness rate 45% < a≤65% is middle sense kind, sickness rate 65% < a≤80% is susceptible variety, sickness rate 80% < a≤100% is high sense kind, wherein immunity or high resistance, disease-resistant, in anti-kind be resistant material.
Result of the test is in Table 1.
The big gold dollar of table 1 Flos Carthami, NC71, K326, Beinhart1000-1 Disease Resistance Identification result
As shown in Table 1, the big gold dollar of Flos Carthami is high sense kind, and NC71, Beinhart1000-1 are disease-resistant varieties, and K326 is anti-kind in being.
NC71, Beinhart1000-1, K326 are resistant materials.
Carrying out the test of 5 different batches, the balck shank sickness rate of the tobacco bred of 5 batches is stable in respective scope, and repeatability meets requirement of experiment, and with 4 kinds disclosed in disease resistance be identical.Result shows that the present invention identifies that the conclusion of the anti-balck shank of Nicotiana tabacum L. is accurately.
Embodiment 5
(1) first stage nursery: by N.rustica, N.nudicaulis, N.longiflora, dark green No. 1, G28, G80, TN86, KY17, the big gold dollar of Flos Carthami, K326, Beinhart1000-1, Coker371, L8, Venus 6007, innovation 3, Yun yan85, little gold 1025, NC89, NC82, Florida301, NC71, NC1071 totally 22 kinds tobacco seed be sowed in the first floating seedlings nursing plate in 325 holes nursery 35 days in order, forge Seedling again 2.5 days, then pick out the seedling of neat and consistent；
(2) second stage nursery: seedling step (1) picked out is transplanted to without in 162 hole the second floating seedling trays of substrate together with substrate, nursery 12 days, seedling culture to fibrous root is entered floating pool through the second floating disc, then reject weak Seedling, screen the tobacco seedlings that fibrous root growing way is consistent, carry out leaf-cutting again, obtain fibrous root tobacco seedlings, in order to inoculation；
(3) preparation of Phytophthora nicotianae zoospore suspension: Phytophthora nicotianae is seeded to OA culture medium flat plate, at 26 DEG C, dark culturing covers with whole flat board to mycelia in 8 days, take mycelia to be cut into small pieces and obtain mycelia block, liquid culture induction produces zoosporangium, low temperature promotes zoosporangium release zoospore, then regulates zoospore concentration to 10 with sterilized water4~6Individual/ml, obtains Phytophthora nicotianae zoospore suspension；
(4) root wound leaching root inoculation: the fibrous root tobacco seedlings that step (2) obtains is taken out dewatering 2 days together with the second floating seedling tray, strike off the fibrous root that the second floating seedlings tray bottom exposes again, the Phytophthora nicotianae zoospore suspension that step (3) obtains is inserted in tray, then the second seedlings nursing plate is dipped in tray, then in tray, tobacco seedlings is inoculated, wherein tray water level 2 centimetres, temperature 26 DEG C, relative humidity more than 75%, under 12h illumination 12h dark alternation condition, in tray, tobacco seedlings is maintained inoculation 28 hours；
(5) Disease Resistance Identification: postvaccinal tobacco seedlings continues floating cultivation, the sickness rate a of tobacco seedlings balck shank of investigation in every 7 days, investigation 3 times altogether, carry out statistics using the highest morbidity survey result as the state of an illness and divide anti-perception, sickness rate a=0 is immunity or high resistance kind, sickness rate 0 < a≤25% is disease-resistant variety, sickness rate 25% < a≤45% be in anti-kind, sickness rate 45% < a≤65% is middle sense kind, sickness rate 65% < a≤80% is susceptible variety, sickness rate 80% < a≤100% is high sense kind, wherein immunity or high resistance, disease-resistant, in anti-kind be resistant material.
Result of the test is in Table 2.
The Disease Resistance Identification result of 22 tobacco breds such as table 2N.rustica
As shown in Table 2, N.nudicaulis is immunological species, totally 1；N.rustica, N.longiflora, Beinhart1000-1, Coker371, L8, innovation 3, NC82, Florida301, NC71, NC1071 are disease-resistant varieties, totally 10；K326, Venus 6007, Yun yan85, NC89 anti-kind in being, totally 4；G28, G80 are susceptible variety, totally 2, dark green No. 1, TN86, KY17, the big gold dollar of Flos Carthami, little gold 1025 be high sense kind, totally 5.
N.nudicaulis, N.rustica, N.longiflora, Beinhart1000-1, Coker371, L8, innovation 3, NC82, Florida301, NC71, NC1071, K326, Venus 6007, Yun yan85, NC89 are resistant material, totally 15.
Consistent by the qualification result of the inventive method qualification result to the anti-balck shank of 22 tobacco breds and 22 published anti-balck shanks of tobacco bred, it was shown that the qualification result of the present invention is accurately.
