Detection of beet soil-borne virus and beet virus Q in sugarbeet in Greece

Sugar beet plants with typical rhizomania symptoms were collected from the five major cultivation zones of Greece. The presence of Beet necrotic yellow vein virus (BNYVV), the primary causal agent of the disease, was ascertained by DAS-ELISA in 38 out of 40 fields surveyed and the positive samples were subsequently examined for the presence of other soil-borne viruses which are frequently associated with rhizomania, using a multiplex RT-PCR assay targeting BNYVV, Beet soilborne virus (BSBV) and Beet virus Q (BVQ). The occurrence of BSBV and BVQ was confirmed in 9 and 23 rhizomania-infected fields, respectively. In contrast to surveys conducted in other countries, the presence of BVQ prevailed throughout Greece in dual infections with BNYVV, whereas BSBV was restricted to rhizomania-infected fields from only two sugarbeet cultivation areas. Nine of the samples tested were infected with all three viruses. BSBV was always found in triple infections. To our knowledge, this is the first report of BSBV and BVQ in Greece. The future assessment of the impact of each of these viruses on sugarbeet could prove significant for bleeding objectives in terms of achieving a more durable resistance to the rhizomania syndrome

Efficiency of Rz-1 based rhizomania resistance and molecular studies on BNYVV isolates from sugar beet cultivation in Geece

A survey was carried out to investigate the current situation concerning rhizomania disease incidence in sugar beet cultivation of Greece. A systematic field evaluation over locations and years revealed a consistent disease severity pattern according to favourable agroclimatic conditions and pointed to the so far effectiveness of the Rz1 gene-based resistance, as no major disease outbreaks were observed. Molecular analyses aiming at the characterization of the type and genetic diversity of the virus further confirmed the widespread occurrence of BNYVV in the country, as evidenced by RT-PCR amplification of all five known genomic molecules and nested-PCR assays. None of the isolates contained an RNA 5, typically found in pathotype P. On the basis of RFLP patterns, all BNYVV isolates analysed were classified as pathotype A. Sequence determination of the full-length RNA 3-encoded p25 protein, responsible for symptom development, revealed amino acid motifs ACHG/VCHG in the hypervariable region aa67–70. The presence of valine in position 67 did not appear associated with increased pathogenicity and resistance breaking properties, as earlier reported

BNYVV-derived dsRNA confers resistance to rhizomania disease of sugar beet as evidenced by a novel transgenic hairy root approach

Agrobacterium rhizogenes-transformed sugar beet hairy roots, expressing dsRNA from the Beet necrotic yellow vein virus replicase gene, were used as a novel approach to assess the efficacy of three intron-hairpin constructs at conferring resistance to rhizomania disease. Genetically engineered roots were similar in morphology to wild type roots but were characterized by a profound abundancy, rapid growth rate and, in some cases, plagiotropic development. Upon challenge inoculation, seedlings showed a considerable delay in symptom development compared to untransformed or vector-transformed seedlings, expressing dsRNA from an unrelated source. The transgenic root system of almost all seedlings contained no or very low virus titer while the non-transformed aerial parts of the same plants were found infected, leading to the conclusion that the hairy roots studied were effectively protected against the virus. This readily applicable novel method forms a plausible approach to preliminarily evaluate transgenic rhizomania resistance before proceeding in transformation and whole plant regeneration of sugar beet, a tedious and time consuming process for such a recalcitrant crop species

Engineering transgenic rhizomania resistance

The only practical means to ensure viability and profitability of the sugar beet crop is to provide efficient protection against rhizomania, caused by beet necrotic yellow vein virus (BNYVV), through the use of varieties, specifically bred as resistant to the disease. Although breeding ingenuity has to date achieved successful control of the disease throughout the world, resistant varieties may still suffer significant losses. At the same time, evolutionary changes in the pathogen continuously pose new challenges and require adjustments in relevant breeding programs if they were to keep providing a durable crop protection through the use of better resisting varieties. Given the fact that acquiring resistance from the repertoire of the crops’ gene pool is delimited by the scarcity of natural genetic sources of resistance to BNYVV, transgenesis offers the possibility to broaden the options for rhizomania resistance. Initial attempts to generate transgenic rhizomania resistance were based on the pathogen-derived resistance (PDR) concept. Recent understanding of the aspects underlying the antiviral pathways of RNA silencing however, has placed the focus of interest on generating rhizomania resistance based on the exploitation of the discovered innate defense mechanism. Alternative resistance strategies involved the employment of genes originating from nonviral sources. This chapter reviews the latest advances in breeding for rhizomania resistance in transgenic sugar beet plants. © 2016 Springer International Publishing Switzerland

