Wheat Spindle Streak Research Articles

Rapid and specific detection of wheat spindle streak mosaic virus using RT-LAMP in durum wheat crude leaf extract.

To accurately determine the spread of any pathogen, including plant viruses, a quick, sensitive, cost-effective, point-of-care diagnostic assay is necessary. Wheat spindle streak mosaic virus (WSSMV) is a Bymovirus, transmitted by the plasmodiophorid Polymyxa graminis Led, which causes yellow mosaic and reduces the grain yield in wheat. Currently, detection protocols for WSSMV use ELISA or more sensitive PCR-based approaches requiring specialized laboratory and personnel. A protocol for reverse transcription loop mediated isothermal amplification (RT-LAMP) has been developed and optimized for the rapid detection of viruses using crude extracts from wheat leaves. The protocol was specific for WSSMV detection, while no reaction was observed with SBCMV or SBWMV, the non-target viruses transmitted by the same vector. The RT-LAMP assay was shown to be as sensitive as the one-step WSSMV specific RT-PCR. The RT-LAMP assay can be performed under field conditions using a portable instrument, and can help the actual spread of WSSMV, an aspect of this virus not yet well understood, to be explored.

Occurrence and diversity of barley yellow dwarf virus in Algeria

AbstractCereals are prone to viral infections and the economic impact of these has increased in recent years. Among these diseases barley yellow dwarf (BYD) is one of the most destructive diseases of cereals today. For three consecutive years (2014–2015–2016) surveys were carried out in order to search for BYDV species (BYDV-PAV and -MAV) as well as other cereal viruses, wheat spindle streak mosaic virus (WSSMV), southern bean mosaic virus (SBMV) and barley stripe mosaic virus (BSMV) in seven regions of Algeria (Algiers, Boumerdes, Tipaza Médéa, Adrar, Khenchla and Batna).Targeted samples were taken randomly from plants of different cereal species (wheat, barley, oats). The sample were analyzed by DAS-ELISA and RT-PCR.The results of ELISA and PCR tests showed the presence of BYDV-PAV in barley, durum wheat, soft wheat and oats. Thus, this viral species were found in all the cereal regions surveyed (North, South, East and West).Seven samples (durum wheat, barley and oats) were sequenced and phylogenetic analyses performed revealed that the Algerian sequences clustered in group I and group II.

Development of a Multiplex RT-PCR Assay for Simultaneous Detection of Ten Major Viral Pathogens of Wheat

Triticum mosaic virus (TriMV) and High plains virus (HPV), identified recently, have been considered among the major viruses that affect wheat. Carried by the same vector, wheat curl mite, both of these viruses cause yellowing and stunting of plants which are very similar to many other viruses attacking wheat. This makes it difficult to detect these viruses in different wheat lines, posing a major problem in the yield. This paper highlights the addition of these two viruses to a multiplex RT-PCR based method which already detected the presence of barley and cereal yellow dwarf viruses (B/CYDVs), soil-borne wheat mosaic virus (SBWMV), wheat spindle streak mosaic virus (WSSMV), and wheat streak mosaic virus (WSMV). The method uses specific sets of primers that detect the target viruses TriMV and HPV at 560 bp and 490 bp, respectively, in the presence of other distinct viruses such as B/CYDVs -PAV, -MAV, -SGV, -RPV, -RMV, WSSMV, SBWMV, and WSMV at 295, 175, 237, 400, 365, 154, 219, and 193 bp, respectively. The forward primer for each specific virus was fluorescently tagged to detect it in a higher throughput manner in capillary electrophoresis. All ten viruses may be viewed as peaks in an electropherogram from the capillary electrophoresis corresponding to their product sizes in base pairs. This advancement in the protocol allows detection of all ten wheat viruses in a single test, thus improving the diagnostic capability with only a slight increase in cost.

CRISPR/Cas9-guided knockout of eIF4E improves Wheat yellow mosaic virus resistance without yield penalty.

CRISPR/Cas9-guided knockout of eIF4E improves Wheat yellow mosaic virus resistance without yield penalty.

Wheat spindle streak mosaic virus

This datasheet on Wheat spindle streak mosaic virus covers Identity, Overview, Distribution, Dispersal, Hosts/Species Affected, Diagnosis, Biology & Ecology, Impacts, Prevention/Control, Further Information.

