Soil-borne Cereal Mosaic Virus Research Articles

Dissecting the genetic basis of resistance to Soil-borne cereal mosaic virus (SBCMV) in durum wheat by bi-parental mapping and GWAS

Soil-borne cereal mosaic virus (SBCMV), the causative agent of wheat mosaic, is a Furovirus challenging wheat production all over Europe. Differently from bread wheat, durum wheat shows greater susceptibility and stronger yield penalties, so identification and genetic characterization of resistance sources are major targets for durum genetics and breeding. The Sbm1 locus providing high level of resistance to SBCMV was mapped in bread wheat to the 5DL chromosome arm (Bass in Genome 49:1140–1148, 2006). This excluded the direct use of Sbm1 for durum wheat improvement. Only one major QTL has been mapped in durum wheat, namely QSbm.ubo-2B, on the 2BS chromosome region coincident with Sbm2, already known in bread wheat as reported (Bayles in HGCA Project Report, 2007). Therefore, QSbm.ubo-2B = Sbm2 is considered a pillar for growing durum in SBCMV-affected areas. Herein, we report the fine mapping of Sbm2 based on bi-parental mapping and GWAS, using the Infinium 90 K SNP array and high-throughput KASP®. Fine mapping pointed out a critical haploblock of 3.2 Mb defined by concatenated SNPs successfully converted to high-throughput KASP® markers coded as KUBO. The combination of KUBO-27, wPt-2106-ASO/HRM, KUBO-29, and KUBO-1 allows unequivocal tracing of the Sbm2-resistant haplotype. The interval harbors 52 high- and 41 low-confidence genes, encoding 17 cytochrome p450, three receptor kinases, two defensins, and three NBS-LRR genes. These results pave the way for Sbm2 positional cloning. Importantly, the development of Sbm2 haplotype tagging KASP® provides a valuable case study for improving efficacy of the European variety testing system and, ultimately, the decision-making process related to varietal characterization and choice.

Nutrients and soil structure influence furovirus infection of wheat.

Soil-borne wheat mosaic virus (SBWMV) and Soil-borne cereal mosaic virus (SBCMV), genus Furovirus, family Virgaviridae, cause significant crop losses in cereals. The viruses are transmitted by the soil-borne plasmodiophorid Polymyxa graminis. Inside P. graminis resting spores, the viruses persist in the soil for long time, which makes the disease difficult to combat. To open up novel possibilities for virus control, we explored the influence of physical and chemical soil properties on infection of wheat with SBWMV and SBCMV. Moreover, we investigated, whether infection rates are influenced by the nutritional state of the plants. Infection rates of susceptible wheat lines were correlated to soil structure parameters and nutrient contents in soil and plants. Our results show that SBWMV and SBCMV infection rates decrease the more water-impermeable the soil is and that virus transmission depends on pH. Moreover, we found that contents of several nutrients in the soil (e.g. phosphorous, magnesium, zinc) and in planta (e.g. nitrogen, carbon, boron, sulfur, calcium) affect SBWMV and SBCMV infection rates. The knowledge generated may help paving the way towards development of a microenvironment-adapted agriculture.

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.

Oligogenic control of resistance to soil‐borne viruses SBCMV and WSSMV in rye (Secale cereale L.)

AbstractRye production in European growing areas is constrained by the soilborne cereal mosaic virus (SBCMV) and the wheat spindle streak mosaic virus (WSSMV). To date, no European rye cultivars are known to exhibit resistance against these viruses. In this study, we pursued a quantitative trait locus (QTL) mapping strategy to identify genomic regions for resistance to SBCMV and WSSMV in rye. Three populations, each comprising 100 lines segregating for resistance to SBCMV and/or WSSMV, were evaluated for disease response at two years in three locations in Germany where soils are naturally infested with SBCMV and WSSMV. In the combined analysis across environments, one QTL for SBCMV resistance on chromosome 5R explained 31.9% of the phenotypic variation in one of the populations. For WSSMV resistance, one QTL explaining up to 64.0% of the phenotypic variation was detected on chromosome 7R in each of the three populations. On the Triticeae homoeologous group 5, we found evidence for synteny of the major QTL for SBCMV resistance between the wheat and rye genomes.

