CN105724001A - Indoor high-flux maize rough dwarf disease artificial inoculation identification method - Google Patents
Indoor high-flux maize rough dwarf disease artificial inoculation identification method Download PDFInfo
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- CN105724001A CN105724001A CN201610058533.4A CN201610058533A CN105724001A CN 105724001 A CN105724001 A CN 105724001A CN 201610058533 A CN201610058533 A CN 201610058533A CN 105724001 A CN105724001 A CN 105724001A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
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Abstract
The invention provides an indoor high-flux maize rough dwarf disease artificial inoculation identification method. The method includes the steps that inoculum poisoned laodelphax striatellus and a certain scale of maize seeds are prepared, inocula of the corresponding number are inoculated in the early germination stage, after certain time, the incidence is investigated, and the resistance level is evaluated. Because of the unique rice black-streaked dwarf virus indoor living body preservation technology of the method, the requirement for needed number of laodelphax striatellus for indoor inoculation is greatly met; because of precise experiment design, instability of natural inoculation is overcome, variety resistance identification time is prolonged, and the resistance identification experiment is further made standard; the inoculation scale is broken through, and experiment cost is greatly saved. The method can be used for maize variety resource exploration and breeding, resistance evaluation, resistance inheritance and numerous fields.
Description
Technical field:
The present invention relates to a kind of indoor high flux MRDV artificial infection idenfication method and in application agriculturally, belong to technical field of agriculture science.
Background technology:
MRDV is the worldwide virus disease of one of harm Semen Maydis, rice black-streaked dwarf virus (the Riceblackstreakeddwarfvirus of (Fjivirus) is belonged to by Reoviridae (Reoviridae) Fijivirus, RBSDV), Madrid Yue Ketuo virus (MaldeRioCuartovirus, MRCV) cause with three kinds of viruses of Maize rough dwarf virus (Maizeroughdwarfvirus, MRDV).Through China's MRDV separator order-checking Comparison Study is confirmed that the pathogen causing China's MRDV is rice black-streaked dwarf virus (RBSDV), this disease repeatedly outbreak of epidemic on China's Semen Maydis once in history, according to statistics, particularly overlapped in the summer corn district of the provinces such as Hebei, Shandong, Shanxi, Jiangsu since 2005 and broadcast or late spring is broadcast, early occurrence injury is serious on Summer Maize；Within 2008, Shandong Province's occurring area reaches 73.3 ten thousand hm2, have no harvest 1.67 ten thousand hm2；The same year is also large area generation in Daliang City, and severe disease field sickness rate reaches 60%-80%, causes huge economic loss, and at present, rough dwarf disease has become as one of Major Diseases on China's Semen Maydis, and Maize Production is constituted serious threat.
The major measure of current MRDV preventing and treating is to adjust date of seeding, and it has played important function in disease control.But date of seeding adjustment has its limitation between interzone, kind, especially owing to the market demand limits the space of date of seeding adjustment on fresh edible maize, therefore cultivate and utilize disease-resistant variety to become the new demand in MRDV prevention and control.Large-scale Screening corn germ plasm resource is the basis cultivating resistant variety, and it is must measure that candidate's germ plasm resource carries out Resistance Identification.
MRDV is carried out germplasm identification mainly natural appraisal method and indoors artificial inoculated identification method two kinds at present.The former is subject to the constraint of natural conditions because of it, easily migrated quantity, impact with factors such as poison rate differences by environmental condition, small brown rice planthopper population, qualification result is differed greatly between interzone and year, accurate result must could be obtained through multiple years repetitive identified, not only add workload, and increase breeding time；The latter is on the former basis, a kind of indoors artificial inoculation identification method that the feature inhaled by small brown rice planthopper thorn according to this disease and propagate with persistent fashion interval grows up, and owing to the qualification of germ plasm resource needs the substantial amounts of poison small brown rice planthopper that passes, small brown rice planthopper artificial breeding's cost is high, raise that poison step is complicated and malicious source obtains limited time and causes that band poison small brown rice planthopper is difficult to obtain, it is difficult to meet the scale amount demand of Resistance Identification, thus greatly limit the popularization and application of small brown rice planthopper artificial infection idenfication.This laboratory is through research to small brown rice planthopper and transmitted virus thereof for many years, invent a kind of rice black-streaked dwarf virus indoor living store method (patent No.: ZL201210385208.0), the method can uninterruptedly obtain a large amount of high-band poison rate small brown rice planthopper throughout the year, meets the demand of a large amount of inoculum of indoors artificial inoculated identification；Additionally create a set of high flux indoors artificial inoculation identification method about this disease through groping, the method has grasped the sensitive period of Semen Maydis inoculation effectively, determine applicable inoculum concentration and appropriate inoculation time, thus substantially increasing inoculation efficiency and precision, save experimentation cost, screened for the Resource exploitation of the anti-rough dwarf disease of Semen Maydis, breed breeding and genetics of resistance law study etc. provide effective technological means.
