Detection of Beet black scorch virus and other Associated Soil-borne Viruses and Determining some of their Molecular Aspects

Soil-borne sugar beet viruses are responsible for the destructive diseases of sugar beet. They can cause significant yield losses worldwide. Four of the soil-borne sugar beet viruses consisted of Beet necrotic yellow vein virus (BNYVV), Beet soil-borne mosaic virus (BSBMV), Beet soil-borne virus (BSBV) and Beet virus Q (BVQ) are transmitted by the protis Polymyxa betae, a common root infecting parasite which ensures the long-term persistence of the viruses in the soils and one consisted of Beet black scorch virus (BBSV), is transmitting by Olpidium brassicae. So far, four out of five aforementioned viruses have been reported (except for BSBMV) in Iran. BNYVV is the causal agent of the rhizomania disease which has been reported in mixed-infections with BSBV, BVQ and/or BBSV. In previous studies, BSBV, BVQ and BBSV have been found alongside the BNYVV in some root samples. Nevertheless, up to now, no field has been spotted with black scorch symptoms of sugar beet leaves. Recently, we observed symptoms of burns on leaves and similar signs of rhizomania disease in the roots of some sugar beet cultivars in the fields of Khorasan Razavi province. The purpose of this study was to detect soil-borne viruses of sugar beet and determine some of their molecular aspects.

In this study, sugar beet with bearded roots and black scorching of leaves symptoms were collected from Mashhad, Fariman, Chenaran, Jolge-Rokh, and Jovein sugar beet fields. Total RNA was extracted from 100 mg of rootlets using RNeasy Mini Kit (Qiagen-Germany) based on the manufacturer's protocol. The presence of three viruses of BSBV, BVQ and BNYVV were analyzed by multiplex reverse transcription-polymerase chain reaction (mRT-PCR). However, For BBSV detection, simplex RT-PCR was used to detect of the 3′-UTR of the genomic RNA. For the molecular characterization of the BNYVV isolates, the BNYVV type (A or B) was determined with duplex RT-PCR (dRT-PCR), using A/B-type-specific primers pairs for the triple gene block (TGB) gene in RNA-2 and the partial p25 gene of the RNA-3 segment of the virus. RT-PCR was done using the PrimeScript™ Reverse Transcriptase kits and the PrimeSTAR GXL DNA Polymerase (Takara, Japan). The PCR products were cloned in pGEM®-T Easy Vector (Promega-USA). The recombinant plasmids were extracted using PrimPrep Plasmid DNA isolation kit (GenetBio-Korea) and then sequenced (Macrogene, South Korea). Nucleotide sequences data were analyzed using Chromas (version 1.45) and MEGA7 softwares.

The results showed that in the three samples with bearded root symptoms (Mashhad, Jovein, and Jolge-rokh), only BNYVV (A-type) was present and there were no BSBV and BVQ in the tested samples. In addition, in the two samples (Fariman and Chenaran), none of the three viruses was detected. The results showed that the two BNYVV isolates had ‘ACHG’ (Mashhad and Jolge-Rokh isolates) or ‘AHHG’ (Jovein isolate) residues in the tetrad position. So that the amino acid cysteine (C) in a68 position was converted to histidine (H). Although this A-type tetrad has been previously reported by Mehrvar et al. (2009) in Khorasan Razavi and Northern Khorasan, Semnan, Qazvin, Zanjan, Ilam, Hamedan, and West Azarbaijan provinces. In this study, BBSV was detected in all samples. In Mashhad, Jolge-rokh, and Jovein samples, BNYVV was present accompanied by BBSV. However, BBSV was detected alone from Fariman (holding black scorching of the leaves and vascular necrosis of root symptoms) and Chenaran (black scorching of the leaves) samples. These results are consistent with the results of other researchers from Spain and the United States who reported the presence of rhizomnia symptoms in the BBSV infected roots.

While most of  the farmers in Khorasan province cultivate resistant cultivars of sugar beet carrying the Rz1 gene for successive years in a field, the breakdown of the resistance and emerging of new resistance breaking (RB) variant of the virus have occurred via amino acid changes. However, more research on BNYVV pathogenicity by the use of additional sources of resistance and alternative disease control majors is needed to have a suitable conclusion. In addition, the results of this study showed that the presence of both viruses (BBSV and BNYVV) together could exacerbate the Rhizomania syndrome symptoms while single infection by BBSV could just cause vascular necrosis in the root and black scorching symptoms of the leaves. This could be very important in symptoms based diagnosing of the disease and preventing errors in evaluating the resistance of sugar beet cultivars in the field.