Polymyxa graminis Isolates from Australia: Identification in Wheat Roots and Soil, Molecular Characterization, and Wide Genetic Diversity.

Polymyxa graminis is an obligate parasite of roots and an important vector of viruses that damage cereal crops in different parts of the world. In 2011 and 2012, P. graminis was identified infecting 11 wheat root samples from three widely dispersed locations in southwest Australia. Its presence was detected by polymerase chain reaction (PCR) and confirmed by DNA sequencing of the transcribed regions of its ribosomal RNA genes (rDNA) and observing sporosori of characteristic morphology and size in stained wheat roots. Also, when soil samples were collected from two locations where P. graminis was found and wheat bait plants grown in them, P. graminis was detected in their roots by PCR. Ribosomal DNA sequences of six southwest Australian isolates were obtained from wheat roots, and one northeast Australian isolate from barley roots. When these seven P. graminis sequences were compared with others from GenBank by phylogenetic analysis, three southwest Australian isolates were classified as P. graminis f. sp. temperata (ribotypes Ia and Ib), and three as f. sp. tepida (ribotypes IIa and IIb). P. graminis f. sp. temperata and tepida both occur in temperate growing regions of other continents and are associated with transmission of soil-borne viruses to cereal crops. The P. graminis isolate from northeast Australia was sufficiently distinct from the five existing sequence groups for it to be placed into a newly proposed grouping, ribotype VI, which also included an isolate from tropical West Africa. However, when randomly collected wheat leaf samples from 39 field crops from 27 widely dispersed locations, 21 individual wheat plant samples collected from low lying areas within 21 fields at 11 locations, and wheat bait plants growing in five soil samples from two locations were tested by reverse transcription (RT)-PCR for the presence of Soil-borne wheat mosaic virus, Soil-borne cereal mosaic virus, Wheat spindle streak mosaic virus, and furoviruses in general, no virus infection was detected. These findings suggest at least three P. graminis introductions into Australia, and the occurrence of f. sp. temperata (ribotype I) and f. sp. tepida (ribotype II) suggests that, if not already present, soil-borne cereal viruses are likely to become established should they become introduced to the continent in the future.

Quantification of Wheat spindle streak mosaic virus and Soil borne cereal mosaic virus in resistance testing of field samples of triticale using real-time RT-PCR

Triticale, a cross between rye and wheat, is a crop important for animal feed and the production of biogas and ethanol. Soil-borne viruses found in wheat and rye, such as Furoviruses and Bymoviruses, also infect triticale. In order to evaluate resistance/tolerance it is necessary to accurately quantify virus content in the plants. We have developed RT-qPCR assays for the quantitative detection of the Bymovirus Wheat spindle streak mosaic Virus (WSSMV) and the Furovirus Soil-borne cereal mosaic virus (SBCMV) in field samples. Both a SYBR Green and a hydrolysis probe approach were tested. The RT-qPCR approach allows the quantitative evaluation and differentiation of triticale resistance to WSSMV and SBCMV. The reproducible large-scale analysis of field samples is feasible.

SYBR Green I based RT-qPCR assays for the detection of RNA viruses of cereals and grasses

Less prevalent viruses of family Poaceae are usually excluded from the focus of interest, even though they represent a possible threat to agricultural production. We designed and validated a set of primer pairs suitable for detection and quantification of five RNA viruses, Lolium latent virus (LoLV), Oat necrosis mottle virus (ONMV), Ryegrass mosaic virus (RgMV), Soil-borne cereal mosaic virus (SBCMV), and Spartina mottle virus (SpMV), by means of one-step RT-qPCR based on SYBR Green I. These primers were used together with primers for Brome mosaic virus (BMV) and Wheat streak mosaic virus (WSMV) described elsewhere to screen grass and cereal samples from the Czech Republic. The results revealed a high prevalence of WSMV and RgMV, which pointed to possible local epidemics. We also make the first report of LoLV presence in the Czech Republic.

