First Report of Beet western yellows virus Infecting Epiphyllum spp

Abstract

Epiphyllum spp. (orchid cactus) is a group of epiphytic plants in the cactus family grown as flowering ornamentals. Symptoms of virus infections are frequently observed in them (Chessin and Hammond 2002). In 2015, an Epiphyllum hybrid with chlorotic spots was found in California. Twelve more Epiphyllum hybrids (six with chlorotic ring spots, mottle, or mosaic and six asymptomatic) were obtained from a commercial nursery in New York. These plants were tested by RT-PCR/PCR using specific primers for Cactus mild mottle virus, Cactus virus X (CVX), Impatiens necrotic spot virus, Saguaro cactus virus, Zygocactus virus X (ZVX), or group primers for badnaviruses and potyviruses. Two potexviruses, CVX and ZVX, were detected from three and one symptomatic hybrids, respectively. The California hybrid and seven New York hybrids (two symptomatic and five asymptomatic) tested positive for a badnavirus. However, no target viruses were detected in two of the seven symptomatic hybrids. To further investigate these observations, total RNAs were extracted from stem tissue of four symptomatic hybrids (one CVX-positive, one ZVX-positive, and two PCR-negative plants) and one asymptomatic hybrid (cv. Professor Ebert) using RNeasy Plant Mini Kit (Qiagen). A pooled extract from the five samples was subjected to high-throughput RNA sequencing (SeqMatic). Analysis of the RNA reads revealed the presence of Alternanthera mosaic virus, Beet western yellows virus (BWYV), and five new viruses (one ampelovirus, one badnavirus, two distinct carlaviruses, and one caulimovirus) in addition to CVX and ZVX. Virus infections in all 13 cacti were further tested for the seven viruses (two known and five new viruses identified in this study) by RT-PCR/PCR using virus-specific primers. Results showed that the symptomless hybrid Professor Ebert was infected with five viruses (BWYV, an ampelovirus, two carlaviruses, and a caulimovirus), the most among the all hybrids. BWYV was detected by RT-PCR using primers BWYVf4972 (5′-TCTGAAGGTCGGTGAAGGAGG-3′) and BWYVr5665 (5′-ACAATGGGTAACACTCGTTGC-3′) only in Professor Ebert. To obtain the genomic sequence of this BWYV isolate, RT-PCR was conducted using seven overlapping primer pairs designed according to the alignment of the BWYV type species (GenBank no. NC_004756) and the contig obtained in this study. The RT-PCR amplicons were cloned into pGEM-T easy vector (Promega) and sequenced (MCLAB). A nearly full genomic sequence of 5,458 nt (MF543022) was assembled from seven overlapped amplicon sequences. BLAST search showed that this isolate had the highest nt sequence identity of 93% with a BWYV isolate infecting Nepenthes mirabilis (KU521325) (Miguel et al. 2016). Stem tips from the Professor Ebert plant were grafted onto four plants of hybrid King’s Ransom, which tested negative for the nine viruses included in this study. Three plants showed mild chlorotic speckling 6 months after grafting. BWYV was detected in new growth of the two grafted plants, indicating the virus was graft transmissible. Twenty additional Epiphyllum hybrids were tested by RT-PCR, and BWYV was detected from hybrids Tele Ann with mottle symptom and Vista Sun without symptoms. Thus, BWYV was detected in three different hybrids of Epiphyllum spp., an observation validated by RT-PCR, sequencing, and graft transmission. BWYV is an aphid-transmitted species of the genus Polerovirus in the family Luteoviridae. It has an extremely wide host range including legumes, crucifers, lettuce, and many weed species and can cause diseases in several crops worldwide (Duffus 1972). To our knowledge, this is the first report of BWYV in orchid cactus. It remains unclear whether BWYV causes symptoms in either single or mixed infections in other species and hybrids of Epiphyllum spp. However, these hosts can serve as a reservoir for the virus. We are currently characterizing the novel viruses detected in the hybrid Professor Ebert.