Quantification of rhizomania virus by automated RNA isolation and PCR based methods in sugar beet

Abstract

Rhizomania is a grave disease affecting sugar beet (Beta vulgaris L.). It is caused by the Beet Necrotic Yellow Vein Virus (BNYVV), an RNA virus transmitted by the plasmodiophorid vector Polymyxa betae. Genetic resistance to the virus has been accomplished mostly using phenotype-genotype association studies. As yet, the most convenient method to ascertain plant resistance has been the quantification of viral titer in roots through the ELISA test. This method is particularly time-consuming and clashes with the necessities of modern plant breeding. Here, we propose an alternative and successful phenotyping method based on the automatic extraction of the viral RNA from sugar beet roots and its relative and absolute quantification by quantitative real-time PCR (qRT-PCR) and digital PCR (dPCR), respectively. Such a method enables an improved standardization of the study, as well as an accurate quantification of the virus also in those samples presenting low virus titer, with respect to the ELISA test. Keywords Sugar beet Á Rhizomania virus Á Highthroughput phenotyping Á Quantitative real-time PCR Á Digital PCR Abbreviations qRT-PCR Quantitative real-time PCR dPCR Digital PCR RT-PCR Reverse transcriptase PCR Rhizomania is the most destructive disease affecting sugar beet: it can decrease sugar yield by up to 70% [24, 31, 33]. Rhizomania is caused by the Beet Necrotic Yellow Vein Virus (BNYVV), a virus transmitted by the soil-borne plasmodiophorid vector Polymyxa betae. The BNYVV is a single-stranded rod-shaped virus consisting of 4 or 5 ssRNAs [28]. According to the RNA structure, the A, B, and P forms of BNYVV have been distinguished. Strains A and B have four RNAs while the P-type also has a fifth RNA strand. The function of each RNA strand has been well characterized [11]. In particular, the third one is involved in the long-distance movement of the virus in the plant [12]. The P25 virulence factor is located on this strand and is responsible for the development of one of the main root symptoms: the extensive proliferation of lateral roots [10, 32]. This gene is the one displaying the highest variability: mutations at positions 67 to 70 (hypervariable tetrad) are unique for each strain of the virus [22]. The variability within the P25 gene allows the virus to bypass the resistance. Koenig et al. [16] described the most common mutations of this tetrad in each strain: in the A-type ACHG, AHHG, AFHG, ALHG, AYHG, VCHG; in the B-type AYHR, AHHR; in the P-type SYHG have been identified. More recently, a new A-strain has been characterized, where the hypervariable tetrad presents the AYPR motif. This strain is less common than the others but more aggressive [4]. Rhizomania virus is spread worldwide, and it causes enormous damage to the crop. The only approach to control the disease is the use of host genetic resistance. The