CN102754623A - Method for acquiring ricedrawfvirus (RDV) by utilizing nephotettix cincticeps under artificial conditions and application of RDV - Google Patents
Method for acquiring ricedrawfvirus (RDV) by utilizing nephotettix cincticeps under artificial conditions and application of RDV Download PDFInfo
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Abstract
The invention discloses a method for acquiring ricedrawfvivus (RDV) by utilizing nephotettix cincticeps under artificial conditions and an application of RDV. Cultured rice infected with the RDV or cryopreserved rice bacterial leaf is used for feeding nephotettix cincticeps, thus the nephotettix cincticeps is infected with the RDV; then the nephotettix cincticeps is transferred to healthy rice to be fed until a circulative period of virus is finished, and virus carrying condition of the nephotettix cincticeps is identified by adopting an RT-PCR (reverse transcription-polymerase chain reaction) technology and a serology method. The nephotettix cincticeps carrying the RDV, which is obtained by utilizing the invention, can be used for screening a monoclonal antibody which is resistant to the RDV, thus serology methods such as dot-ELISA (enzyme-linked immunosorbent assay) and TAS-ELISA (triple antibody sandwich-enzyme-linked immunosorbent assay), which take the monoclonal antibody as a core, are established, and monitoring on occurrence and epidemic of the viral disease is carried out; besides, the nephotettix cincticeps carrying the RDV can be subjected to rice inoculation experiment to identify resistance of a rice variety, thus a disease-resistant variety can be screened; and the nephotettix cincticeps carrying the RDV also can be used for researching interaction among the RDV, a host and a virus media, and theoretical basis is provided for elaborating the pathogenesis of the viral disease and providing an effective control strategy.
Description
Technical field
Field under the present invention is a biological technical field, especially relates to the method and the application thereof of rice leafhopper acquisition fractilinea oryzae under a kind of artificial condition.
Background technology
(Rice drawf virus is one of a kind of common paddy rice virus disease RDV) to fractilinea oryzae, belongs to Reoviridae (Reoviridae), and the plant reovirus belongs to (Phytoreovirus).This virus disease finds in Xichang, Sichuan Province that early than nineteen thirty-nine the later stage in the sixties comes into vogue in China, mainly is popular in paddy rice producing regions such as Japan, Korea, Southeast Asia and China southeast now.This virus disease works the mischief to Rice Production in some paddy rice producing regions of China at present.The rice dwarf virus disease viral disease also endangers gramineous plantses such as barley, wheat except that hazard rice.Paddy rice is from this disease of susceptible in seedling stage to tillering stage, susceptible in the meantime after, whole symptom show as plant stunt, tiller increase, leaf dark green, stiff.The growth later stage, susceptible paddy rice can not ear solidly, and sick leaf symptom then shows and is divided into two kinds of white point type and meander configurations.White point type symptom show as plant stunt, tiller increase, the leaf look dark green; The dotted line shape yellowish-white color dot streak parallel with vein appears on blade or the leaf sheath, and the most obvious with base portion; The diseased plant root system development is bad; The bar scab only appears putting clearly in the morbidity of booting later stage on sword-like leave, leaf sheath; The heading sequela only shows scab on the sword-like leave sheath.The meander configuration symptom shows as the diseased plant poor growth, significantly stunt, tiller increase, leaf dark green, under the not enough situation of illumination, lobus cardiacus is extracted out and is distorted shape, stretches with lobus cardiacus, wavy incising appears in blade edge, color and luster is yellowish; Booting stage, how the white point bar appearred in morbidity on sword-like leave blade and leaf sheath, and the fringe constriction is short, formed the bag neck or partly wrapped the neck fringe.In recent years this virus disease in Fujian, rice districts such as Zhejiang, Guangxi, Guizhou, Henan take place; And the morbidity of the paddy rice producing region of Xun Dianxian, Songming County is more general in Yunnan, and also all there is generation in the part paddy rice producing region of the Qujing in the Kunming at middle part, northeast district and the Jinghong in Zhaotong, south and the west and south and Dehong.
