CN105794561B

CN105794561B - Rape element azoles improves the application and method of Rice Resistance rice black-streaked dwarf virus disease

Info

Publication number: CN105794561B
Application number: CN201610167152.XA
Authority: CN
Inventors: 孙宗涛; 何宇青; 张合红; 李俊敏; 洪高洁; 陈剑平
Original assignee: Zhejiang Academy of Agricultural Sciences
Current assignee: Zhejiang Academy of Agricultural Sciences
Priority date: 2016-03-23
Filing date: 2016-03-23
Publication date: 2019-01-01
Anticipated expiration: 2036-03-23
Also published as: CN105794561A

Abstract

Present invention firstly discovers that rape element azoles can be used for improving Rice Resistance rice black-streaked dwarf virus disease, specific method is that paddy growth is cultivated in 1000mL beaker to two ~ tri-leaf period, and every glass of rice has 40 or so rice seedlings, shares four glasss.By 0.1% tritonX-100 solution and 1 μM of rape element azoles is diluted to 0.1% tritonX-100 being uniformly sprayed at small watering can on the rice leaf in two ~ tri-leaf period, primary every spray in 2 days, coprocessing is three times respectively.

Description

Rape element azoles improves the application and method of Rice Resistance rice black-streaked dwarf virus disease

Technical field

It is specifically a kind of to improve Rice Resistance rice black-streaked dwarf virus disease using rape element azoles the present invention relates to agriculture field Application and method.

Technical background

Rice black-streaked dwarf virus (Rice black-streaked dwarf virus, RBSDV) is Reoviridae (Reoviridae) Fijivirus category (Fijivirus) a kind of diplornavirus.Complete RBSDV particle is icosahedral structure of virus, Appearance is spherical in shape, diameter about 75nm, has double capsid.Respectively there is 12 protrusions, the viral genome in capsid in interior outer capsid It is made of 10 line style double-stranded RNAs (double strand RNA, dsRNA), by its electrophoretic mobility on gel by slowly extremely Successively it is known as S1~S10 fastly.RBSDV full-length genome is about 30kb, includes 12 open reading frame (open reading Frame, ORF), wherein this 6 albumen of P1, P2, P3, P4, P8 and P10 are the structural proteins of virion.P5-1, P6, P7-1, This 6 albumen of P7-2, P9-1, P9-2 are non-structural proteins.Rice black-streaked dwarf virus is mainly distributed on China, Japan and Korea Equal East Asia Regions.The virus mainly by small brown rice planthopper (Laodelphax Striatellus) as vector with persistence without Ovum mode is propagated, without soil, branches and leaves friction and seed dispersal.RBSDV natural host is in extensive range, mainly includes rice, jade More than totally 60 kinds of cereal crops and the gramineae weed such as staple food crops such as rice, wheat, barley.Pole is brought to agricultural production Big harm.RBSDV can cause plant teratogenesis after infecting plant host, and cardinal symptom shows as plant tillering and increases leaf color Occur white strumae on dark green and blade back and stalk, after become the billet warty protuberance of brown, fringe very little, setting percentage It is low.The virus causes rice black-streaked dwarf virus sick on the south the Changjiang river, causes corn rough dwarf in North of Yangtze River.In recent years, with Warming for weather, the population number for propagating the mediator insect small brown rice planthopper of RBSDV be significantly increased, cause black streaked dwarf virus of rice quick-fried Hair is popular, seriously endangers the safety in production of China cereal crops.Currently, the control method to black streaked dwarf virus of rice mainly sprays Insecticide is spilt to reduce the insect density for passing virus mediator small brown rice planthopper, but control efficiency is not very ideal, and caused potential Pesticide Residue, therefore find effective preparation and formulate reasonable measure to prevent and treat the disease very urgent.

Rape element azoles (Brassinazole, Brz) is a kind of specific inhibitor of BR synthesis, inhibits rape element sterol It is catalyzed 6- oxygen-campestanol in (Brassinosteroid, BR) route of synthesis and generates the activity for blocking the monooxygenase of its sterone. Rape element sterol is the important growth promoted type sterol parahormone of one kind of plant endogenous synthesis.Rape element sterol earliest by Michelle was extracted from rape pollen in 1970 a kind of can promote plant stem elongation and fissional high activity Substance.The rape element sterol of very low concentrations can induced strong growth and differentiation.Rape element azoles is mainly used in blocking plant BR route of synthesis shows corresponding metamorphosis for studying after plant lacks BR.However, there is no oily about utilizing at present The report of dish element azoles raising Rice Resistance RBSDV.

