阅读说明：本技术 柑橘黄化脉明病毒弱毒分离株及其应用 (Citrus yellowed vein clearing virus attenuated isolate and application thereof ) 是由 宋震 宾羽 崔甜甜 周常勇 于 2019-09-30 设计创作，主要内容包括：本发明公开了一种柑橘黄化脉明病毒弱毒分离株,该弱毒株相对于强毒株产生的基因突变点是：TGB区域的T<Sup>5306</Sup>→C,A<Sup>5482</Sup>→G,T<Sup>5792</Sup>→C,A<Sup>6058</Sup>→G,C<Sup>6096</Sup>→T,以及位于CP基因上的T<Sup>6817</Sup>→C。还公开了该弱毒分离株在防治柑橘黄化脉明病毒强毒株系侵染方面的应用。本发明获得了柑橘黄化脉明病毒弱毒分离株AY001,AY001可有效保护植株免受CYVCV强毒株系的侵染。(The invention discloses a citrus yellow vein clearing virus attenuated isolate, wherein the gene mutation points of the attenuated strain relative to the virulent strain are as follows: t of TGB region 5306 →C，A 5482 →G，T 5792 →C，A 6058 →G，C 6096 → T, and T located in CP Gene 6817 → C. Also discloses application of the attenuated isolate in preventing and treating infection of citrus yellow vein clearing virus virulent strains. The invention obtains the separated strain AY001 of citrus yellow vein clearing virus with weak toxicity, and AY001 can effectively protect the plant from being infected by CYVCV virulent strain.)

1. A citrus yellow vein clearing virus attenuated isolate is characterized in that gene mutation points generated by the attenuated strain relative to a virulent strain are as follows: t of TGB region⁵³⁰⁶→C，A⁵⁴⁸²→G，T⁵⁷⁹²→C，A⁶⁰⁵⁸→G，C⁶⁰⁹⁶→ T, and T located in CP Gene⁶⁸¹⁷→C。

2. The attenuated isolate of citrus yellow vein clearing virus according to claim 1, which is preserved in the China general microbiological culture Collection center with the preservation registration number of CGMCC No. 18177.

3. Use of the weakly virulent isolate of citrus yellow vein clearing virus according to claim 1 or 2 for controlling infection by a strongly virulent strain of citrus yellow vein clearing virus.

Technical Field

The invention belongs to the technical field of viruses, and particularly relates to a citrus yellow vein clearing virus attenuated isolate and application thereof.

Background

Cross Protection (MSCP) refers to the protection of a plant from a different strain of the same virus or from another virus with a close relationship (i.e., a virulent or challenge strain) after infection with a strain of the virus (usually a lentogenic strain). The cross protection effect degrees of the plant viruses are different, if the challenge strains are directly inoculated and should show local necrosis symptoms, the asymptomatic expression of the challenge strains is called as complete protection effect after the interference of the viruses inoculated firstly; challenge strains are slightly proliferated and slight symptoms appear, so that the challenge strains belong to incomplete protection; if the symptoms are identical to those of the challenge strain when infected alone, the protection effect is completely non-protective. At present, the cross protection effect is mainly expressed in four aspects of inhibiting the replication of challenge viruses, limiting the movement of the challenge viruses, delaying the onset time of plants and reducing or not onset symptoms. In productive practice, cross-protection is premised on the availability of a virulent strain.

There are three main traditional methods for obtaining attenuated strains: the plants with better growth conditions in the diseased groups are selected from plants with natural diseases, most probably infected with the attenuated strains to be protected, and the attenuated strains for preventing and treating citrus tristeza virus disease are discovered in the way; obtained by high-temperature mutagenesis; chemical mutagenesis. With the development of the plant virus infectious clone construction technology, researchers can obtain attenuated strains through site-directed mutagenesis on the basis of determining the virulence and genome functions of viruses. Weak-virulent strains such as ZYMV, PRSV, Clover yellow vein virus (CIYVV) and Bean Yellow Mosaic Virus (BYMV) constructed by the method are widely applied to practical production. In addition, the gene of the virus can be cloned and expressed by utilizing the infectivity of the plant virus, and the cross protection effect on the virus can be mediated. For example, Culver et al insert the CP gene sequence of TMV into a PVX infectious cDNA clone, and the recombinant plasmid, when inoculated into tobacco, can confer protection against subsequent challenge strain inoculation with TMV.