Under field conditions (factors) fractilinea oryzae through rice leafhopper (
Nephotetix cincticeps) and recilia dorsalis (
Recilia dorsalia) propagate, can in insect bodies, duplicate, in case obtain poison, promptly lifelong band poison, and can pass to the next generation through worm's ovum, nymph and adult all can pass poison, and the efficient poison that passes.Rice leafhopper inhales on sick rice that juice is the shortest to obtain the 5 minutes time of poison, and obtaining needs to follow back the phase through one section behind the poison and could pass poison, is 17 days when following back 20 ℃ of phases, is 12.4 days in the time of 29.2 ℃.After leafhopper was obtained malicious 15-20 days, body inner virus concentration increased sharply, and passed poison with the persistence mode afterwards and gave host plant, and have the tangible malicious characteristic that intermittently passes.Virus can be survived the winter in the rice leafhopper body; Rice leafhopper survives the winter with the nymph form on amur foxtail, and next spring sprouts wings and moves back to the rice field and cause harm, after the early rice harvesting; Adjourn on the late rice and cause harm; After the late rice results, adjourn on 38 kinds of gramineous plantses such as amur foxtail, winter rice and survive the winter, band poisonous insect amount is this pathogenetic main factor of influence.So early monitoring and early warning for rice leafhopper and fractilinea oryzae are very necessary.With the monoclonal antibody be serological method that core is set up be most economical, the most effectively detect in the rice leafhopper body and paddy rice in the high-throughout detection method of RDV, thereby it is particularly important to screen suitable antibody; Screen disease-resistant variety in addition and be most economical, the effective method of control paddy rice virus disease.Through utilizing the sick leaf of the paddy rice rice leafhopper of feeding, obtain the rice leafhopper of carrying RDV, can be used for the screening of anti-RDV monoclonal antibody, in order to monoclonal antibody be the serological method set up of core to the generation popularity of this virus disease forecast, early warning; And can inoculate rice strain, and help the rice varieties of the anti-rice dwarf virus disease viral disease of Rapid identification, be excavation, the resistant variety seed selection of the paddy rice resource of anti-rice dwarf virus disease viral disease; The acquisition of carrying the rice leafhopper of RDV can provide material base for the mutual do research between RDV and host and the vector insect thereof.
Summary of the invention
The purpose of this invention is to provide the method and the application thereof of rice leafhopper acquisition fractilinea oryzae under a kind of artificial condition.
Rice leafhopper obtains the method for fractilinea oryzae under artificial feeding's condition, it is characterized in that comprising the steps:
1) acquisition of rice leafhopper:
Take place to raise at indoor nontoxic rice seedling after rice leafhopper is caught in regional rice field collection from non-rice dwarf virus disease; Treat that rice leafhopper adult post-coitum single head worm lays eggs separately; Randomly draw in the colony 10% rice leafhopper and detect the malicious situation of being with, keep not with the offspring of malicious female worm and set up indoor population with the RT-PCR method;
2) rice leafhopper obtain poison:
Rice leafhopper is obtained poison from the infection RDV paddy rice of freezing preservation or the susceptible paddy rice diseased plant that lives:
Before raising poison, rice leafhopper in the beaker that filter paper is preserved moisture hungry 1～3 hour; Preserve the diseased plant of taking out freezing preservation the refrigerator from-10 ℃ to-80 ℃, move in the beaker, each beaker is put 1～10 strain diseased plant; Insert 20～300 3～4 age nontoxic rice leafhopper nymph, beaker seals with nylon gauze; Put into the greenhouse to beaker or growth chamber is raised, temperature is 26～30 ℃, and relative moisture is 60 %～75 %, and the photoperiod is 16L:8D; Raise poison and after 1～2 day the rice leafhopper of survival moved on the healthy water rice seedling grow in greenhouse or growth chamber and feed, temperature is 26～30 ℃, and relative moisture is 60 %～75 %, and the photoperiod is 16L:8D, follows back the phase, about 15～20 days until what tide over virus; The single head rice leafhopper of raising behind the poison lays eggs separately on healthy paddy rice; Randomly draw thereafter for 10% rice leafhopper in the colony and detect the malicious situation of being with the RT-PCR method; The offspring of the female worm of leave strip poison also sets up indoor band seed culture of viruses crowd, promptly obtains the rice leafhopper of carrying fractilinea oryzae;
Perhaps, rice leafhopper is obtained poison from the paddy rice diseased plant of planting in greenhouse or growth chamber:
Before raising poison, rice leafhopper in the beaker that filter paper is preserved moisture hungry 1～3 hour; Infect the paddy rice diseased plant of fractilinea oryzae, the circular open plastic containers that seal with nylon gauze are enclosed in the growth chamber of Temperature and Humidity Control and cultivate, and each plastic containers is put 1～3 strain diseased plant plant; Each the device in put into 20～200 3～4 age nontoxic rice leafhopper nymph, the cultivation temperature of feeding is 26～30 ℃, relative moisture is 60 %～75 %, the photoperiod is 16L:8D; Raising poison moves into the rice leafhopper of survival to raise on the fresh and healthy paddy rice seedling until what tide over virus after 1～3 day and follows back the phase, about 15～20 days; The single head rice leafhopper of raising behind the poison lays eggs separately on healthy paddy rice; Randomly draw thereafter for 10% rice leafhopper in the colony and detect the malicious situation of being with the RT-PCR method; The offspring of the female worm of leave strip poison also sets up indoor band seed culture of viruses crowd, promptly obtains the rice leafhopper of carrying fractilinea oryzae.
