Potato spindle tuber viroid detected in seed of uncultivated Solanum anguivi, S. coagulans and S. dasyphyllum collected from Ghana, Kenya and Uganda

Adapting Agriculture to Climate Change (Crop Wild Relatives) is a project, jointly managed by the Global Crop Diversity Trust and the Millennium Seed Bank, Royal Botanic Gardens, Kew (RBG Kew). As part of this project, seed of 30 wild taxa related to Solanum melongena (aubergine) were collected from seven countries. These Solanum species grow wild in Africa and are used for food and/or medicinal purposes. No virus or viroid symptoms were seen on the plants at collection. Initial cleaning and drying of collected seed was carried out in-country before being shipped to RBG Kew, where debris was removed, and the samples x-rayed, counted and sealed in universal glass bottles and stored at -20°C.

In June 2018, 98 seed samples (100 seeds per sample) of the solanaceous species were sent from RBG Kew to Fera Science Ltd. to be tested for Potato spindle tuber viroid (PSTVd) to facilitate export to the World Vegetable Center, Taiwan for pre-breeding research. This pre-breeding research is the initial step aimed at identifying important genetic diversity within crop wild relatives to enable subsequent breeding of domesticated crops that are better adapted to future climatic conditions. Seed samples were of eight species, namely Solanum anguivi, S. anomalum, S. cerasiferum, S. coagulans, S. dasyphyllum, S. incanum, S. macrocarpon and S. virginianum. Solanum anguivi and S. dasyphyllum were considered priority species in the current project, due to the lack of existing collections.

Due to the small consignment size, an initial screen was done on a subsample of 10 seeds per sample. RNA was extracted from the seeds using a magnetic bead extraction method and tested by real-time RT-PCR using an assay that detects PSTVd, Tomato chlorotic dwarf viroid and Tomato planta macho viroid (Boonham et al., 2004). Eight of the samples tested positive using this assay: five samples of S. anguivi, two of S. dasyphyllum and one of S. coagulans. To identify the species of pospiviroid detected, RNA extracts were tested by RT-PCR with a Verso 1-step RT- PCR Reddymix kit (Thermo Scientific, USA) using 3H1-F and 2H1-R primers (Shamloul et al., 1997) and pospi 1 FW and pospi 1 RE primers (Verhoeven et al., 2004).  Bands were detected of the correct size (c. 196 bp) for all eight samples using the Verhoeven et al. (2004) primers and for three samples using the Shamloul et al. (1997) primers (c. 360 bp). The resulting PCR products were sent for sequencing using the above primer sets (Sequiserve, Germany). PSTVd was confirmed in all eight samples and the sequences added to GenBank (Table 1).

Herbarium specimens collected alongside the seed collections were also tested by real-time RT-PCR using the Boonham et al. (2004) assay. Two of the herbarium samples tested positive and were tested by RT-PCR using the Verhoeven et al. (2004) assay. The resulting PCR products were sent for sequencing, PSTVd was identified and sequences added to GenBank (Table 1). The available sequence for the related herbarium and seed samples shared 98-99% homology.

This is the first report of a pospiviroid being detected in S. anguivi, S. coagulans and S. dasyphyllum, confirming the potential for pospiviroids to be distributed through non-commercial seed. There are very few reports of PSTVd in Africa; the viroid was detected in Egypt in potato and in Nigeria (CABI, 2018) and in Ghana in tomato (Batuman et al., 2019). These data indicate that PSTVd may be more widely distributed than suggested by current reports. PSTVd-infected seed collections and related herbarium specimens were destroyed or transferred under quarantine licence to Fera Science Ltd. for research purposes.