Wheat yellow mosaic bymovirus (wheat spindle streak mosaic)

Wheat yellow mosaic bymovirus (wheat spindle streak mosaic)

Efficient CRISPR/Cas-Mediated Targeted Mutagenesis in Spring and Winter Wheat Varieties.

CRISPR/Cas technology has recently become the molecular tool of choice for gene function studies in plants as well as crop improvement. Wheat is a globally important staple crop with a well annotated genome and there is plenty of scope for improving its agriculturally important traits using genome editing technologies, such as CRISPR/Cas. As part of this study we targeted three different genes in hexaploid wheat Triticum aestivum: TaBAK1-2 in the spring cultivar Cadenza as well as Ta-eIF4E and Ta-eIF(iso)4E in winter cultivars Cezanne, Goncourt and Prevert. Primary transgenic lines carrying CRISPR/Cas-induced indels were successfully generated for all targeted genes. While BAK1 is an important regulator of plant immunity and development, Ta-eIF4E and Ta-eIF(iso)4E act as susceptibility (S) factors required for plant viruses from the Potyviridae family to complete their life cycle. We anticipate the resultant homozygous tabak1-2 mutant lines will facilitate studies on the involvement of BAK1 in immune responses in wheat, while ta-eif4e and ta-eif(iso)4e mutant lines have the potential to become a source of resistance to wheat spindle streak mosaic virus (WSSMV) and wheat yellow mosaic virus (WYMV), both of which are important pathogens of wheat. As winter wheat varieties are generally less amenable to genetic transformation, the successful experimental methodology for transformation and genome editing in winter wheat presented in this study will be of interest to the research community working with this crop.

Screening and genetic studies on resistance to Soil-born Cereal Mosaic Virus (SBWMV) in rye

Due to several reasons soil-borne viruses such as the furoviruses, i. e., cereal mosaic virus (SBCMV) and wheat mosaic virus (SBWMV) as well as the bymovirus wheat spindle streak mosaic virus (WSSMV) gained importance in cereal breeding including rye. High yield losses are recorded, today. Since there is no or little resistance to these viruses in modern rye cultivars, an extended screening for resistance was initiated. In addition to earlier screenings, 37 rye genotypes were tested for resistance. Among them, three genotypes were found with persistent resistance to SBCMV. They belong to Secale montanum and S. vavilovii species, i. e., wild types of rye. One accession, PC2243 (S. montanum), was used as a resistance donor for the present genetic study. In F2 generation, it was observed that resistance to SBCMV is independently inherited from WSSMV. The evaluation of the ELISA values pointed to a 3:1 distribution assuming duplicate dominant epistasis. Molecular marker analysis supports this segregation pattern. By composite interval mapping a QTL on chromosome 2R could be detected. It can be assumed that there is a DNA region of about 13 cM on the long arm of chromosome 2R (2RL) harboring SBCMV resistance with the closest markers “C9654_1947” and “isotig11640”.Moreover, genotypes with a yellow seed coat showed practically no infection with SBCMV. Thus, the resistance gene could be linked to the allele an1 determining non expression of anthocyanins. This locus was also mapped earlier on chromosome 2R.

Occurrence and High-Throughput Sequencing of Viruses in Ohio Wheat.

Ohio is a leading producer of soft red winter wheat in the United States. Many viruses impact wheat production, but there is a lack of contemporary information on the distribution and potential impact of wheat viruses in Ohio. To address this knowledge gap, we created a comprehensive dataset of viruses identified by high-throughput sequencing (HTS) and their incidence in field sites sampled across the state. Samples were collected from 103 field sites in surveys conducted in 2012, 2016, and 2017 and subjected to RNA HTS, reverse transcription (RT) PCR, or enzyme-linked immunosorbent assay to assess virus sequence diversity, prevalence, and incidence within fields. Partial and complete virus sequences were assembled and detection validated by RT-PCR. Assembled sequences were compared with previously known virus sequences, and novel sequences were validated by Sanger sequencing. The viruses detected most often included barley yellow dwarf virus (BYDV), cereal yellow dwarf virus (CYDV), wheat streak mosaic virus (WSMV), and wheat spindle streak mosaic virus (WSSMV). These viruses were detected at 67, 69, 55, and 28% of the field sites sampled, with mean incidences of 18, 19, 20, and 49%, respectively, within fields where they were detected. Brome mosaic virus (BMV) and cocksfoot mottle virus (CfMV) were also viruses of potential importance detected in Ohio, found in 26 and 17% of the field sites sampled, respectively. Based on results from logistic regression analyses, the presence of BYDV, CYDV, WSMV, and WSSMV was associated with the presence of volunteer wheat, BYDV and CfMV with monocots as the previous crop, and BMV with the presence of nearby corn fields (P < 0.10). For six viruses, there was evidence of spatial clustering in at least one field site and the variance of mean incidence was higher at the county level than at the regional spatial level. This finding suggests that county- and site-specific factors influenced the incidence and spatial pattern of some viruses. The results of this study provide a snapshot of viruses present in Ohio wheat and insights into their biology, potential risks to wheat production, and possible management strategies.