Summary of the invention:
The present invention is directed to that current MRDV nature inoculated identification accuracy is low and artificial infection idenfication band poison small brown rice planthopper scale amount such as is difficult at the difficult problem, it is provided that a kind of indoor high flux MRDV artificial infection idenfication method.
It is a feature of the present invention that: in room conditions, utilizing the band poison small brown rice planthopper that long-term indoors artificial is raised is inoculum, the corn seed of certain scale quantity is in a kind of specific inoculator, inoculum in rudiment initial vaccination respective amount, after inoculation certain time, transplant and be cultured to the aobvious disease of morbidity in basin alms bowl or big Tanaka, evaluate the corn variety to be measured resistance level to MRDV with sickness rate height.
In the above-mentioned methods, " long-term indoors artificial raise with poison small brown rice planthopper " be the method long-term artificial breeding in indoor according to patent " rice black-streaked dwarf virus indoor living store method (patent No.: ZL201210385208.0) " with poison rate small brown rice planthopper colony more than 30%.
Described " corn seed of certain scale quantity " refers to and broadcasts effective corn seed 10～100 in single container.
Described " a kind of specific inoculator " refers to the circular of internal diameter 4.5～10cm, high 6～14cm or square light transmission container.
Before in described " inoculum in rudiment initial vaccination respective amount ", " rudiment initial stage " refer to that corn seed is sprouted and ejected bud scale to first blade；When " inoculum of respective amount " is according to inoculation, the quantity of seed germination time length and corn seed accesses the small brown rice planthopper of respective numbers, and average inoculation band poisonous insect amount is 0.1 head/plant to 0.9 head/plant.
Described " inoculation certain time " refers to inoculation Semen Maydis 49～72 hours.
Compared with existing MRDV identification technology, a kind of indoor high flux MRDV artificial infection idenfication method provided by the invention, it has plurality of advantages.First, the preservation of the indoor long-term live body of inoculum a large amount of high-band poison small brown rice planthopper effectively overcomes small brown rice planthopper and obtains, from ill plant, the technical bottleneck that its plant of poison is difficult to preserve for a long time, has widened the time identified, has met the demand of the germplasm identification inoculation scale of construction；Secondly, specific inoculation container " the circular or square light transmission container of internal diameter 4.5～10cm, high 6～14cm " ensure that the healthy growth of certain scale corn seed because of its stronger light transmission；Again, repetition test has grasped the sensitive period that the Semen Maydis rudiment initial stage is inoculation, it is determined that effectively inoculation worm amount is 0.1 head/plant～0.9 head/plant, and inoculation time is 49～72 hours, substantially increases inoculation efficiency.This authentication method not only achieves the high flux demand of MRDV indoors artificial inoculated identification, it is applied to excavation and selection-breeding, the resistance applicating evaluating of corn resources, apply also for the various fields such as phenotypic evaluation and genetics of resistance law study of localization of disease resistance genes, thus providing strong technical support for Semen Maydis research.
Detailed description of the invention:
Further illustrating the present invention below by specific embodiment, method therefor is conventional method if no special instructions.
The infrastructure raising small brown rice planthopper and artificial vaccination in following example is as follows:
There is the insectary of homoiothermic and light irradiation apparatus, keep the temperature between 25～28 DEG C and ensure the light application time of 12h every day；The glass beaker of 50～1000mL, nylon gauze (mesh specification 1mm), rustless steel support worm frame, microcomputer time-controlled switch, the Oryza sativa L. feeding small brown rice planthopper and wheat seed (Oryza sativa L.: Wu-Yu-Geng 3；Semen Tritici aestivi: Yangmai No.158)；The transfer black cloth of small brown rice planthopper, brush pen, pest sucking device etc..
Embodiment 1, the corn variety indoors artificial inoculation identification method to MRDV resistance
1. the acquisition of certain scale high-band poison (RBSDV) small brown rice planthopper.