The rice leafhopper of the carrying fractilinea oryzae healthy water rice seedling that is used to feed is identified and is used for the resistance of rice varieties the disease-resistant variety of Screening of Rice, and is anti-fractilinea oryzae breeding for disease resistance service.
The rice leafhopper of the carrying fractilinea oryzae healthy water rice seedling that is used to feed identifies that the method for rice varieties resistance is: the rice leafhopper that will carry fractilinea oryzae moves into plantation to be identified in the greenhouse or the 2-5 leaf phase paddy rice seedling of growth chamber; Adopt single seedling 10 cephalont methods to inoculate rice varieties to be identified; Raise and shift out whole rice leafhoppers after 3-7 days; Rice seedling is transplanted to greenhouse or growth chamber; Cultivation temperature is 26-30 ℃, and relative moisture is 65 %-75 %, and the photoperiod is 16L:8D; Began to observe viral symptom in 7 days later on, continuous observation 20-50 days; Detect paddy rice with RT-PCR and dot-ELISA method; Connect viral symptom of malicious paddy rice and the resistance of RT-PCR and dot-ELISA testing result judgement paddy rice thereof according to feeding.
The rice leafhopper of carrying fractilinea oryzae is used to screen special, the sensitive monoclonal antibody of anti-fractilinea oryzae, is the serological method of fractilinea oryzae in detection paddy rice and the rice leafhopper body of core thereby set up with the monoclonal antibody.
The rice leafhopper of carrying fractilinea oryzae is used to study providing material base as mechanism mutually between fractilinea oryzae and paddy rice host and the amboceptor insect thereof.
The beneficial effect that the present invention compared with prior art has is: 1) under artificial feeding's condition, can make rice leafhopper obtain fractilinea oryzae, solve the problem that ill plant is difficult to long preservation; The rice leafhopper of 2) carrying fractilinea oryzae is screened the monoclonal antibody of anti-RDV, thereby lays the first stone for setting up the serological method that detects RDV; 3) set up the method for the rice varieties of the anti-rice dwarf virus disease viral disease of Rapid identification under the artificial condition, be excavation, the resistant variety seed selection of the paddy rice resource of anti-rice dwarf virus disease viral disease; The acquisition of 4) carrying the rice leafhopper of RDV can provide material base for the research of the mutual do between RDV and host and the vector insect thereof.
Description of drawings
Fig. 1 is the result that the RT-PCR method detects RDV in the paddy rice sample;
Fig. 2 is the result that the Dot-ELISA method detects RDV in the paddy rice sample;
Fig. 3 be refrigerated diseased leaves feed rice leafhopper obtain the poison installation drawing;
Fig. 4 is the result that the Dot-ELISA method detects RDV in the rice leafhopper sample that refrigerated diseased leaves feeds;
Fig. 5 be fresh sick leaf feed rice leafhopper obtain the poison installation drawing;
Fig. 6 is the result that the RT-PCR method detects RDV in the rice leafhopper sample that fresh sick leaf feeds;
Fig. 7 is that rice leafhopper passes malicious Experimental equipment;
Fig. 8 Dot-ELISA method detects the result of RDV in the inoculation paddy rice;
Fig. 9 obtains malicious rice leafhopper to be used for Screening of Rice resistance installation drawing.
Embodiment
The monoclonal antibody that the rice leafhopper that fractilinea oryzae is carried in utilization of the present invention can be screened anti-RDV is used for the detection of virus and the authentication method of setting up the varietal resistance of rice dwarf virus disease viral disease; For excavation, the resistant variety seed selection of the paddy rice resource of anti-rice dwarf virus disease viral disease provides the research means on basis, also can be further research RDV and provide material base with mutual work research between host and the vector insect thereof.