Resistance of cereals to soil-borne virusesOdporność zbóż na wirusy odglebowe

Soil-borne viruses, belonging to the genera Furovirus and Bymovirus, are serious pathogens of winter cereals, transmitted by rootinhabiting fungal-like plasmodiophorid Polymyxa graminis. The vector is widely spread through in Asia, North America and Europe. In Europe there are two main cereal diseases caused by soil-borne viruses: barley yellow mosaic and soil-borne cereal mosaic. Plant protection against this kind of viruses is based exclusively on the cultivation of resistant or tolerant cultivars, as chemical control remains ineffective regarding the biological properties of the vector and unacceptable from the ecological point of view. Symptoms of barley yellow mosaic are caused by two closely related viruses: Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), which infect only winter barley. Genetic studies led to the identification of sixteen genes carrying the resistance feature to the barley yellow mosaic viruses in barley. Soil-borne cereal mosaic virus (SBCMV), the causal agent of soil-borne cereal mosaic, can infect different cereal species, including wheat, triticale, rye and barley. Investigations on the resistance of wheat to SBCMV revealed that diverse mechanisms are involved in the reaction of plants and final effect of infection is dependent on many factors including climatic conditions.

A major QTL for resistance to soil-borne cereal mosaic virus derived from an old Italian durum wheat cultivar

The genetic basis of resistance to soil-borne cereal mosaic virus (SBCMV) in the Triticum turgidum L. var. durum cv. Neodur was analyzed in this study, using a linkage mapping approach. We performed phenotypic and molecular analyses of 146 recombinant inbred lines derived from the cross Cirillo (highly susceptible)×Neodur (highly resistant). A major quantitative trait locus (QTL) that explained up to 87% of the observed variability for symptom severity was identified on the short arm of chromosome 2B, within the 40-cM interval between the markers Xwmc764 and Xgwm1128, with wPt-2106 as the peak marker. Three minor QTLs were found on chromosomes 3B and 7B. Two markers coding for resistance proteins co-segregate with the major QTL on chromosome 2B and the minor QTL on chromosome 3B, representing potential candidate genes for the two resistance loci. Microsatellite markers flanking the major QTL were evaluated on a set of 25 durum wheat genotypes that were previously characterized for SBCMV resistance. The allelic composition of the genotypes at these loci, together with pedigree data, suggests that the old Italian cultivar Cappelli provided the SBCMV-resistance determinants to durum cultivars that have been independently bred in different countries over the last century.

Genetic analysis of Soil-Borne Cereal Mosaic Virus response in durum wheat: evidence for the role of the major quantitative trait locus QSbm.ubo-2BS and of minor quantitative trait loci

Genetic analysis of Soil-Borne Cereal Mosaic Virus (SBCMV) resistance in durum wheat was carried out using a population of 180 recombinant inbred lines (RILs) obtained from Simeto (susceptible) × Levante (resistant). The RILs were characterized for SBCMV response in the field under severe and uniform SBCMV infection in two growing seasons and genotyped with simple sequence repeat (SSR) and Diversity Arrays Technology® markers. Transgressive segregation was observed for disease reaction as estimated by symptom severity scores and virus concentration in leaves. Heritability of the disease response was high, with h2 values consistently above 80%. A major quantitative trait locus (QTL) (QSbm.ubo-2BS) in the distal telomeric region of chromosome 2BS accounted for 60–70% of the phenotypic variation for symptom severity, 40–55% for virus concentration and 15–30% for grain yield. The favorable allele was contributed by Levante. Seven additional QTL influenced SBCMV resistance, with the low-susceptibility allele contributed by Levante at five QTL and by Simeto at the remaining two. The meta-QTL analysis carried out using the data from two mapping populations (Simeto × Levante and Meridiano × Claudio) suggests that in both populations SBCMV resistance is likely controlled by QSbm.ubo-2BS. Our results confine QSbm.ubo-2BS to a c. 2-cM-wide interval flanked by SSR markers that are already being used for marker-assisted selection.

