Diagnosis of Potato Mop-Top Virus in Soil Using Bait Plant Bioassay and RT-PCR-microplate Hybridization

Abstract

Spraing (brown rings or arcs) of potato caused by Potato mop-top virus (PMTV) occurred in a field of Tokachi, Hokkaido, Japan in 2005. To monitor the expansion of spraing-affected areas, we developed a soil diagnostic method that consisted of a bioassay using tomato as bait plant to trap the vector of PMTV, Spongospora subterranea, the causal agent of powdery scab of potatoes, followed by reverse transcription-polymerase chain reaction-microplate hybridization (RT-PCR-MPH) to detect the virus from roots of bait plants. After incubation of tomato seedlings grown with their roots immersed in a soil suspension at 18°C for 9 days, total RNA extracted from bait roots was analyzed by RT-PCR-MPH using PMTV-specific primers and a digoxigenin (DIG)-labeled probe. Soil diagnosis using the present method in an infested area revealed 137 of 224 fields (61.2%) were infested by PMTV although tubers harvested from only one of these fields had spraing.Resumen En Hokkaido, Japon, en 2005, se presentaron en el campo estriados (anillos o arcos cafés) en papa, causados por el virus de trapeador apical (Potato mop-top virus, PMTV). Para monitorear la expansión de las áreas afectadas por el estriado, desarrollamos un método de diagnostico al suelo, consistente en un bioensayo usando el tomate como una planta trampa para atrapar al vector del PMTV, Spongospora subterranea, el agente causal de la roña polvorienta de las papas, seguido por una hibridación en microplaca de la reacción en cadena de la polimerasa de reverso-transcripción (RT-PCR-MPH) para detectar al virus de las raíces de las plantas trampa. Después de la incubación de las plántulas de tomate crecidas con sus raíces inmersas en una suspensión de suelo a 18°C por 9 días, se analizó el RNA total de las raíces-trampa con RT-PCR-MPH, utilizando iniciadores específicos para PMTV y una sonda marcada con digoxigenina (DIG). El diagnóstico del suelo utilizando este método en un área infestada reveló que 137 de 224 campos (61.2%) estaban infestados por PMTV, aunque solo los tubérculos cosechados de uno de estos campos tuvieron el estriado.

Detection, distribution and control of Potato mop‐top virus, a soil‐borne virus, in northern Europe

“…Double‐antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA), immunocapture reverse transcription polymerase chain reaction (IC‐RT‐PCR) using PMTV coat protein (CP)‐specific polyclonal or monoclonal antibodies and virus‐specific primers (Sokmen et al , 1998; Sandgren et al , 2001; Germundsson et al , 2002; Latvala‐Kilby et al , 2009), RT‐PCR, or real‐time RT‐PCR using CP ‐specific primers (see below) are all suitable methods for PMTV detection. Furthermore, other RT‐PCR‐based methods such as qualitative amplification‐based specific hybridisation (FLASH‐PCR) (Ryazantsev & Zavriev, 2009) and RT‐PCR‐microplate hybridisation (Nakayama et al , 2010) have been applied to detect PMTV.…”

“…This virus has also been detected in Costa Rica in Central America (Montero‐Astúa et al , 2008) and the United States and Canada in North America (Xu et al , 2004; P. Hamm, Oregon State University, personal communication ). In Asia, PMTV occurs on Hokkaido Island in Japan (Imoto et al , 1981; Nakayama et al , 2010). In Europe, PMTV was first detected in Ireland, England and Scotland (Calvert & Harrison, 1966) and in the Netherlands (van Hoof & Rozendaal, 1969) but has not been reported in the Netherlands ever since.…”

A Review Paper on Potato Mop-Top Virus (Pmtv): Occurrence, Properties and Management

“…Later PMTV was detected in potato tubers of England, Scotland and Ireland (Calvert and Harrison, 1966). In Asia, PMTV was detected in Hokkaido islands of Japan (Nakayama et al, 2010) and in China. PMTV has been detected from the Czech Republic and Switzerland (Schwärzel, 2002).…”

“…Therefore, for detection of PMTV, virus-specific methods such as PMTV coat protein (CP) specific antibodies, DAS-ELISA, immune capture reverse transcription polymerase chain reaction (IC-RT-PCR) or RT-PCR using PMTV specific primers techniques are used (Sandgren et al, 2001). Modern methods such as RT-PCR micro-plates hybridization and fluorescent amplification based specific hybridization (FLASH-PCR) have also been applied to improve PMTV detection ( (Nakayama et al, 2010) Enzyme linked immunusorbent Assay (ELISA) is reported by several researchers as a best option for detection PMTV (Sokmen et al, 1998). RT-PCR having fluorescent assays have increased accuracy in diagnosis of PMTV (Mumford et al, 2000).…”

“…Similarly, in the Narino region of Columbia another virus 'soil born virus 2' (SbV2) was detected along with the PMTV (Gil et al, 2016). RT-PCR along with the baiting methods to trapped Sss have proven effective in the detection of PMTV (Nakayama et al, 2010). For successful detection of PMTV from the soil and plant samples a combination of infectivity assays, ELISA and RT-PCR has recently been reported by a researcher (Arif et al, 2014).PMTV and Vector: S. subterraneaf.…”