Comparing RT-PCR of individual samples with high throughput sequencing of pooled plant samples for field-level surveillance of viruses in blackberry and wild Rubus.

Blackberry production is increasing in the southeastern U.S. with the availability of new cultivars. In addition to high production costs, growers are challenged by virus diseases. Blackberry yellow vein disease (BVYD) significantly limits blackberry production. BYVD is associated with the crinivirus blackberry yellow vein-associated virus (BYVaV) in mixed infections with other viruses. The specific disease etiology and ecological factors underlying BYVD are not well understood and rely on the effective diagnosis of several viruses involved in the complex. In 2021, we collected samples from blackberry plants showing BYVD symptoms, asymptomatic blackberry plants, and wild Rosaceae spp. from nine farms across South Carolina, for a total of 372 individual plant samples. RNA from individual samples was isolated and pooled into sample groups (i.e., symptomatic, asymptomatic, and wild) from each farm for a total of 24 pooled samples. We sequenced the pooled RNA using Illumina and analyzed sequence profiles using the Virtool bioinformatics application. We also tested each plant for six viruses by RT-PCR or RT-qPCR and compared plant (PCR)-level and field (high throughput sequencing (HTS))-level data. Virtool detected 17 known viruses in the pooled samples, including 11 blackberry viruses. PCR testing was mostly consistent with HTS, with some notable disagreements for specific viruses. Our study demonstrates that HTS could be used as an efficient tool to detect viruses in bulked samples in blackberry fields, though limitations to using HTS for field-level surveillance are also discussed here.

First report of Alternaria cinerariae causing leaf blight on Farfugium japonicum in South Carolina, USA.

Farfugium japonicum, commonly known as leopard plant, is a popular perennial used in landscapes in the Southeastern U.S. In March 2022, leaf blight was observed on 20 leopard plants at a landscape site in Georgetown Co., SC. Almost all leaves were infected. Symptoms included purple to brown necrotic leaf spots and blighted petioles. Large spots had concentric circles and coalesced causing entire leaves to blight. Leaf pieces surrounding necrotic spots were excised, sterilized in 10% bleach for 1 min, rinsed in sterile water, placed onto potato dextrose agar (PDA), and incubated at 25°C. A total of three Alternaria isolates, 22-094-A, 22-094-B, and 22-094-C were obtained by transferring hyphal tips to new plates. All isolates had identical morphological traits. Colonies on PDA were blackish at the center and brownish at the edge. Conidia were produced using a technique described by Shahin and Shepard (1979). Conidiophores were mostly short and unbranched. They were characterized by solitary conidia or short chains of two to three conidia. Conidia (n=30) were obpyriform to obclavate and averaged 88.5 ± 26.1 µm in body length, 118.4 ± 36.3 µm in total length, and 23.9 ± 5.9 µm in width. They had 3 to 7 transverse septa and 0 to 4 longitudinal septa. Beaks were broadly tapered. Sequence of the internal transcript spacer (ITS) region of isolate 22-094-A (GenBank Accession No. OP481973) had 100% homology to that of CBS 116495 (KC584190), a representative strain of A. cinerariae (Woudenberg et al. 2013). Based on the morphological and sequence characters, the casual fungus was identified as A. cinerariae. Pathogenicity confirmation was done in two separate assays. In a detached-leaf assay, mature leaves were collected from 5-year-old F. japonicum 'Gigantea' plants. Five leaves (abaxial surface) were sprayed with a mixture of conidial suspensions of the three isolates at 300 conidia per mL and 1.5 mL per leaf, while sterile water was used for a non-inoculated control leaf. Leaves were placed in a plastic tray with wet paper towels. The tray was placed at 22°C for an 8-h photoperiod and covered for 3 days to maintain moisture. Small purple to brown spots were visible on inoculated leaves 2 days after inoculation (DAI). More than 90% of inoculated leaf areas were blighted 10 DAI, whereas the control leaf remained asymptomatic. In a whole-plant assay, three F. japonicum 'Argenteo Marginata' plants grown in 10-inch pots were placed in a plastic tray and sprayed with a conidial suspension of 22-094-A onto both abaxial and adaxial surfaces at 300 conidia per mL and 40 mL per plant. The tray was maintained as described above. Sterile water was used for a non-inoculated control plant. Small leaf spots appeared on the inoculated plants 2 DAI. Large necrotic areas developed on leaves and girdled petioles causing aboveground tissues to collapse 4 DAI. All inoculated leaves were blighted 7 to 10 DAI, while the non-inoculated control plant remained healthy. Each assay was repeated once. Alternaria cinerariae, identified by distinct morphology traits (Nishikawa and Nakashima 2015), was consistently re-isolated from inoculated leaves in both assays. Leaf spot on F. japonicum caused by A. cinerariae has been reported in CA, USA (Woudenberg et al. 2013) and Japan (Sakoda et al. 2010). This is the first report in SC, USA. This fungus also infects at least 25 other hosts (Farr and Rossman 2022). This disease may pose a threat to leopard plants in nurseries and landscapes under conducive conditions. Disease management strategies are warranted.