Resistance to Soil-borne cereal mosaic virus in durum wheat is controlled by a major QTL on chromosome arm 2BS and minor loci

Soil-borne cereal mosaic (SBCM) is a viral disease, which seriously affects hexaploid as well as tetraploid wheat crops in Europe. In durum wheat (Triticum durum Desf.), the elite germplasm is characterized by a wide range of responses to SBCMV, from susceptibility to almost complete resistance. In this study, the genetic analysis of SBCMV resistance was carried out using a population of 181 durum wheat recombinant inbred lines (RILs) obtained from Meridiano (resistant) × Claudio (moderately susceptible), which were profiled with SSR and DArT markers. The RILs were characterized for SBCMV response in the field under severe and uniform SBCMV infection during 2007 and 2008. A wide range of disease reactions (as estimated by symptom severity and DAS-ELISA) was observed. A large portion of the variability for SBCMV response was explained by a major QTL (QSbm.ubo-2BS) located in the distal telomeric region of chromosome 2BS near the marker triplet Xbarc35-Xwmc661-Xgwm210, with R(2) values ranging from 51.6 to 91.6%. The favorable allele was contributed by Meridiano. Several QTLs with minor effects on SBCMV response were also detected. Consistently with the observed transgressive segregation, the resistance alleles at minor QTLs were contributed by both parents. The presence and effects of QSbm.ubo-2BS were validated through association mapping in a panel of 111 elite durum wheat accessions.

Investigation of soilborne mosaic virus diseases transmitted by Polymyxa graminis in cereal production areas of the Anatolian part of Turkey

Polymyxa graminis is the vector of several important viruses, including Soilborne cereal mosaic virus, Wheat spindle streak mosaic virus, Barley yellow mosaic virus and Barley mild mosaic virus, of winter cereals worldwide. Surveys were carried out to detect these viruses and their vector P. graminis in 300 soil samples from the main wheat and barley production areas of the Anatolian part of Turkey collected in May 2002, June 2004 and May 2005. For these surveys, various susceptible wheat and barley cultivars were pot grown in the collected soil samples in a greenhouse and then analysed using ELISA and RT-PCR to detect the presence of different virus species. In addition, a combination of light microscopy following roots staining with acid fuchsin and PCR was used for detection of P. graminis. All soil samples analysed were found to be free of these soilborne viruses and their vector.

Studies on Cereal Soil-Borne Viruses in Poland

Studies on Cereal Soil-Borne Viruses in PolandFour soil-borne cereal viruses have been identified in Poland, so far:Soil-borne cereal mosaic virus(SBCMV),Wheat spindle streak mosaic virus(WSSMV),Barley yellow mosaic virus(BaYMV) andBarley mild mosaic virus(BaMMV). SBCMV was identified in 1993 as a dangerous pathogen of winter cereals and became the object of special interest. Studies on the virus included its biological and molecular characterization, and investigations of the response of winter wheat and winter triticale cultivars on the SBCMV infection. Results of preliminary experiments aiming at the evaluation of the response of winter barley cultivars on barley yellow mosaic viruses were also presented.

Characterization of Two Unusual Features of Resistance to Soilborne cereal mosaic virus in Hexaploid Wheat: Leakiness and Gradual Elimination of Viral Coat Protein from Infected Root Tissues

