First Report of Citrus yellow vein clearing virus on Lemon in Yunnan, China

Eureka lemon (Citrus limon) is an economically important crop in China. In 2009, a new disease was observed on Eureka lemon in an orchard of Ruili city in Yunnan Province. Nearly 10% of the plants were symptomatic with yellow vein clearing on lateral veins, combined with leaf crinkling and warping of young leaves. Water-soaked appearances were also seen on the adaxial leaf veins. The symptoms were present on mature leaves, resulting in almost 20% yield reduction. The symptoms on leaves were more severe in spring and autumn than in summer. This disease was transmitted by grafting from infected Eureka lemon to six lemon (C. limon) varieties (Femminello, Fino, Villafranca, Verna, Kutdiken, and Monachello) and two sour orange (C. aurantium) varieties (Daidai, Morocco), with inoculated plants showing similar symptoms. Variable symptoms were observed on other hosts, including severe yellowing and vein clearing on Dweet tangor (C. reticulata × C. sinensis), slight vein clearing on Tongshui 72-1 sweet orange (C. sinensis) and Marsh grapefruit (C. paradisi), but no symptoms were observed on Etrog citron (C. medica), Mexican lime (C. aurantifolia), or Ponkan (C. reticulata). When infected branches from Etrog citron, Mexican lime, and Ponkan were grafted onto healthy Morocco sour orange, typical yellowing and vein clearing symptoms were observed. In the mechanical transmission trials using eight herbaceous plant species, leaf rolling, shrinking, and pointed necrosis were observed on Vigna unguiculata, slight leaf rolling symptoms were observed on Capsicum frutescens, whereas Lycopersicum esculenum, Nicotiana benthamiana, Momordica charantia, Solanum melongena, Cucumis sativus, and Sesamum indicum remained symptomless. Symptom expression was influenced by temperature, being visible between 18 and 24°C but disappearing over 32°C. Crude extracts from young symptomatic lemon leaves were negatively stained for transmission electron microscopy observation (1) and showed filamentous virus particles of 13 to 15 × 400 to 1,000 nm. The described combination of symptoms in citrus species and herbaceous indicators, and virus morphology revealed by electron microscopy, suggested presence of Citrus yellow vein clearing virus (CYVCV). RNA extractions were made from 52 samples of Eureka lemon with yellow vein clearing symptoms. A one-step RT-PCR using primer pair (sense: 5′-TACCGCAGCTATCCATTTCC-3′ and antisense: 5′-GCAGAAATCCCGAACCACTA-3′), designed from coat protein gene of CYVCV genome (GenBank Accession No. JX040635) (1), was performed. All of the symptomatic samples yielded the expected cDNA fragment and no amplicon was obtained from healthy plants. Three PCR amplicons were selected for cloning and sequencing. BLAST analysis showed that these nucleotide sequences (KJ859679 to 81) had greater than 97% nucleotide identity with the corresponding region of CYVCV reference genome (2), and therefore, this disease was associated with CYVCV. Symptoms resembling CYVCV infection were observed in China before (3); however, to our knowledge, this is the first proven report of CYVCV present in China. The identification of the pathogen now allows for appropriate field management and control measures. References: (1) V. Grimaldi et al. 13th IOCV Conference, 343-345, 1996. (2) G. Loconsole et al. Phytopathology 102:1168, 2012. (3) C. Zhou et al. 18th IOCV Conference, 44, 2010.