In November 2012, plants of Russell prairie gentian (Eustoma grandiflorum, Lisianthus russellianus) were collected from a commercial greenhouse in Atibaia, SP, Brazil, displaying necrotic spots on leaves and necrosis on stems, followed by generalized systemic necrosis. Disease symptom incidence was estimated at 10%. Preliminary electron microscopy observations of negatively stained leaf extracts prepared from those lesions revealed the presence of a large number of spherical tospovirus-like, approximately 100 nm in diameter. Samples of infected leaves were ground in 0.01 M phosphate buffer containing 0.5% sodium sulphide and mechanically inoculated in six plants of each species of Nicotiana glutinosa, N. tabacum cv. White Burley, N. megalosiphon, N. debneyii, Datura stramonium, Chenopodium amaranticolor, C. quinoa, and E. grandiflorum. All inoculated plants displayed local lesions 4 to 5 days after inoculation, while N. debneyii and D. stramonium showed systemic symptoms, typical of Tospovirus infection. In addition, E. grandiflorum reproduced the original symptoms. Total RNA was extracted from infected E. grandiflorum and D. stramonium, and reverse transcription (RT)-PCR was performed using universal primers BR60 and BR65 (2) targeting conserved regions of the nucleocapsid gene (N). The amplification products of approximately 450 bp were purified, cloned, and sequenced. The unknown virus was identified as Chrysanthemum stem necrosis virus (CSNV-Lis) based on host range and nucleotide sequence (Genbank Accession No. KC894721) and showed 99% identity with a CSNV chrysanthemum isolate from Japan (AB600872). Maximum likelihood phylogenetic analysis using nine homologous CSNV sequences available in GenBank classified CSNV-Lis into a monophyletic group formed by chrysanthemum isolates from Japan and China while a Japanese lisianthus isolate was separately clustered. CSNV is a member of the genus Tospovirus (Bunyaviridae) and was first reported on chrysanthemum in Brazil (1) and later in the Netherlands, Slovenia, United Kingdom, and Japan (3). Despite scattered recent reports of CSNV, the simultaneous production of chrysanthemum and lisianthus crops along the year by Brazilian farmers has contributed to the virus maintenance in the field. The high identity between Brazilian and Japanese isolates of CSNV suggest a possible reintroduction of the virus through exchange of vegetative propagating material. References: (1) L. M. L. Duarte et al. J. Phytopathol. 143:569, 1995. (2) M. Eiras et al. Fitopatol. Bras. 26:170, 2001. (3) K. Momonoi et al. J. Gen. Plant Pathol. 77:142, 2011.
Callus culture and micropropagation of Eustoma grandiflorum Shinn.
