Development of a Full-Length Infectious cDNA Clone of the Grapevine Berry Inner Necrosis Virus

Xudong Fan,Baodong Zhang,Fang Ren,Chen Li,Zunping Zhang,Yafeng Dong,Guojun Hu

doi:10.3390/plants9101340

Abstract

Grapevine berry inner necrosis virus (GINV) belongs to the genus Trichovirus in the family Betaflexiviridae. The GINV isolate LN_BETA_RS was obtained from a “Beta” grapevine (Vitis riparia × Vitis labrusca) exhibiting chlorotic mottling and ring spot in Xingcheng, Liaoning Province, China. To verify the correlation between GINV and grapevine chlorotic mottling and ring spot disease, we constructed an infectious cDNA clone of GINV isolate LN_BETA_RS using the seamless assembly approach. Applied treatments of agroinfiltration infectious cDNA confirmed systemic GINV infection of the Nicotiana occidentalis 37B by reverse transcription polymerase chain reaction (RT-PCR) and transmission electron microscopy, exhibiting chlorotic mottling symptoms on leaves. Infectious cDNA was also transmitted to new healthy N. occidentalis plants through rub-inoculation. Moreover, the cDNA clone was agroinfiltrated into “Beta” and “Thompson Seedless” grapevine plantlets, and the inoculated grapevines exhibited leaf chlorotic mottling and ringspot during the two years of observation. GINV-inoculated “Beta” grapevines had serious leaf chlorotic mottling and ringspot symptoms on the whole plant, while relatively few symptoms were observed on the leaves of agroinoculated “Thompson Seedless” grapevines in early spring and only weak ring spot gradually appeared later in the top young leaves. Our experiments fulfilled Koch’s postulates and revealed the causative role of GINV in grapevine chlorotic mottling and ring spot disease.

Highlights

China has one of the world’s leading grape-production areas, which was recently estimated to cover 725,100 ha
The fused fragments were transformed into competent Escherichia coli DH5a cells, and single clones were grown on the Luria Broth (LB) liquid medium supplemented with 50 mg/L kanamycin
Grapevine berry inner necrosis virus (GINV) was associated with the occurrence of chlorotic mottling and ring spot symptoms in most diseased grapevine cultivars in China [23], which satisfied the first of Koch’s postulates, in which the microorganism must be present in most, if not all, cases of the disease

Summary

Introduction

China has one of the world’s leading grape-production areas, which was recently estimated to cover 725,100 ha. Grapes are widely favored by consumers and fruit growers because they have high nutritional and economic values. Grapevines can be infected by close to 80 viral species, some of which affect vine health and cause serious economic losses [1,2]. 20 species of grapevine viruses have been reported in China, including grapevine leafroll-associated virus 1–4, 7 and 13, grapevine rupestris stem pitting-associated virus, grapevine fleck virus, grapevine fanleaf virus, grapevine virus A, grapevine virus B, grapevine virus E, grapevine virus T, grapevine Pinot gris virus (GPGV), grapevine berry inner necrosis virus (GINV), grapevine fabavirus (GFabV), grapevine rupestris vein feathering virus, grapevine geminivirus A, grapevine Syrah virus-1. Our previous studies, based on symptom investigations and virus identification in a large number of grapevine samples, showed that

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Conclusion