High-Throughput Sequencing of Small RNAs for Diagnostics of Grapevine Viruses and Viroids in Russia

The use of high-throughput sequencing (HTS) technology has led to significant progress in the identification of many viruses and their genetic variants. In this study, we used the HTS platform to sequence small RNAs (sRNAs) of grapevine to study the virome. Isolation of RNA was performed using symptomatic grapevines collected from commercial vineyards in Krasnodar Krai in 2017–2018. To determine the viromes of vineyards, we used an integrated approach that included a bioinformatic analysis of the results of sRNA HTS and the molecular method RT-PCR, which made it possible to identify 13 viruses and 4 viroids. Grapevine leafroll-associated virus 4 (GLRaV-4), Grapevine Syrah Virus-1 (GSyV-1), Raspberry bushy dwarf virus (RBDV), Australian grapevine viroid (AGVd), and Grapevine yellow speckle viroid 2 (GYSVd-2) were identified for the first time in Russia. Out of 38 samples analyzed, 37 had mixed infections with 4–11 viruses, indicating a high viral load. Analysis of the obtained sequences of fragments of virus genomes made it possible to identify recombination events in GLRaV-1, GLRaV-2, GLRaV-3, GLRaV-4, GVT, GPGV, GRSPaV, GVA, and GFLV. The obtained results indicate a wide spread of the viruses and a high genetic diversity in the vineyards of Krasnodar Krai and emphasize the urgent need to develop and implement long-term strategies for the control of viral grapevine diseases.

Raspberry bushy dwarf virus in Slovenia - geographic distribution, genetic diversity and population structure

Raspberry bushy dwarf virus (RBDV) is a long-known virus naturally infecting Rubus and grapevine. It is also one of the economically most important viruses of raspberries, but there are only a limited number of sequences covering a substantial part of the genome available in the databases. The aim of this study was: i) to study the geographic distribution of RBDV in Slovenia, and ii) to sequence RNA2 of several red raspberry and grapevine RBDV isolates and study their phylogeny and population structure. Geographic distribution studies were performed over a period of 13 years in three wine-growing regions of Slovenia (Primorska, Podravje and Posavje). The highest incidence of RBDV was found in Podravje (58.8%) and the lowest in Primorska (5.1%). Big differences were observed between Vipavska dolina (10.2%) and three other wine-growing districts of Primorska region (0.4–1.2%). Almost complete RNA2 sequences were obtained for four red raspberry isolates and seven grapevine isolates. Additionally, only coat protein sequences were obtained for three red raspberry isolates. Phylogenetic and population diversity analyses were performed on all available RBDV sequences. Phylogenetic analysis has shown clear differences in sequences from Rubus and grapevine that form two highly supported clades. In RNA2 analysis additional two sub-clades were found in grapevine clade. Two major subclades were identified also in the Rubus clade with further differentiation within these subclades. Purifying or stabilizing selection was found to be acting on both, CP and MP genes while few codons were found to be under positive selection.

Late-summer Disease Symptoms in Western Washington Red Raspberry Fields Associated with Co-Occurrence of Phytophthora rubi, Verticillium dahliae, and Pratylenchus penetrans, but not Raspberry bushy dwarf virus

Sixty percent of the $109 million processed red raspberry industry of the United States occurs in northern Washington State. In 2012, late-summer symptoms of vascular wilt and root disease were observed in many raspberry plantings. These symptoms were initially attributed to Verticillium dahliae. However, diagnostic tests for the pathogen were often contradictory and other soilborne pathogens (Phytophthora rubi and Pratylenchus penetrans) or Raspberry bushy dwarf virus (RBDV) might also have been involved. Therefore, a survey was conducted in 2013 and 2014 to (i) establish the incidence and soil population levels of V. dahliae in red raspberry production fields, (ii) compare among diagnostic methods and laboratories for detecting and quantifying V. dahliae from raspberry field soil, and (iii) assess which pathogens are associated with late-summer disease symptoms of raspberry. Plant and soil samples were collected from 51 disease sites and 20 healthy sites located in 24 production fields. Samples were analyzed for the presence and quantity of each pathogen using traditional plating and extraction methods (V. dahliae, P. rubi, and P. penetrans), quantitative polymerase chain reaction (qPCR) (V. dahliae and P. rubi), and enzyme-linked immunosorbent assay (RBDV). Results showed that V. dahliae was present in 88% of the production fields and that detection of the pathogen differed by method and by laboratory: qPCR detected V. dahliae in the soil from approximately three times as many sites (51 of 71 total sites) as by plating on NP10 semi-selective medium (15 of 71 total sites). Soil populations of V. dahliae were slightly greater at disease sites, but the pathogen was detected with similar frequency from healthy sites and it was rarely isolated from diseased plants (4%). P. rubi, P. penetrans, and RBDV were also common in production fields (79, 91, and 53% of fields, respectively). Both P. rubi (soil and root samples) and P. penetrans (root populations only), but not RBDV, were more frequently found at disease sites than healthy sites, and the amount of P. rubi detected by qPCR was greater from disease sites than healthy sites. In addition, P. rubi was isolated from 27% of the symptomatic plants located at disease sites. Regardless of detection method, V. dahliae, P. rubi, and P. penetrans, either with or without RBDV, were more likely to co-occur at disease sites (73%) than healthy sites (35%), suggesting that a soilborne disease complex is present in raspberry production fields. Results indicate that P. rubi is the primary pathogen most strongly associated with late-summer symptoms of disease, but root populations of P. penetrans and higher soil populations of V. dahliae may also be of concern. Therefore, disease control methods should focus on all three soilborne pathogens.