In insectary, obtain high-band poison rate small brown rice planthopper (band poison rate 30% more than) according to the method in patent " rice black-streaked dwarf virus indoor living store method (patent No.: ZL201210385208.0) " throughout the year, " a kind of method (patent No.: ZL201410298198.6) of indoor mass rearing small brown rice planthopper raises high-band poison small brown rice planthopper on a large scale to recycling patent in indoor, can obtain high-band poison (RBSDV) small brown rice planthopper of certain scale.Research shows 10m2Insectary can indoor a collection of raising about 50000, with the small brown rice planthopper of poison rate more than 30%.
2. the determination of utensil is inoculated
Adopt 100,200 and 300mL plastic cup, 50,250,500 and 1000mL glass beaker etc. carry out corn seed seedling growth test, variant through comparing corn seed germination rate in different vessels, but difference is not notable, therefore can select as required, namely selecting internal diameter 4.5～10cm, the transparent vessel of high 6～14cm is nursery inoculation utensil.
3. the determination of inoculum concentration
Inoculation intensity arranges 0.1～0.9 head/plant of totally 9 process by effectively inoculation worm amount (effectively inoculation worm amount=inoculation worm amount × band poison rate), repeats 3 times.36 seeds of susceptible variety Soviet Union 951 are soaked in 250mL beaker, after 24h, water are outwelled and rinse seed 2～3 times with clear water, be sowed in the 50mL beaker being covered with 1/4 fine sand, 12/glass, it is placed in temperature 25 DEG C, the illumination box of illumination 12h grows.Inoculated identification (eliminating the seed that growing way is weak before inoculation, select 10 consistent strains of growth for inoculated identification) is carried out when corn seed is sprouted and ejected bud scale to first blade.Access and catch up with every day after small brown rice planthopper worm 2 times to guarantee that tested Semen Maydis uniformly obtains poison, remove small brown rice planthopper after inoculation 49h, tested maize seedling is transplanted to land for growing field crops, normal water and fertilizer management.Institute an inquiry sickness rate after 30 days, within every 7 days, investigate once, successive survey 3 times.Symptom main manifestations is plant dwarfing, and leaf color is dark green, and blade is stiff, wide short and thick, and lobus cardiacus can not normally launch, and blade back vein produces wax protuberance striped, touches with hands and have obvious harsh feeling.Experiments show that incidence such as table 1 of corn variety Soviet Union 951 under different vaccination intensity.Along with the increase connecing worm intensity, the sickness rate of Soviet Union 951 increases, and effectively inoculation intensity is do not have notable difference between 0.1～0.9 head/plant of process, inoculates the seedling age phase at this, and inoculum concentration can be identified as 0.1～0.9 head/plant.
The incidence of corn variety Soviet Union 951 under table 1 different vaccination intensity
Note: different letter representation variance analyses difference in 1% (capitalization) level extremely notable (table 2, table 3 and table 4 are same).
4. the determination of seedling age is inoculated
Inoculation seedling age arranges bud scale phase (Seed sprouting ejects bud scale to first blade), 2 leaf 1 heart stages, 4 leaf 1 heart stage 3 process, selects susceptible variety Soviet Union 951, each process effective Seedling 10 strain, repeats 3 times.Inoculation intensity is effectively inoculation worm amount 0.1 head/plant, 0.5 head/plant, 1 head/plant and 2 heads/plant 4 process, does an orthogonal test.Inoculation and investigation method are ibid.Small brown rice planthopper incidence such as table 2 of Semen Maydis different larval instar inoculation varying number.When Semen Maydis inoculation age is in the bud scale phase, when inoculation intensity is 0.1 head/plant, 0.5 head/plant, 1 head/plant and 2 heads/plant 4 process, sickness rate is more than 90%, is absent from significant difference between variant inoculation intensity；And along with the increase of inoculation seedling age, inoculation intensity also needs corresponding increase.The sensitive period considering seedling age and the principle saving worm amount determine that inoculation seedling age is the bud scale phase, and it is comparatively suitable namely to inoculate when Seed sprouting to first blade ejects bud scale.
