灰飞虱传播的一种小麦病毒病鉴定

Identification of a virus transmitted by small brown plant hopper in wheat

在实验室中利用灰飞虱接种小麦时出现一种新的病毒病症状，鉴定表明其病原为大麦黄条点花叶病毒（Barleyyellowstriatemosaicvirus，BYSMV）。采用生物学测定、电镜观察、RT—PCR检测和序列分析的方法，明确了该病毒的粒体特性、危害症状及田间发生情况。接种试验表明该病毒通过灰飞虱传播，接种7-10d后小麦新生叶片出现黄色斑点、斑驳，继而发展成黄色条纹，叶片对生且细而窄，重病株新叶扭曲，叶鞘不能伸长，病株矮化。对小麦病叶超薄切片电镜观察，发现细胞质中存在大量弹状病毒粒子，病毒粒体大小为（315—353）mm×（46～57）nm。利用特异性引物从病株总RNA中扩增出565bp基因片段，序列同源性分析显示与大麦黄条点花叶病毒（Barleyyellowstriatemosaicvirus，BYSMV）Zanjan-1分离物聚合酶（L）基因对应序列的一致性为97％，与北方禾谷花叶病毒（Northerncerealmosaicvirus，NCMV）L基因对应序列的一致性为78％～79％。对采自河北邯郸、石家庄、保定、唐山的31株样品进行RT—PCR检测，25株检测到BYSMV，7株检测到水稻黑条矮缩病毒（Riceblackstreakeddwarfvirus，RBSDV），其中5株为2种病毒复合侵染，结果表明BYSMV的田间分布较广。系统发育分析表明BYSMV—Lab／TS／ZX／QY4个分离物与本研究的BYSMV亲缘关系密切。BYSMV是我国小麦上新发现的一种弹状病毒，并已形成危害，暂定名为小麦黄条纹矮缩病，应加强流行动态监测。

Distinct viral symptoms were observed on wheat inoculated with small brown plant hopper （SBPH） （Laodelphax striatellus Fallen, Homoptera： Delphacidae） in greenhouse, and the virus was identified as Barley yellow striate mosaic virus （BYSMV）. In this paper, biological assays and transmission electron microscopy observation were used to determine the characteristics of disease symptoms and viral particle morphology. Specific primer pairs were designed to identify the pathogen through RT-PCR assays and sequences were aligned with reported gene sequence of Barley yellow striate mosaic virus （BYSMV）, Rice stripe virus （RSV）, Rice black streaked dwarf virus （RBSDV） and Northern cereal mosaic virus （NCMV）. Further inoculation tests confirmed that BYSMV virus is transmitted by SBPH. The aviruliferous SBPH were fed on diseased wheat for 3 days and then transferred onto fresh wheat seedlings for about a 10-day incubation period and then were exposed to 2-3 leaf stage wheat seedlings for inoculation. The infected wheat plants developed typical symptoms including chlo- rotic spots, mosaic and yellow striation along the veins at 7 days post inoculation （dpi）. Most BYSMV-infected leaves presented opposite and narrow, and the stem elongation was also inhibited, resulting in plant stunting, leaves twisting and even the disappearance of aerial stems. Rhabdovirus-like particles were observed in the cyto- plasm through transmission electron microscopy of ultrathin sections of symptomatic wheat leaves, the identified particles were about （315-353）mm×（46-57）nm in size. The gene fragment was about 565 bp amplified with the primer pair BYSMV-L3F/L3R by RT-PCR. BLAST analysis showed that the sequences shared 97% nucleotide identity with the polymerase L gene sequence of BYSMV （GenBank Acc. No. FJ665628） isolate Zanjan-1, whereas only 78%-79% identity with NCMV （GenBank Acc. No. AB030277 and GU985153）. In recent years, plant stunting and flag leaf yellowing at the time of heading have been observed in many wheat fields in Hebei province. The samples with viral symptoms were collected from the wheat fields in Handan, Shijiazhuang, Baoding and Tangshan areas. RT-PCR detection revealed that 25 and 7 in the total 31 samples, respectively, were found to be infected with BYSMV and RBSDV. In addition, 5 samples presented a mixed infection with both viruses. Phylogenetic analysis of protein sequences using Neighbor-Joining （N-J） method indicated that the four isolates BYSMV-Lab/TS/ZX/QY were closely related to BYSMV isolate Zanjan-1. Our results indicated that BYSMV caused a severe disease in wheat and were widely distributed in these areas. Barley yellow striate mosaic virus induced a new disease in China and has led to the economic losses of wheat. It could be named tentatively wheat yellow striate dwarf disease and epidemic dynamic monitoring should be strengthened in the future.