First report of Prunus necrotic ringspot virus and Mulberry cryptic virus 1 in mulberry (Morus alba) in the United Kingdom

In September 2017, a sample of mulberry leaf (Morus alba cv. Capsrum) was submitted from a nursery in Worcestershire, to Fera Science Ltd. Over 100 trees were affected with a chlorotic oak leaf line pattern on the fully developed leaves which was consistent with viral infection (Fig. 1).

The sample was tested by ELISA for Arabis mosaic virus, Raspberry ringspot virus, Tomato black ring virus (TBRV), Tomato spotted wilt virus (all antisera provided by DSMZ, Germany), Impatiens necrotic spot virus, Strawberry latent ringspot virus, TBRV (Bioreba, Switzerland) and Cucumber mosaic virus (CMV) (Agdia, USA). Healthy control mulberry was not available, so a composite of several plants was used, including tomato and Nicotiana tabacum. ELISA testing was negative except for a positive reaction for CMV. To confirm the CMV positive result, the mulberry was inoculated onto Chenopodium quinoa, N. glutinosa, N. hesperis, N. occidentalis P1 and N. tabacum. Twenty-one days post inoculation no virus symptoms were seen. The sample was also tested by real-time PCR for CMV (Table 1), CMV was not detected. After the initial testing asymptomatic mulberry leaves were also tested by ELISA for CMV and again a positive ELISA reaction was obtained.

To further investigate the virus infection status, the sample was screened using an Illumina MiSeq as described by Adams et al. (2014). From these data the presence of Prunus necrotic ringspot virus (PNRSV, genus Ilarvirus, family Bromoviridae) and Mulberry cryptic virus 1 (suggested acronym MuCV1, tentative member of the family Partitiviridae) was inferred. The sequences were submitted to GenBank, Accession Nos. MH282499 and MH282498, respectively. No CMV sequences were detected. Considered with the real-time PCR and sap inoculation results, the CMV ELISA reaction was an erroneous result. Given the likely cross reaction of mulberry leaf homogenate with the CMV antisera, mulberry may be a problematic host for ELISA testing. To confirm the PNRSV finding the sample was tested by RT-PCR using primers C and D (Sanchez-Navarro et al, 1997) and the resulting PCR product was sent for sequencing. PNRSV was confirmed (MH282500).

Cryptic viruses generally induce no or only very mild symptoms (Boccardo et al, 1987), therefore the oak leaf-like pattern seen may be caused by PNRSV, or by a synergistic effect with Mulberry cryptic virus 1. This is the first report of PNRSV in mulberry. As the only other report of Mulberry cryptic virus 1 is an incidental detection from mulberry in Fayetteville, Arkansas, USA (GU145316.1) (I. Tzanetakis, pers. comm.), this is the first report of MuCV1 in Europe.