The method of rice leafhopper acquisition fractilinea oryzae comprises the steps: under artificial feeding's condition
1) acquisition of rice leafhopper:
Take place to raise at indoor nontoxic rice seedling after rice leafhopper is caught in regional rice field collection from non-rice dwarf virus disease; Treat that rice leafhopper adult post-coitum single head worm lays eggs separately; Randomly draw in the colony 10% rice leafhopper and detect the malicious situation of being with, keep not with the offspring of malicious female worm and set up indoor population with the RT-PCR method;
2) rice leafhopper obtain poison:
Rice leafhopper is obtained poison from the infection RDV paddy rice of freezing preservation or the susceptible paddy rice diseased plant that lives:
Before raising poison, rice leafhopper in the beaker that filter paper is preserved moisture hungry 1～3 hour; Preserve the diseased plant of taking out freezing preservation the refrigerator from-10 ℃ to-80 ℃, move in the beaker, each beaker is put 1～10 strain diseased plant; Insert 20～300 3～4 age nontoxic rice leafhopper nymph, beaker seals with nylon gauze; Put into the greenhouse to beaker or growth chamber is raised, temperature is 26～30 ℃, and relative moisture is 60 %～75 %, and the photoperiod is 16L:8D; Raise poison and after 1～2 day the rice leafhopper of survival moved on the healthy water rice seedling grow in greenhouse or growth chamber and feed, temperature is 26～30 ℃, and relative moisture is 60 %～75 %, and the photoperiod is 16L:8D, follows back the phase, about 15～20 days until what tide over virus; The single head rice leafhopper of raising behind the poison lays eggs separately on healthy paddy rice; Randomly draw thereafter for 10% rice leafhopper in the colony and detect the malicious situation of being with the RT-PCR method; The offspring of the female worm of leave strip poison also sets up indoor band seed culture of viruses crowd, promptly obtains the rice leafhopper of carrying fractilinea oryzae;
Perhaps, rice leafhopper is obtained poison from the paddy rice diseased plant of planting in greenhouse or growth chamber:
Before raising poison, rice leafhopper in the beaker that filter paper is preserved moisture hungry 1～3 hour; Infect the paddy rice diseased plant of fractilinea oryzae, the circular open plastic containers that seal with nylon gauze are enclosed in the growth chamber of Temperature and Humidity Control and cultivate, and each plastic containers is put 1～3 strain diseased plant plant; Each the device in put into 20～200 3～4 age nontoxic rice leafhopper nymph, the cultivation temperature of feeding is 26～30 ℃, relative moisture is 60 %～75 %, the photoperiod is 16L:8D; Raising poison moves into the rice leafhopper of survival to raise on the fresh and healthy paddy rice seedling until what tide over virus after 1～3 day and follows back the phase, about 15～20 days; The single head rice leafhopper of raising behind the poison lays eggs separately on healthy paddy rice; Randomly draw thereafter for 10% rice leafhopper in the colony and detect the malicious situation of being with the RT-PCR method; The offspring of the female worm of leave strip poison also sets up indoor band seed culture of viruses crowd, promptly obtains the rice leafhopper of carrying fractilinea oryzae.
The rice leafhopper of the carrying fractilinea oryzae healthy water rice seedling that is used to feed is identified and is used for the resistance of rice varieties the disease-resistant variety of Screening of Rice, and is anti-fractilinea oryzae breeding for disease resistance service.
The rice leafhopper of the carrying fractilinea oryzae healthy water rice seedling that is used to feed identifies that the method for rice varieties resistance is: the rice leafhopper that will carry fractilinea oryzae moves into plantation to be identified in the greenhouse or the 2-5 leaf phase paddy rice seedling of growth chamber; Adopt single seedling 10 cephalont methods to inoculate rice varieties to be identified; Raise and shift out whole rice leafhoppers after 3-7 days; Rice seedling is transplanted to greenhouse or growth chamber; Cultivation temperature is 26-30 ℃, and relative moisture is 65 %-75 %, and the photoperiod is 16L:8D; Began to observe viral symptom in 7 days later on, continuous observation 20-50 days; Detect paddy rice with RT-PCR and dot-ELISA method; Connect viral symptom of malicious paddy rice and the resistance of RT-PCR and dot-ELISA testing result judgement paddy rice thereof according to feeding.