The small brown rice planthopper incidence of table 2 different larval instar Semen Maydis inoculation varying number
5. the determination of inoculation time
Inoculation time arranges 24h, 48h, 72h and 96h totally 4 process, selects susceptible variety Soviet Union 951, each process effective Seedling 10 strain, repeats 3 times.Inoculation intensity is worm amount 0.5 head/plant of effectively inoculating, and inoculation seedling age is the bud scale phase, and inoculation and investigation method are ibid.Incidence such as table 3 of kind Soviet Union 951 under the different vaccination time.Along with the sickness rate increasing Soviet Union 951 connecing the worm time also improves constantly, result shows do not have significant difference between inoculation three time period sickness rate of 48h, 72h and 96h, so inoculation time is defined as 49～72h during the bud scale phase.
The incidence of corn variety Soviet Union 951 under the table 3 different vaccination time
6. scale is inoculated
One batch, the existing insectary, this room according to mentioning in 1. can raise about 50000, small brown rice planthopper with poison rate more than 30%, in conjunction with the inoculation seedling age bud scale phase 2., 3., 4. determined, inoculation intensity 0.1 head/plant, inoculation time 72h calculates, and can inoculate 50000 (head) × 30% (band poison rate) ÷ 0.1 (head/plant)=150000 strain maize seedlings for every batch.
The present invention serves primarily in the Resistance resource carried out for MRDV and screens excavation, the qualification of disease-resistant variety, evaluation and selection-breeding.This technology also in the research work of the hereditism of disease resistance of plant and anti-rice black-streaked dwarf virus transgenic, as biological detection method accurately, can will can greatly speed up the process of this area research simultaneously.
The comparison of embodiment 2, artificial infection idenfication and severe disease district field test effect
Select severe disease district, Kaifeng as identifying garden, in seeding corn kind Zheng Dan 958 on May 23rd, 2015.On Maize Seedling, small brown rice planthopper worm amount is 420/100 strain, band poison rate 3.5%.In field, the 1st onset peak period investigation incidence, 7 days repeated surveys 1 time, calculate sickness rate.Artificial infection idenfication and field test result are in Table 4.Without significant difference between the artificial infection idenfication of corn variety Zheng Dan 958 and severe disease district field test result, illustrate that the artificial infection idenfication method that this research is set up can simulate severe disease district field test effect, its evaluation of resistance reliable results.
Table 4 artificial infection idenfication and field test result
Above-mentioned enforcement does not limit the present invention in any form.
Claims (6)
1. an indoor high flux MRDV artificial infection idenfication method, it is characterized in that: in room conditions, utilizing the band poison small brown rice planthopper that long-term indoors artificial is raised is inoculum, the corn seed of certain scale quantity is in a kind of specific inoculator, inoculum in rudiment initial vaccination respective amount, after inoculation certain time, transplant and be cultured to the aobvious disease of morbidity in basin alms bowl or big Tanaka, evaluate the corn variety to be measured resistance level to MRDV with sickness rate height.
2. a kind of indoor high flux MRDV artificial infection idenfication method according to claim 1, it is characterised in that described long-term indoors artificial raise with poison small brown rice planthopper be method long-term artificial breeding in indoor according to patent " rice black-streaked dwarf virus indoor living store method (patent No.: ZL201210385208.0) " with poison rate small brown rice planthopper colony more than 30%.
3. a kind of indoor high flux MRDV artificial infection idenfication method according to claim 1, it is characterised in that the corn seed of described certain scale quantity refers to broadcasts effective corn seed 10～100 in single container.
4. a kind of indoor high flux MRDV artificial infection idenfication method according to claim 1, it is characterised in that the specific inoculator of described one refers to the circular of internal diameter 4.5～10cm, high 6～14cm or square light transmission container.
5. a kind of indoor high flux MRDV artificial infection idenfication method according to claim 1, it is characterised in that before the described rudiment initial stage in the inoculum of rudiment initial vaccination respective amount refers to that corn seed is sprouted and ejected bud scale to first blade；When the inoculum of respective amount is according to inoculation, the quantity of seed germination time length and corn seed accesses the small brown rice planthopper of respective numbers, and average inoculation band poisonous insect amount is 0.1 head/plant to 0.9 head/plant.
6. a kind of indoor high flux MRDV artificial infection idenfication method according to claim 1, it is characterised in that described inoculation certain time refers to inoculation Semen Maydis 49～72 hours.
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| CN109971823A (en) * | 2019-03-28 | 2019-07-05 | 江苏省农业科学院 | A kind of hole tray ridging method corn rough dwarf artificial infection idenfication method |
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Application publication date: 20160706 |