Claims (10)
1. the method for a Phytophthora nicotianae inoculation wound tobacco root system, it is characterized in that: include (1) first stage nursery, (2) second stage nursery, the preparation of (3) Phytophthora nicotianae zoospore suspension, the leaching root inoculation of (4) root wound, (5) Disease Resistance Identification, specifically comprise the following steps that
(1) first stage nursery: tobacco seed is sowed in the first floating seedling tray of carrying substrates nursery 30 ~ 40 days, then forge Seedling 2 ~ 3 days, then pick out the seedling of neat and consistent；
(2) second stage nursery: seedling step (1) picked out is transplanted to without in the second floating seedling tray of substrate together with substrate, nursery 10 ~ 20 days, seedling culture to fibrous root is entered floating pool through the second floating disc, then weak Seedling is rejected, the tobacco seedlings that screening fibrous root growing way is consistent, carry out leaf-cutting again, obtain fibrous root tobacco seedlings, in order to inoculation；
(3) preparation of Phytophthora nicotianae zoospore suspension: Phytophthora nicotianae is seeded to OA culture medium flat plate, at 25 ~ 28 DEG C, dark culturing covers with whole flat board to mycelia in 7 ~ 10 days, take mycelia to be cut into small pieces and obtain mycelia block, mycelia block produces zoosporangium through liquid culture induction, then promote zoosporangium release zoospore through K cryogenic treatment, then regulate zoospore concentration to 10 with sterilized water4~6Individual/ml, obtains Phytophthora nicotianae zoospore suspension；
(4) root wound leaching root inoculation: the fibrous root tobacco seedlings that step (2) obtains is taken out dewatering 1 ~ 2 day together with the second floating seedling tray, strike off the fibrous root that the second floating seedlings tray bottom exposes again, the Phytophthora nicotianae zoospore suspension that step (3) obtains is inserted in tray, then the second seedlings nursing plate is dipped in tray, under 12h illumination 12h dark alternation condition, in tray, tobacco seedlings is maintained inoculation 24 ~ 36 hours；
(5) Disease Resistance Identification: postvaccinal tobacco seedlings continues floating cultivation, the sickness rate a of tobacco seedlings balck shank of investigation in every 7 days, investigate 3 ~ 4 times altogether, carry out statistics using the highest morbidity survey result as the state of an illness and divide anti-perception, sickness rate a=0 is immunity or high resistance kind, sickness rate 0 < a≤25% is disease-resistant variety, sickness rate 25% < a≤45% be in anti-kind, sickness rate 45% < a≤65% is middle sense kind, sickness rate 65% < a≤80% is susceptible variety, sickness rate 80% < a≤100% is high sense kind, wherein immunity or high resistance, disease-resistant, in anti-kind be resistant material.
2. the method for Phytophthora nicotianae according to claim 1 inoculation wound tobacco root system, is characterized in that: the tobacco seedlings that screening fibrous root growing way is consistent in described step (2) is have 8 ~ 16 through the fibrous root quantity of floating disc, fibrous root length 4 ~ 5 centimetres, and in pure white.
3. the method for Phytophthora nicotianae according to claim 1 inoculation wound tobacco root system, it is characterized in that: the first described floating seedling tray is 300 ~ 400 hole floating seedling trays, second floating seedling tray is 100 ~ 200 hole floating seedling trays, and the area equation of the area of the first floating seedling tray and the second floating seedling tray.
4. the method for Phytophthora nicotianae according to claim 1 inoculation wound tobacco root system, it is characterized in that: after the tobacco seedlings that in described step (2), screening fibrous root growing way is consistent, quantity at least 12 strain of each kind tobacco seedlings in one the second floating disc, and second the quantity of floating disc no less than 3, namely the total quantity of each kind tobacco seedlings is no less than 36 strains.
5. the method for Phytophthora nicotianae according to claim 1 inoculation wound tobacco root system, is characterized in that: in described step (4), the water level of tray is 1 ~ 2cm, and temperature is 25 ~ 28 DEG C, relative humidity more than 75%.
6. the method for Phytophthora nicotianae according to claim 1 inoculation wound tobacco root system, it is characterized in that: in described step (2), leaf-cutting is to wipe out along tobacco seedlings blade tip to launch the 1/3 ~ 1/2 of blade, then cuts off the 2-4 sheet Huang leaf of cigarette strain bottom and whole leaves of Lao Ye and petiole.
7. the method for Phytophthora nicotianae according to claim 1 inoculation wound tobacco root system, it is characterized in that: in described step (3), liquid culture is to be joined by mycelia block in the culture dish of 10%V8 juice fluid medium, every ware adds mycelia block 20 ~ 40, continue at dark culturing 3 ~ 4 days at 25 ~ 28 DEG C, incline culture fluid, rinse 3 ~ 5 times with aquesterilisa again, then change aquesterilisa every day 1 ~ 3 time, the generation of continuous 2 ~ 3 days induction zoosporangiums.
8. the method for Phytophthora nicotianae according to claim 1 inoculation wound tobacco root system, is characterized in that: in described step (3), K cryogenic treatment is when mycelia block zoosporangium is 104/cm2Time more than individual, in 5 ~ 8 DEG C of K cryogenic treatment 20 ~ 30min, take out and be placed in room temperature 15 ~ 30min induction zoosporangium release zoospore.
9. the method for Phytophthora nicotianae according to claim 1 inoculation wound tobacco root system, is characterized in that: in described step (5), the temperature of floating cultivation is 22 ~ 30 DEG C.
10. the method for Phytophthora nicotianae according to claim 1 inoculation wound tobacco root system, is characterized in that: in described step (1), forging Seedling is to be taken out from floating pool by the first floating disc, ventilation dewatering 2 ~ 3 days.
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