The rice leafhopper of carrying fractilinea oryzae is used to screen special, the sensitive monoclonal antibody of anti-fractilinea oryzae, is the serological method of fractilinea oryzae in detection paddy rice and the rice leafhopper body of core thereby set up with the monoclonal antibody.
The rice leafhopper of carrying fractilinea oryzae is used to study providing material base as mechanism mutually between fractilinea oryzae and paddy rice host and the amboceptor insect thereof.
Below in conjunction with embodiment and accompanying drawing the present invention is described further.
1, infects the acquisition of RDV paddy rice
2011 in southern Xun Dian county the collection of fractilinea oryzae generating region have plant stunt, tiller increase, the leaf look dark green; Appearance dotted line shape yellowish-white color dot streak and the lobus cardiacus extraction parallel with vein is distorted shape on blade or the leaf sheath, and the sick appearance of wavy paddy rice of incising appears in blade edge.
(1) the RT-PCR method detects paddy rice and whether is with poison
Extract total RNA of the sick appearance of paddy rice with reference to Trizol reagent; According to capsid protein gene (CP gene) sequences Design primer: the RDV-CP-F:TACTTT/CTCCGGGCTCACAACAGG (corresponding CP gene 84-101 position) and the RDV-CP-R:CCCCGCAACAGACCGAAACAA (corresponding CP gene 466-486 position) of the fractilinea oryzae of reporting among the GenBank, and synthetic by Shanghai Ying Jun Bioisystech Co., Ltd.Adopt the RT-PCR method to detect and confirm that sick appearance is infected fractilinea oryzae.Reverse transcription is carried out with reference to M-MLV reverse transcriptase (Promega Company products) specification; The PCR reaction system is: i.e. cDNA template 1 μ l; 5 * PrimeSTARTM Buffer (containing Mg2+), 10 μ l, dNTP Mix 4 μ l, PrimeSTARTM DNA Polymerase (2.5 U/ μ L) 0.5 μ l; Each 1 μ l of upstream and downstream primer, it is 50 μ l that last two steaming sterile waters are supplied the reaction final volume.The PCR reaction system is following: preparatory 94 ℃ of 2 min of sex change, and 94 ℃ of 45 s of sex change, 57 ℃ of 45 s that anneal extends 72 ℃ of 1 min30s, and cyclic amplification 35 times extends 72 ℃ of 10 min at last.Amplified production carries out electrophoretic analysis (Fig. 1) in 0.8% Ago-Gel, and reclaims kit (AxyGEN) with the PCR gel and reclaim dna fragmentation, and concrete operations reference reagent box specification carries out.The PCR product end of purifying is added A to be connected with cloning vector pMD-18T vector; Recombinant plasmid called after pMD18-T-CP; And be transformed in the competent cell of Escherichia coli DH 5 α; Extract recombinant plasmid with plasmid extraction kit (AxyGEN), the recombinant plasmid that extracts is carried out PCR and double digestion evaluation, and through the CP gene order entrained among the sequence verification recombinant cloning vector pMD18-T-CP and the correctness of reading frame; Sequence analysis software is DNAstar, NCBI-BLAST, and used database is GeneBank etc.Confirm the malicious in spite of illness RDV of being of paddy rice sample institute.
(2) Dot-ELISA detects paddy rice and whether is with poison
The liquid nitrogen grinding powdered use in the rice leaf back of weighing, and (w/v, g/mL) add 0.01 mol/L PBS (pH7.4) grinding afterwards press 1:10～30; Centrifugal 3 min of sick juice 5000 rpm; Get on the 3 μ l and check on the NC, health and susceptible paddy rice leaf juice are set simultaneously respectively as feminine gender and positive control; Drying at room temperature 10-20 min; The NC film is immersed in PBST (the 0.01 mol/L PBS that the contains 0.05% Tween-20) confining liquid that contains 5% skim milk powder room temperature and seals 30 min; The NC film is put into the monoclonal antibody incubated at room 30-60 min that 1:5000 doubly dilutes; Wash film 3～4 times with PBST, each 3 min; The NC film is put into AP enzyme labeling sheep anti-mouse igg two anti-incubated at room 30 ~ 60 min that 1:8000 doubly dilutes; PBST washes film 4～5 times, each 3 min; 66 μ L NBT and 33 μ L BCIP substrates (Promega) join 10 ml substrate buffer solutions (0.1 mol/L Tris Cl, 0.1 mol/L NaCl, 0.025 mol/L MgCl, pH9.5) mixing, and film is put into substrate solution and reacted, the perusal result.Treat the positive control colour developing obviously, and feminine gender has no when colour developing running water rinsing cessation reaction, the Taking Pictures recording result, the result is as shown in Figure 2, confirms that further paddy rice infects RDV.
