Fast and Sensitive Detection of Soil-Borne Cereal Mosaic Virus in Leaf Crude Extract of Durum Wheat

Soil-borne cereal mosaic virus (SBCMV) is a furovirus with rigid rod-shaped particles containing an ssRNA genome, transmitted by Polymyxa graminis Led., a plasmodiophorid that can persist in soil for up to 20 years. SBCMV was reported on common and durum wheat and it can cause yield losses of up to 70%. Detection protocols currently available are costly and time-consuming (real-time PCR) or have limited sensitivity (ELISA). To facilitate an efficient investigation of the real dispersal of SBCMV, it is necessary to develop a new detection tool with the following characteristics: no extraction steps, very fast results, and high sensitivity to allow pooling of a large number of samples. In the present work, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) protocol with such characteristics, and we have compared it with real-time PCR. Our results show that the sensitivity of LAMP and real-time PCR on cDNA and RT-LAMP on crude extracts are comparable, with the obvious advantage that RT-LAMP produces results in minutes rather than hours. This paves the way for extensive field surveys, leading to a better knowledge of the impact of this virus on wheat health and yield

Characterisation of two tomato lines highly resistant to TSWV following transformation with the viral nucleoprotein gene.

Tomato spotted wilt virus (TSWV) is a serious threat to both horticultural and ornamental crops. Three fresh-market tomato (Lycopersicon esculentum Mill.) lines were transformed with the nucleoprotein gene of TSWV by Agrobacterium tumefaciens. Primary transformants for each line were analysed for transgene integration and expression. Results showed that inserted transgenic DNA was often rearranged. Progeny of selected primary transformants were obtained by self-pollination and tested for resistance to TSWV. One completely resistant line was identified having a single integration locus with multiple rearranged transgene copies and a true-to-type phenotype. It will be further tested for possible inclusion in breeding program