Spatiotemporal infection patterns of Soilborne cereal mosaic virus (SBCMV) were compared between resistant and susceptible wheat cultivars to elucidate disease resistance mechanisms. Resistance to SBCMV was manifested by a gradual disappearance of the viral coat protein (CP) from the roots following an initial short period of steady accumulation. Interestingly, viral RNA persisted in the roots of resistant cultivars even after the CP had disappeared. Traces of viral RNA were also detected in the uninoculated leaves of the resistant cv. Cadenza. These findings suggest that the resistance mechanism to SBCMV in wheat involves the efficient disassembly of virus particles and either an inhibition of further synthesis of viral CP or its proteolytic degradation. SBCMV accumulated in the leaves of a small proportion of individual plants of Cadenza and other recognized resistant cultivars, highlighting the leaky nature of the resistance, but the roots of these plants were often devoid of viral CP. Increasing or decreasing the concentration of the inocula had no effect on the incidence rate of such "resistance breakdown"; however, a positive correlation was found between the incidence rate of resistance breakdown and the percentage of systemically infected individuals of recognized susceptible cultivars in each separate experiment.

Broad-spectrum detection and quantitation methods of Soil-borne cereal mosaic virus isolates

A broad-spectrum reverse transcription-polymerase chain reaction (RT-PCR) protocol was developed for detecting Soil-borne cereal mosaic virus (SBCMV) isolates, responsible for mosaic diseases in Europe, using primers targeting the highly conserved 3′-untranslated region of RNA-1 and RNA-2 of SBCMV. The 3′-end region is a privileged target for the detection of a wide range of isolates, because of sequence conservation, of the tRNA-like structure, the major role in viral replication and the signal amplification due to the presence of numerous genomic and subgenomic RNAs. The primers were also designed for virus quantitation using real-time RT-PCR with SYBR-Green chemistry. No cross-reaction with Wheat spindle streak mosaic virus, frequently associated with SBCMV, was observed. The use of RT-PCR and real-time quantitative RT-PCR allowed a more sensitive detection and quantitation of SBCMV to be made than was the case with ELISA. The methods enabled European isolates of SBCMV from Belgium, France, Germany, Italy and the UK to be detected and quantified. Real-time RT-PCR represents a new tool for comparing soil inoculum potential as well as cultivar resistance to SBCMV.

Genetic analysis of resistance to soil-borne wheat mosaic virus derived from Aegilops tauschii

Genetic Analysis of Resistance to Soil-Borne Wheat Mosaic Virus Derived from Aegilops tauschii. Euphytica. Soil-Borne Wheat Mosaic Virus (SBWMV), vectored by the soil inhabiting organism Polymyxa graminis, causes damage to wheat (Triticum aestivum) yields in most of the wheat growing regions of the world. In localized fields, the entire crop may be lost to the virus. Although many winter wheat cultivars contain resistance to SBWMV, the inheritance of resistance is poorly understood. A linkage analysis of a segregating recombinant inbred line population from the cross KS96WGRC40 × Wichita identified a gene of major effect conferring resistance to SBWMV in the germplasm KS96WGRC40. The SBWMV resistance gene within KS96WGRC40 was derived from accession TA2397 of Aegilops taushcii and is located on the long arm of chromosome 5D, flanked by microsatellite markers Xcfd10 and Xbarc144. The relationship of this locus with a previously identified QTL for SBWMV resistance and the Sbm1 gene conferring resistance to soil-borne cereal mosaic virus is not known, but suggests that a gene on 5DL conferring resistance to both viruses may be present in T. aestivum, as well as the D-genome donor Ae. tauschii.

Mapping and diagnostic marker development for Soil-borne cereal mosaic virus resistance in bread wheat

Monogenically-inherited resistance to Soil-borne cereal mosaic virus (SBCMV) in hexaploid bread wheat cultivars ‘Tremie’ and ‘Claire’ was mapped on chromosome 5D. The two closest flanking markers identified in the Claire-derived mapping population, Xgwm469-5D and E37M49, are linked to the resistance locus at distances of 1 and 9 cm, respectively. Xgwm469-5D co-segregated with the SBCMV resistance in the Tremie-derived population and with the recently identified Sbm1 locus in the cv. Cadenza. This suggested that Tremie and Claire carry a resistance gene allelic to Sbm1, or one closely linked to it. The diagnostic value of Xgwm469-5D was assessed using a collection of SBCMV resistant and susceptible cultivars. Importantly, all susceptible genotypes carried a null allele of Xgwm469-5D, whereas resistant genotypes presumably related to either Claire and Tremie or Cadenza revealed a 152 or 154 bp allele of Xgwm469-5D, respectively. Therefore, Xgwm469-5D is well suited for marker assisted selection for SBCMV resistance.