2, the acquisition of rice leafhopper
Gather the rice leafhopper nymph that catches from field, non-virus district; On the paddy rice susceptible variety, raise, post-coitum single head worm lays eggs separately, randomly draws in the colony 10% rice leafhopper and detects this worm with RT-PCR and Dot-ELISA method and be with malicious situation; Keep not offspring with malicious female worm; In later per generation, randomly drawed in the colony 10% rice leafhopper and confirms it with RT-PCR and Dot-ELISA method and be not with poison really, promptly obtains to pollute for avoiding passing virus mediator with the rice leafhopper of RDV; Raise at every turn and get 100 rice leafhoppers before the poison and detect, determine that it is no poisonous insect.
3, rice leafhopper is obtained poison from the paddy rice that infects RDV
(1) rice leafhopper is obtained poison from the sick leaf of freezing paddy rice
Take out refrigerated diseased leaves from-20 ℃ (perhaps-70 ℃), place after 2 hours for 4 ℃, place in the greenhouse; Move in the beaker after 3 hours, because freezing blade is dry, available cotton of soaking is fixed in the blade bottom; And add low amounts of water and avoid blade too dried, the toxic effect fruit is raised in influence.(beaker seals with nylon gauze, and existing air preferably flows and to make the rice leafhopper survival, can avoid rice leafhopper to escape again) (Fig. 3), each beaker insert 60 2-3 age nontoxic rice leafhopper, make its hungry 3 hours before raising poison.Raise poison and fed about 15-20 days on the rice leafhopper immigration healthy water rice seedling with survival after 48 hours, that tides over virus follows back the phase, and the rice leafhopper quantity of record survival.Afterwards rice leafhopper is inoculated susceptible rice varieties, observe the symptom whether paddy rice shows rice dwarf virus disease, and verify through immunological method and molecular engineering.30 left and right sides rice leafhoppers that shift out are carried out RT-PCR in the inoculation back and Dot-ELISA detects, and the rice leafhopper average band poison rate that freezing blade is raised poison is 55% (Fig. 4).The single head rice leafhopper of raising behind the poison lays eggs separately on healthy paddy rice; Randomly draw thereafter for 10% rice leafhopper in the colony and detect the malicious situation of being with the RT-PCR method; The offspring of the female worm of leave strip poison also sets up indoor band seed culture of viruses crowd, promptly obtains the rice leafhopper of carrying fractilinea oryzae.
(2) rice leafhopper is obtained poison from the sick plant of the fresh paddy rice of cultivating
The circular open plastic containers that the sick plant of paddy rice is sealed with nylon gauze are enclosed in and cultivate (Fig. 5) in the Temperature and Humidity Control incubator, and good air flows and guarantees the survival of plant and rice leafhopper, avoids the escape of rice leafhopper again.The same, put in each device insert 60 2-3 age nontoxic rice leafhopper, make its hungry 3 hours before raising poison.Raise after malicious 2-5 days rice leafhopper with survival and move into to feed on the fresh and healthy paddy rice seedling and follow back the phase until what tide over virus, and the record rice leafhopper quantity of surviving.Afterwards rice leafhopper is inoculated susceptible rice varieties, whether the observed and recorded paddy rice shows the symptom of rice dwarf virus disease, and verifies through immunological method and molecular engineering.30 left and right sides rice leafhoppers that shift out are carried out RT-PCR in the inoculation back and Dot-ELISA detects, and it is with malicious rate is 75% (Fig. 6).The single head rice leafhopper of raising behind the poison lays eggs separately on healthy paddy rice; Randomly draw thereafter for 10% rice leafhopper in the colony and detect the malicious situation of being with the RT-PCR method; The offspring of the female worm of leave strip poison also sets up indoor band seed culture of viruses crowd, promptly obtains the rice leafhopper of carrying fractilinea oryzae.
