First Report of Basella rugose mosaic virus Infecting Four O'Clock (Mirabilis jalapa) in China.

Abstract

Four o'clock (Mirabilis jalapa) and M. himalaica var. chinensis are members of the family Nyctaginaceae and are widely distributed weeds in Yunnan Province, China. In 2009, mosaic and malformation symptoms were observed on leaves of the four o'clock on the campus of Yunnan Agricultural University and in the Black Dragon Pool Park in Kunming City, China. More than 30% of the four o'clock plants showed symptoms of the disease. Sap from leaves of symptomatic four o'clock plants caused local chlorotic and necrotic lesions in inoculated Chenopodium amaranticolor after 7 to 10 days and systemic mosaic symptoms in C. quinoa and Nicotiana benthamiana after 10 to 12 days. No symptoms were observed following inoculation of sap from asymptomatic plants. A pure virus isolate (MJ) was obtained after three successive single-lesion transfers from C. amaranticolor. Following mechanical inoculation of the MJ isolate, seedlings of indicator plants, N. benthamiana, displayed mosaic symptoms. Moreover, back transmission to healthy four o'clock seedlings by leaf extracts from systemically infected N. benthamiana plants caused similar mosaic and malformation symptoms. Flexuous, filamentous particles (650 to 700 nm long and 13 nm wide) and cytoplasmic laminar aggregates and pinwheel inclusions typical of members of the genus Potyvirus were observed in infected four o'clock leaves by electron microscopy. No other virus particles were observed. Serological testing of 10 symptomatic and healthy plants using a monoclonal antibody specific for Potyvirus group members in an indirect ELISA (Agdia Inc., Elkhart, IN) also resulted in positive reactions in infected leaves, however, all healthy seedlings tested were negative. Total RNAs were extracted from infected four o'clock leaves with the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) and the 3'-terminal portion of the viral genome (including part of the NIb polymerase, the entire coat protein (CP), and 3'-UTR) was then amplified by reverse transcription-PCR with a universal Potyviridae primer Sprimer/M4 and an M4T as the initial primer (2). A fragment of 1,720 nucleotides long were separated, purified, and cloned and three independent clones were sequenced (GenBank Accession No. JN250997). Nucleotide and amino acid sequence analysis of the putative CP gene, respectively, revealed 75.1 to 76.3% and 80.3 to 82.1% identity with the Basella rugose mosaic virus (BaRMV) (GenBank Accession Nos. DQ821938, DQ394891, and DQ821939), 77.4 and 81.0% identity with Peace lily mosaic virus (GenBank Accession No. DQ851494), and 76.0 and 81.7% identity with the Phalaenopsis chlorotic spot virus (GenBank Accession No. HM021142). However, on the basis of the CP gene sequence analyses, these three viruses shared high (>88.5 and >94.3%) CP nucleotide and amino acid identity and should be classified as the same Potyvirus species. According to the species demarcation criteria for the Potyviridae (1), the pathogen causing mosaic and malformation symptoms on four o'clock was BaRMV (3). To our knowledge, this is the first report of BaRMV in four o'clock.