Soil-borne viruses affecting cereals—Known for long but still a threat

A number of furo- and bymoviruses, which are naturally transmitted by the ubiquitous soil-borne protozoon Polymyxa graminis are of great economic importance for autumn-sown wheat, barley, rye, triticale, and oats. The two barley-affecting bymoviruses, Barley yellow mosaic virus and Barley mild mosaic virus, are widespread in Europe and East Asia. Production of wheat, rye, and triticale is threatened by three related furoviruses and two bymoviruses. Soil-borne wheat mosaic virus mainly occurs in North America and Chinese wheat mosaic virus in Asia, whereas Soil-borne cereal mosaic virus predominates in Europe. The bymovirus Wheat yellow mosaic virus so far is restricted to Asia; Wheat spindle streak mosaic virus occurs in North America and Europe. Oat plants can be separately or mixed-infected by a bymo- and a furovirus, named Oat mosaic virus and Oat golden stripe virus, respectively. Because P. graminis cannot be controlled by chemicals and the viruses retain their infectivity in the resting spores for many years cultivation of resistant plants is the only means of preventing severe losses. The genetic variability of the known viruses and the sporadic reports of newly emerging pathogens, such as Aubian wheat mosaic virus, make continued resistance breeding and careful monitoring of field crops necessary.

Identification and Characterization of a Novel Efficient Resistance Response to the Furoviruses SBWMV and SBCMV in Barley

The interaction between the furoviruses Soilborne cereal mosaic virus (SBCMV) and Soilborne wheat mosaic virus (SBWMV) and their main host wheat is well documented; however, to date, only a few reports have addressed the response of other cereal species to these viruses. Here, we show that, in contrast to wheat, barley germplasm is a rich source of resistance to furoviruses. Moreover, we demonstrate that barley genotypes respond differentially to SBCMV and SBWMV, thereby providing an additional biological basis for classification of these viruses as two separate species. Following natural (soil) inoculation, some barley genotypes permitted foliar infection by SBWMV, whereas all 22 genotypes tested were resistant to SBCMV. Resistance is unlikely to be directed toward the virus vector, because Polymyxa graminis DNA was detected in the roots of all tested genotypes. Resistance to SBCMV in some barley genotypes was overcome by artificial virus inoculation onto the leaves, suggesting a block on virus translocation from roots to shoots as in resistant wheat genotypes. However, other genotypes were fully resistant following both inoculation techniques. One barley genotype, 'Dayton,' exhibited extreme resistance to both furoviruses. Further molecular analyses suggested that this novel and highly efficient resistance to furoviruses in barley operates by limiting virus spread from the primary inoculated cells.

RNA2 of Soil-borne cereal mosaic virus is detectable in plants of winter wheat grown from infected seeds

Soil-borne cereal mosaic virus (SBCMV) causes a severe disease in susceptible cultivars of winter wheat. The virus is vectored by the soil-borne protist Polymyxa graminis. Experiments were conducted to investigate whether SBCMV RNA2 could persist in seed from SBCMV-infected susceptible cultivars of winter wheat. Over 7,000 seedlings were generated from seed collected from two cultivars of SBCMV-infected winter wheat. Seedlings were grown in a glasshouse compartment and batch tested for the presence of SBCMV using real-time RT-PCR. The majority of batches tested positive for SBCMV, indicating an RNA2 transmission rate of 1.8–9.4% in wheat. The presence of the virus was confirmed by amplifying and sequencing a larger (400 bp) fragment of viral RNA2 in a sub-set of the seedlings testing positive by real-time RT-PCR. Root extracts from this sub-set tested negative for P. graminis using real-time PCR. The implications for disease epidemiology of this virus are discussed.