4, obtain the application of malicious rice leafhopper in the monoclonal antibody screening operation
The single head rice leafhopper is put into the centrifuge tube of the eppendorf of 1.5 mL; And add 100 μ L PBS (0.01mol/L; PH7.4); After mashing rice leafhopper with toothpick, centrifugal 3 min of 5000 rpm, get on the 3 μ l and check on the NC, be provided with simultaneously healthy be with malicious rice leafhopper respectively as feminine gender and positive control; Drying at room temperature 10-20 min; The NC film is immersed in PBST (the 0.01 mol/L PBS that the contains 0.05% Tween-20) confining liquid that contains 5% skim milk powder room temperature and seals 30 min; The NC film is put into the monoclonal antibody incubated at room 30-60 min of appropriateness dilution; Wash film 3～4 times with PBST, each 3 min; The NC film is put into HRP enzyme labeling sheep anti-mouse igg two anti-incubated at room 30 ~ 60 min of appropriateness dilution; PBST washes film 4～5 times, each 3 min; The A chromogenic substrate is the chromogenic substrate of HRP, promptly like TMB sedimentation type chromogenic substrate etc.If positive reaction is arranged, explain that then antibody can be used for the detection of virus, thereby be that core is set up suitable serological method the paddy rice and the rice leafhopper in field detected with this monoclonal antibody, predict early warning to the generation of RDV is popular.
5, obtain malicious the reaching of biography of malicious rice leafhopper in rice varieties resistance application in identification
Select susceptible rice varieties to pass the poison experiment; 5-10 is only taken the single seedling inoculation of malicious rice leafhopper, in the circular open plastic containers that nylon gauze seals, cultivate (Fig. 7), seedling age is in one heart stage of two leaves; After passing malicious 2-3 d rice leafhopper is shifted out, observe and write down the incidence of paddy rice then.The investigation standard is with reference to the classical symptom of rice dwarf virus disease: plant stunts, tiller increase, the leaf look dark green, occurs the yellowish-white color dot streak of dotted line shape parallel with vein and lobus cardiacus on blade or the leaf sheath and extract out and be distorted shape, the blade edge appearance is wavy incises etc.Detect postvaccinal rice leaf (Fig. 8) with the Dot-ELISA method; There is the paddy rice of classical symptom all can reveal purple dot; The paddy rice of no disease symptom keeps color constant, and this shows that rice leafhopper obtains can be seeded on the rice varieties behind the fractilinea oryzae from refrigerated diseased leaves and fresh sick leaf.
Obtain the resistance evaluation that malicious rice leafhopper can be used for rice varieties; Place and inoculate paddy rice (Fig. 9) in the insect protected case tiding over the malicious rice leafhopper of obtaining of the phase followed back; Inoculation paddy rice seedling age is one heart stages of two leaves, and every seedling is with malicious leafhopper l-2 head, lets it pass poison and gets food 5-7 d; Catch up with worm 2-3 time with bamboo pole every day in cylinder mould, guarantee to pass the consistent relatively of poison as far as possible.Then postvaccinal rice seedling is transplanted in earth, fly net on the cover is observed rice pathogenesis situation and record data and the statistics incidence of disease, and is detected paddy rice with RT-PCR and dot-ELISA method and whether infect RDV.According to the unified resistance grade scale of formulating of national paddy rice virus disease scientific research cooperative groups, be 100% with the incidence of disease of check variety cave 136-12, add up the relative incidence rate of each kind. divide resistance grade, totally 5 grades according to the incidence of disease.0 grade: (immunity IM), does not fall ill in immunity; 1 grade: high resisting (highly resistant, HR), the incidence of disease is 0.10%-5.00%; 2 grades: in anti-(moderately resistant, MR), the incidence of disease is 5.10%-30.00%; 3 grades: middle sense (moderately susceptible, MS), the incidence of disease is 30.10%-60.00%; 4 grades: (highly susceptible, HS), the incidence of disease is more than 60.10% in high sense.