Summary of the invention

Rice Resistance rice black-streaked dwarf virus disease is effectively improved using rape element azoles the object of the present invention is to provide a kind of Using.

Application of the present invention is specially that rape element azoles dilution is sprayed on rice leaf.

Preferably, the concentration of the rape element azoles dilution is 1 μM.

Preferably, the rice leaf is the rice leaf for being in for two~tri-leaf period.

Preferably, the rape element azoles dilution is diluted with 0.1% tritonX-100.

Preferably, the period is sprayed are as follows: primary every spray in 2 days, coprocessing is three times.

Preferably, spraying capacity is to soak entire blade, i.e., until starting to drip on blade.Present invention firstly discovers that rape Plain azoles can be used for improving Rice Resistance rice black-streaked dwarf virus disease, and speculate to its mechanism, it is believed that external source rape element azoles is logical The resistance for improving rice itself is crossed, to generate resistance to RBSDV.

Detailed description of the invention

The rice plant (right side) that the rice plant (left side) and RBSDV of Fig. 1 health infect；

The detection of 2 rice plant band poison of Fig. 2 control group A；

Fig. 3 processing group B2 rice band poison detection case.

Specific implementation method

Example 1 obtains the method for carrying RBSDV small brown rice planthopper

1. the 5 age striatellus imagos that can be laid eggs of laboratory rearing are swept to 2- of the kind in 1000 milliliters of beakers first It lays eggs on the Wu-Yu-Geng 3 rice seedlings of 3 leaf ages (10-12d), scans out striatellus imago from beaker to come after 3-4 days.

2. 1-2 instar larvae can be hatched after striatellus imago is laid eggs 8-10 days, 1-2 instar larvae is swept into kind 5000 Poison is raised on the diseased plant of infection rice black-streaked dwarf virus in milliliter beaker, poison is raised after 3-4 days to be terminated.It should be noted that water The sick seedling of rice black streak dwarf poison, it is in advance that stem processing is clean, withered leaf sheath is cut, is exposed as far as possible than comparatively fresh Stem, because the stem that small brown rice planthopper mainly passes through drinking water rice sick seedling obtains poison.

3. the small brown rice planthopper for raising malicious 4 days to be transferred to the Wu-Yu-Geng 3 rice seedlings for the 2-3 leaf age (10-12d) broadcast in advance On, allow virus to cross the phase of walking around in small brown rice planthopper body, virus walks around to the time and is generally 12-15 days small brown rice planthopper is intracorporal, that is, obtains Carry the small brown rice planthopper of RBSDV.

Example 2tritonX-100 solution handles rice and raises healthy small brown rice planthopper and carry the small brown rice planthopper of RBSDV

It soaks seed 2 days 1. the rice varieties of poison waiting are placed in culture dish, vernalization 1 day, is sowed at 1L beaker after germination respectively In, 28 plants every glass, totally two glasss.

2. the tritonX-100 solution for preparing 0.1%, is then uniformly sprayed at the rice in two~tri-leaf period with small watering can On (Huaihe River rice No. 5) blade, primary every spray in 2 days, coprocessing is three times.

3. the amount sprayed is that solution soaks entire blade, i.e., until starting to drip on blade.

4. after handling 16 hours, carrying out rice with the small brown rice planthopper of the small brown rice planthopper of health and carrying RBSDV respectively and connecing poison.It presses Small brown rice planthopper is inoculated on No. 5 rice seedlings of Huaihe River rice of 3~4 leaf phases by effective inoculation worm amount of every 4 worm of seedling, and healthy small brown rice planthopper makees For control group A 1, the small brown rice planthopper of RBSDV A2 (table 1) as a control group is carried.

5. clearing away small brown rice planthopper after inoculation 3 days, and rice seedling is transplanted to greenhouse (28 DEG C, 16h illumination 8h is dark) training Support observation.

6. after culture 90 days, observing and counting control group A 1 and A2 rice plant height situation (being shown in Table 1) respectively.

3 rape element azoles of example handles rice and raises healthy small brown rice planthopper and carry the small brown rice planthopper of RBSDV

2. rape element azoles is diluted to 1 μM with 0.1% tritonX-100, then two are uniformly sprayed at small watering can~ On rice (the Huaihe River rice No. 5) blade in tri-leaf period, primary every spray in 2 days, coprocessing is three times.

3. the amount sprayed is that rape element azoles dilution soaks entire blade, i.e., until starting to drip on blade.

