Detection and relative quantitation of Soil-borne cereal mosaic virus (SBCMV) and Polymyxa graminis in winter wheat using real-time PCR (TaqMan^®)

Abstract

Soil-borne cereal mosaic virus (SBCMV) was first reported affecting wheat crops in Italy in 1960 and has spread subsequently to many other European countries, including the UK. SBCMV causes a serious disease of wheat, reducing yield by up to 70%; growing resistant varieties represents the only economical means of control. Real-time RT-PCR and PCR assays based on TaqMan^® chemistry were developed for the detection and quantitation of SBCMV and its vector, Polymyxa graminis. Each assay incorporated an RNA or DNA specific internal control to facilitate quantitation. Nucleic acid extracts from SBCMV-infected plants were diluted in a nucleic acid extract from a healthy plant and amplified by real-time PCR to produce a standard curve. The standard curve was used to quantify the amount of SBCMV and P. graminis in plant samples. The sensitivity of the real-time assays were compared to established serological quantitation and conventional PCR methods by testing a range of SBCMV-infected wheat varieties. The results indicate that real-time assays were a 1000 times more sensitive than ELISA for the quantitation of SBCMV, and a 100 times more sensitive than conventional PCR for the quantitation of P. graminis. Real-time assays enabled sensitive, reproducible and specific detection of both virus and vector in wheat tissues. The real-time assays are potentially useful tools for determining variations in virus and vector concentrations in plant tissue from wheat varieties differing in resistance to SBCMV.

Introduction

Soil-borne wheat mosaic virus (SBWMV) was first described in the USA in 1919 (McKinney, 1925). Sequences obtained from all isolates prior to 1998 were ascribed to this species. More recently sequence analysis (Koenig et al., 1999, Diao et al., 1999, Koenig and Huth, 2000, Yang et al., 2001) has resulted in the International Committee on Taxonomy of Viruses to approve a taxonomic proposal to divide American, European and Chinese isolates into different species within the Furovirus genus. New species have been denominated Soil-borne wheat mosaic virus, Soil-borne cereal mosaic virus (SBCMV) and Chinese wheat mosaic virus (CWMV). On the basis of the new classification, SBWMV has been reported from United States, Brazil and Canada, as well as from Africa (Zambia) and Europe (southern Germany) (Brakke, 1971, Kapooria et al., 2000, Koenig and Huth, 2003); SBCMV from Europe (France, Germany, Italy, Denmark, Poland and UK) (Canova and Quaglia, 1960, Clover et al., 1999, Koenig and Huth, 2000) and CWMV from various regions in Asia (China and Japan) (Diao et al., 1999).

SBCMV is transmitted by the soil-borne plasmodiophorid Polymyxa graminis, virus particles are transported within resting spores and zoospores of the vector. Viruliferous resting spores can survive in the soil for over 10 years (Richard-Molard, 1985) and germinate when suitable environmental conditions arise, releasing primary zoospores. It is not known whether the virus is able to multiply whilst packaged within P. graminis spores. Several studies have investigated genetic resistance to SBCMV, however the results are contradictory, attributing genetic control of resistance to one, two or even three genes (Nakagawa et al., 1959, Modawi et al., 1982, Barbosa et al., 2001). No similar studies have been conducted to evaluate P. graminis resistance on varieties of common or durum wheat.

SBCMV has been shown to cause grain yield reductions of about 50–70% on the most susceptible varieties of common and durum wheat grown in France and Italy (Budge and Henry, 2002, Rubies-Autonell et al., 2003). Due to the persistent nature of the virus within the soil, the only practical and economically viable means of control is the use of resistant varieties. Profiling the resistance of wheat varieties to SBCMV has been conducted for many years by evaluating agronomic performance on infected land and correlating results with virus presence/absence in plant tissues and symptom development (Budge and Henry, 2002, Rubies-Autonell et al., 2003). Such trials have been useful in determining the economic viability of growing individual varieties and identifying useful germplasm for resistance breeding.

As described previously by Clover et al. (2001), several serological and molecular methods for SBWMV/SBCMV detection have been developed, however, the enzyme-linked immunosorbent assay (ELISA) remains the only practical technique for quantitation of SBCMV (Kanyuka et al., 2004). Other studies detecting viral targets have concluded that real-time assays offer greater sensitivity than conventional PCR or serological methods (Mumford et al., 2000, Weller et al., 2000, Boonham et al., 2002, Boonham et al., 2004, Korimbocus et al., 2002). Real-time assays would therefore offer an increased level of detection and potentially accurate quantitation for SBCMV.

The main objective of this study was to develop assays that offer greater sensitivity for the detection and quantitation of SBCMV and P. graminis in plant tissues. In addition, a secondary objective was to demonstrate how real-time assays can be used in epidemiological studies to help determine the resistance of wheat varieties to SBCMV.