Evolutionary insights inferred by molecular analysis of the ITS1-5.8S-ITS2 and IGS Avena sp. sequences

In an attempt to clarify phylogenetic and genome relationships among 35 diploid (A and C genomes), 13 tetraploid (AB and AC genomes) and 6 hexaploid (ACD genome) Avena taxa, 71 clones of the ITS1-5.8S-ITS2 fragment were sequenced, aligned and a network was constructed. In addition, the intergenic spacer (IGS) fragment was fingerprinted by means of a RFLP analysis using three different restriction enzymes. Both approaches led to comparable results. Clustering among the 54 Avena sp. entries was according to karyotype. Major genic divergence between the A and C genomes was revealed, while distinction among the A and B/D genomes was not possible. High affinity among the AB genome tetraploids and the A(s) genome diploid A. lusitanica was found, while AC genome tetraploids and ACD hexaploids were highly affiliated with the A(l) genome diploid A. longiglumis. The possible role of A. longiglumis in Avena sp. evolution is discussed

Exploiting pre- and post-harvest metabolism in sweet sorghum genotypes to promote sustainable bioenergy production

Sweet sorghum's high yielding ability, low-input demands and tolerance to stresses, render it highly suitable for syrup and bioenergy production. Exploiting its biomass, however is hampered by the seasonality of its production and the rapid post-harvest sugar catabolism degrading biomass quality. We aimed at elucidating aspects of sweet sorghum's metabolism at different developmental and post-harvest stages, to investigate possibilities of expanding the harvesting window. GC–MS-mediated metabolic profiling was employed to monitor changes across growth stages and targeted transcriptomic analysis was used to determine the expression of genes involved in sucrose metabolism. Changes were studied both in the leaves and stems before, during and after the stage considered as optimum for harvest, whereas harvested stems were analyzed to determine post-harvest changes. Significant alterations in the levels of sugars, amino acids and organic acids were found, sugar levels attaining a maximum at soft dough stage. Overall data suggest that sugar metabolic content is regulated at transcriptional level, while temporal regulation of development and sucrose metabolism is under strong genotypic dependency. Although total sucrose levels were relatively lower at stems harvested one month past the optimum stage or harvested on time and piled for one month, compared to the optimum stage, losses encountered are adequately compensated by the prolonged period of biomass supply to the processing plants. It becomes evident that fine tuning of crop's harvesting and delivery time could significantly contribute to relaxing seasonality-related bottlenecks, thus upgrading the economic operation of plants utilizing sweet sorghum as a bioenergy crop. © 2020 Elsevier B.V

Differential expression of heat shock protein genes in sorghum (Sorghum bicolor L.) genotypes under heat stress

Abstract Various types of sorghum were subjected to thermal stress to reveal the mode of expression of genes of the heat shock protein (hsp) family. In silico sequence determination of hsp genes in related cereal species led to the selection of appropriate primers for PCR amplification of a segment corresponding to the hsp90 gene from sorghum. Deduced sequence information allowed the design of gene specific primers for quantification of hsp90 gene expression by means of real-time quantitative polymerase chain reaction (RTqPCR). Fourteen days-old plants were exposed to a temperature of 47°C for a time period ranging from 10 to 180 min. Total RNA was extracted from stressed and control plants and subjected to reverse transcription and RT-qPCR analysis. The actin gene was used as an internal standard. Gene expression was assessed by using cDNA from all types of plant material and for all the different durations of heat stress exposure. Data from RT-qPCR analyses were analyzed using REST software. The highest level of hsp90 gene expression was realized upon exposure to heat for either 60 or 30 min, while expression levels differed among the genotypes studied. In addition, overall levels of hsp90 gene expression were significantly different among varieties tested. Information of such genotypic variation in expression levels of hsp90 gene under heat stress, coupled with related field performance data, could potentially be exploited in breeding programs