Citrus Yellow Vein Clearing Virus (CYVCV) is a new virus which is found to infect Citrus in 2009 in China, can cause symptoms such as bright vein, yellowing, leaf rolling and shrinkage of tender leaves of lemon and lime, and can cause tender leaf shedding and vein necrosis in severe cases to cause weak tree vigor and reduced fruit yield, and sometimes even be harvested; in recent years, the generation of the virus in citrus producing areas in China shows an increasing trend year by year. CYVCV can infect not only lemon, lime and citron, causing serious harm, but also sweet orange, citrus with broad peel, grapefruit and other citrus species. Meanwhile, CYVCV has high-efficiency insect vector transmission in nature, and the existence of CYVCV virus is detected in most citrus producing areas in China. Thus, CYVCV has a great risk of outbreak and has become an important problem affecting the citrus industry, especially the lemon industry, in our country.

CYVCV is a positive-sense single-stranded RNA virus, with virus particles in a curved line, belonging to the family of viruses of the family A-type Viridae (Alphaflexividae) genus Citrus indicus (Mandarivirus). CYVCV can be transmitted by Bemisia citrea and Aphis spiraecola (Aphis spiraecola, also can be transmitted by grafting and field farming operation tools). CyVCV genome contains 6 ORFs, wherein ORF2, ORF3 and ORF4 constitute a three-gene linked structure (TGB). at present, the prevention and treatment measures of the disease mainly comprise the application of nontoxic nursery stocks, the strict prevention and control of transmission insect vectors, the strict disinfection of farming tools in farming operations and the like. 1998) In octa-citrus, japan (Sasaki et al,1979), and grapefruit, in the united states (Cost and Muller,1980), australia (Broadbent et al, 1991), and south africa (Van et al, 2000), among others. However, the screening of attenuated strains aiming at CYVCV and the application thereof in cross protection are not reported yet.

Disclosure of Invention

The invention aims to solve the technical problem and provides a citrus yellow vein clearing virus attenuated isolate and application thereof.

The technical scheme of the invention is as follows:

a citrus yellow vein clearing virus attenuated isolate, the gene mutation point of the attenuated strain relative to the virulent strain is: t of TGB region⁵³⁰⁶→C，A⁵⁴⁸²→G，T⁵⁷⁹²→C，A⁶⁰⁵⁸→G，C⁶⁰⁹⁶→ T, and T located in CP Gene⁶⁸¹⁷→C。

The citrus yellow vein clearing virus attenuated isolate is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation registration number is CGMCC No. 18177.

The invention also provides application of the citrus yellow vein clearing virus attenuated isolate in preventing and treating infection of citrus yellow vein clearing virus virulent strains.

The invention has the beneficial effects that: the separated strain AY001 of the citrus yellow vein clearing virus with weak toxicity is obtained, and the AY001 can effectively protect the plant from being infected by CYVCV virulent strains.

Drawings

FIG. 1 is a representation of leaves of Eulek lemon inoculated with CYVCV attenuated strain AY001, wherein A is an inoculation negative control, B is an inoculation positive control, and C is inoculation AY 001.

FIG. 2 is a diagram of the alignment of the segmented multiple sequences of the attenuated strain and the virulent strain and the prediction of the secondary structure of MFE.

FIG. 3 is a graph of a segmented multiple sequence alignment of attenuated strains and virulent strains and MFE secondary structure prediction.

FIG. 4 is a graph of a segmented multiple sequence alignment of attenuated strains and virulent strains and MFE secondary structure prediction.

Detailed Description