Claims (5)
1. rice leafhopper obtains the method for fractilinea oryzae under artificial feeding's condition, it is characterized in that comprising the steps:
1) acquisition of rice leafhopper:
Take place to raise at indoor nontoxic rice seedling after rice leafhopper is caught in regional rice field collection from non-rice dwarf virus disease; Treat that rice leafhopper adult post-coitum single head worm lays eggs separately; Randomly draw in the colony 10% rice leafhopper and detect the malicious situation of being with, keep not with the offspring of malicious female worm and set up indoor population with the RT-PCR method;
2) rice leafhopper obtain poison:
Rice leafhopper is obtained poison from the infection RDV paddy rice of freezing preservation or the susceptible paddy rice diseased plant that lives:
Before raising poison, rice leafhopper in the beaker that filter paper is preserved moisture hungry 1～3 hour; Preserve the diseased plant of taking out freezing preservation the refrigerator from-10 ℃ to-80 ℃, move in the beaker, each beaker is put 1～10 strain diseased plant; Insert 20～300 3～4 age nontoxic rice leafhopper nymph, beaker seals with nylon gauze; Put into the greenhouse to beaker or growth chamber is raised, temperature is 26～30 ℃, and relative moisture is 60 %～75 %, and the photoperiod is 16L:8D; Raise poison and after 1～2 day the rice leafhopper of survival moved on the healthy water rice seedling grow in greenhouse or growth chamber and feed, temperature is 26～30 ℃, and relative moisture is 60 %～75 %, and the photoperiod is 16L:8D, follows back the phase, about 15～20 days until what tide over virus; The single head rice leafhopper of raising behind the poison lays eggs separately on healthy paddy rice; Randomly draw thereafter for 10% rice leafhopper in the colony and detect the malicious situation of being with the RT-PCR method; The offspring of the female worm of leave strip poison also sets up indoor band seed culture of viruses crowd, promptly obtains the rice leafhopper of carrying fractilinea oryzae;
Perhaps, rice leafhopper is obtained poison from the paddy rice diseased plant of planting in greenhouse or growth chamber:
Before raising poison, rice leafhopper in the beaker that filter paper is preserved moisture hungry 1～3 hour; Infect the paddy rice diseased plant of fractilinea oryzae, the circular open plastic containers that seal with nylon gauze are enclosed in the growth chamber of Temperature and Humidity Control and cultivate, and each plastic containers is put 1～3 strain diseased plant plant; Each the device in put into 20～200 3～4 age nontoxic rice leafhopper nymph, the cultivation temperature of feeding is 26～30 ℃, relative moisture is 60 %～75 %, the photoperiod is 16L:8D; Raising poison moves into the rice leafhopper of survival to raise on the fresh and healthy paddy rice seedling until what tide over virus after 1～3 day and follows back the phase, about 15～20 days; The single head rice leafhopper of raising behind the poison lays eggs separately on healthy paddy rice; Randomly draw thereafter for 10% rice leafhopper in the colony and detect the malicious situation of being with the RT-PCR method; The offspring of the female worm of leave strip poison also sets up indoor band seed culture of viruses crowd, promptly obtains the rice leafhopper of carrying fractilinea oryzae.
2. application of the rice leafhopper of carrying fractilinea oryzae of method acquisition according to claim 1; It is characterized in that described rice leafhopper of the carrying fractilinea oryzae healthy water rice seedling that is used to feed identifies the resistance of rice varieties; The disease-resistant variety that is used for Screening of Rice, and be anti-fractilinea oryzae breeding for disease resistance service.
3. a kind of application of carrying the rice leafhopper of fractilinea oryzae as claimed in claim 2; It is characterized in that described rice leafhopper of the carrying fractilinea oryzae healthy water rice seedling that is used to feed identifies that the method for rice varieties resistance is: the rice leafhopper that will carry fractilinea oryzae moves into plantation to be identified in the greenhouse or the 2-5 leaf phase paddy rice seedling of growth chamber; Adopt single seedling 10 cephalont methods to inoculate rice varieties to be identified; Raise and shift out whole rice leafhoppers after 3-7 days, rice seedling is transplanted to greenhouse or growth chamber, cultivation temperature is 26-30 ℃; Relative moisture is 65 %-75 %, and the photoperiod is 16L:8D; Began to observe viral symptom in 7 days later on, continuous observation 20-50 days; Detect paddy rice with RT-PCR and dot-ELISA method; Connect viral symptom of malicious paddy rice and the resistance of RT-PCR and dot-ELISA testing result judgement paddy rice thereof according to feeding.
4. application of the rice leafhopper of carrying fractilinea oryzae of method acquisition according to claim 1; It is characterized in that described rice leafhopper of carrying fractilinea oryzae is used to screen special, the sensitive monoclonal antibody of anti-fractilinea oryzae, is the serological method of fractilinea oryzae in detection paddy rice and the rice leafhopper body of core thereby set up with the monoclonal antibody.
5. the application of the rice leafhopper of carrying fractilinea oryzae of method acquisition according to claim 1 is characterized in that described rice leafhopper of carrying fractilinea oryzae is used to study providing material base as mechanism mutually between fractilinea oryzae and paddy rice host and the amboceptor insect thereof.
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