4. after handling 16 hours, carrying out rice with the small brown rice planthopper of the small brown rice planthopper of health and carrying RBSDV respectively and connecing poison.It presses Small brown rice planthopper is inoculated on No. 5 rice seedlings of Huaihe River rice of 3~4 leaf phases by effective inoculation worm amount of every 4 worm of seedling, and healthy small brown rice planthopper makees For processing group B1, the small brown rice planthopper of RBSDV B2 (table 1) as a control group is carried.

6. after culture 90 days, observation and respectively statistical disposition group B1 and B2 rice plant height situation (being shown in Table 1).

RBSDV viral diagnosis in 4 rice plant of example

The extraction of 4.1RNA and reverse transcription reaction

Connect malicious RBSDV sample be fully ground with liquid nitrogen flash freezer after, be added 1~2mL TRIzol, according to TRIzol reagent Box specification extracts, and appropriate amount of sample is taken to be fully ground with liquid nitrogen flash freezer, and the Trizol that 1~2mL is added continues to grind, and melts It is transferred in the centrifuge tube of 1.5mL afterwards；The chloroform-isoamyl alcohol (24:1) of 0.2mL is added, 5min is stood after mixing, at 4 DEG C 12000g is centrifuged 10min, and isometric chloroform-isoamyl alcohol is added after drawing supernatant, repeats the above steps primary；Supernatant is taken to be added Isometric isopropanol mixing is placed on room temperature 10min, and 12,000g is centrifuged 10min at 4 DEG C；It abandons supernatant and 1mL75% is added Ethyl alcohol is washed, and 12,000g is centrifuged 5min at 4 DEG C, is repeated the above steps primary；After appropriateness to be precipitated is dry, 20-40 is used The RNase-free water of μ L dissolves RNA.Reverse transcription reaction is then carried out using iScript cDNA Synthesis kit, is closed At the first chain cDNA.

Reverse transcription reaction system is as follows:

RNA sample	7.5μL(500ng)
		5x iScript reaction mix	2μL
iScript reverse transcriptase	0.5μL
		Total Volume	10μL

After sample blending, 25 DEG C of 5min, 42 DEG C of 30min, 80 DEG C of 5min, 4 DEG C of preservations.Obtain cDNA template be placed on- 20 DEG C save backup.

4.2RT-PCR detects RBSDV virus

Two pairs of primers are devised with the genome S10 of RBSDV, primer sequence is as follows:

RB-S10-F	AACAACCGACCAACAATCAC
		RB-S10-R	GAGCAGGAACTTCACGACAG

RT-PCR is carried out, according to PCR product size, sequence analysis and infection RBSDV positive control rice plant judgement Whether rice infects RBSDV.PCR system: 10 × buffer 1uL, dNTP 1uL, RB-S4-F and RB-S4-R each 0.3uL of primer, CDNA template 1uL, HiFi enzyme 0.3uL, adds dd H2O to mend to 10uL.PCR amplification condition are as follows: 94 DEG C of 3min；94 DEG C of 30s, 58 DEG C 30s, 72 DEG C of 30s, totally 35 recycle；Last 72 DEG C of 10min extend.

As a result as follows:

At 1.90 days, No. 5 plant A1 groups of Huaihe River rice and B1 group plant growing way are normal, the whole 28 plants of rice plant tables of control A2 group Symptom of now obviously stunting (see Fig. 1), and handling 28 plants of rice of B2 group has 13 plants to occur stunting symptom (being shown in Table 1), this shows rape Rice Resistance RBSDV can be significantly provided after plain azoles processing.

2. detecting rice plant band poison situation using RT-PCR method, the rice plant height in pcr amplification product and table 2 Unanimously, the ratio of A2PCR amplified production is 100% (see Fig. 2, in which: M, Marker；, healthy rice plant control；+, it takes Rice plant positive control with RBSDV；The plant of RBSDV is inoculated with after 1-28,0.1%Triton X-100 processing), B2 amplification Product has 13 plants (sharing 28 plants) (see Fig. 3, in which: M, Marker out；, healthy rice plant control；+, carry RBSDV's Rice plant positive control；1-28 is inoculated with the plant of RBSDV after the processing of rape element azoles).Therefore, there is symptom of stunting in rice As carry RBSDV plant.

1 rape element azoles of table handles rice and connects malicious scheme

Claims

1. rape element azoles improve Rice Resistance rice black-streaked dwarf virus disease application, which is characterized in that the application it is specific Method are as follows: rape element azoles is diluted to 1 μM with 0.1% tritonX-100, is then sprayed at the rice leaf in two~tri-leaf period On, it is primary every spray in 2 days.

2. application according to claim 1, which is characterized in that spraying capacity is to soak entire blade, i.e., starts on blade Until dripping.