First report of the ability of Olpidium virulentus to vector blueberry mosaic associated virus (BlMaV) on southern highbush blueberry in California

Blueberry mosaic associated virus (BlMaV, genus Ophiovirus), the suspected causal agent of blueberry mosaic disease (BMD), is thought to be vectored by fungi in the genus Olpidium (Sekimoto et al. 2011). In 2015, BlMaV was identified in nursery stock of southern highbush blueberry (Vaccinium corymbosum L.) in Watsonville, CA. Yellowish-pink leaf mottling was observed, which BlMaV typically induces. BlMaV was detected in symptomatic leaves using two virus-specific primer sets (targeting the viral coat protein and RNA polymerase) using RT-PCR techniques from Thekke-Veetil et al. (2014); however, no Olpidium resting spores were observed on plant roots. To isolate the fungal vector, we conducted a trap plant study using 1-month-old lettuce, carrot, broccoli, and cucumber plants (n = 5). Virus-free trap plants were grown in soil collected from the field where BlMaV-stock originated. After 8 weeks, only the lettuce plants appeared stressed and stellate Olpidium resting spores were observed on lettuce roots under a light microscope and identified as O. virulentus (OV). Lettuce roots were confirmed to be virus-free by using RT-PCR protocols described above. Lettuce roots were desiccated and used to inoculate six 3-month-old BlMaV- and OV-free blueberry plants (selection 92D9). Each plant was placed in a 4-inch pot containing 400 g of soil mixed with 0.02 g of dried lettuce roots per pot and grown at 23°C with a 16/8 h day/night cycle. These plant roots were tested for OV at weeks 2, 4, and 6 by extracting DNA using phenol/chloroform (n = 6). To increase sensitivity, a nested PCR protocol was performed with internal transcribed spacer region primers (ITS1, ITS4), followed by the multiplex PCR protocol outlined in Herrera-Vásquez et al. (2009). OV resting spores were first detected in the blueberry roots at 6 weeks. A 575-bp PCR product was amplified and sequenced (KY905661), which shared 99% nucleotide homology (530/534 bp) to OV from Japan (AB205203). The infested blueberry roots were dried and 0.02 g per pot was used to inoculate 22 BlMaV-positive blueberry plants using the protocol described above. The roots were found to be positive for OV after 6 weeks using PCR. These BlMaV-positive plants with OV in the roots (treatment 1), healthy negative control plants (treatment 2), and propagated BlMaV-positive plants with no OV in the roots (treatment 3), were grown and deliberately flooded every 10 days for 50 days (n = 22 per treatment). All of the runoff water from each treatment was collected separately and used to inoculate BlMaV- and OV-free blueberries every 10 days (n = 6 per treatment). After 5 weeks, three roots and leaves were tested per plant for the presence of BlMaV and OV using the virus and vector amplification protocols described above. Only plants that received water from treatment 1 (OV + BlMaV) tested positive for the virus and vector and 50% of leaves were positive for BlaMV and 83% of roots were positive for OV and BlMaV. All other plants treated with water from treatments 2 and 3 tested negative; no symptoms were observed on the infected plants. Since BlMaV was only detected in plants that received water containing both the virus and the vector, we have concluded that OV is capable of transmitting BlMaV. These results could be important in production systems where Olpidium spp. and BlMaV are present.