Occurrence and molecular characterization of wheat streak mosaic virus in wheat in Serbia

The wheat streak mosaic virus (WSMV), vectored by the wheat curl mite, is globally distributed and threatens wheat production worldwide. Since its first occurrence in Serbia in the 1960s, WSMV presence has not been monitored. In 2019, a total of 62 samples of fi ve wheat cultivars from eight locations in Serbia were collected and tested for the presence of nine common wheat viruses: WSMV, barley yellow dwarf virus-PAV, -MAV, -SGV, and -RMV, cereal yellow dwarf virus-RPV, wheat spindle streak virus, brome mosaic virus, and soil-borne wheat mosaic virus, using individual or multiplex RT-PCR. WSMV was detected in 58.1% of the tested samples in seven wheat crops at five different locations. Species-specific primers failed to detect the presence of the other eight tested viruses. For further confirmation of WSMV, RT-PCR with the WS8166F/WS8909R primers covering the coat protein (CP) gene was carried out for both amplification and sequencing. The amplified product of the correct predicted size (750 bp) derived from four selected isolates, 98-19, 99-19, 102-19 and 120-19, was sequenced and deposited in GenBank (MT461299, MT461300, MT461301 and MT461302, respectively). Serbian WSMV isolates showed very high nucleotide identity (98.16-99.02%) and shared a deletion of triplet codon GCA at nucleotide position 8412- 8414 resulting in deletion of glycine amino acid (Gly2761). Phylogenetic analysis conducted on CP gene sequences revealed the existence of four clades, named A, B, C and D, and one recently introduced clade B1. All Serbian wheat WSMV isolates grouped into clade B together with other European isolates and one isolate from Iran. The results of this study provide the first insight into molecular characterisation of Serbian WSMV isolates, indicating their close relationship with other European isolates and existence of a single genotype in the country. Phylogenetic analysis also confirms the dispersal of WSMV isolates throughout Europe from a single locus.

Occurrence of Viruses and Clavibacter michiganensis in Winter Wheat in Illinois, 2009 to 2011 and 2019 to 2020

Field surveys in 2009, 2010, 2011, 2019, and 2020 determined the incidence and diversity of viruses present in fields of soft red winter wheat (Triticum aestivum) in Illinois. In addition, the putative presence of the bacterial mosaic pathogen, Clavibacter michiganensis subsp. tessellarius (Cmt), was evaluated. A total of 341 fields were sampled across years, and plants were tested for barley yellow dwarf virus (BYDV-PAV; BYDV-MAV), barley stripe mosaic virus (BSMV), brome mosaic virus (BMV), cereal yellow dwarf virus (CYDV-RPV), High Plains virus (HPV), Potyvirus group (POTY), soil-borne wheat mosaic virus (SBWMV), tobacco mosaic virus (TMV), wheat spindle streak mosaic virus (WSSMV), wheat streak mosaic virus (WSMV), and Cmt using ELISA. Average field incidence across years varied by virus with BYDV-PAV (22%), WSSMV (16%), CYDV-RPV (7%), WSMV (3%), and HPV (0.5%) detected in samples. The bacterial mosaic pathogen (Cmt), or potentially a related species of Clavibacter, was detected the most frequently in each year, with an average incidence of 77%. The consistent detection of Cmt warrants further study to determine the nature of the organisms associated with positive Cmt tests and their role in Illinois wheat production systems.

A novel putative member of the family Benyviridae is associated with soilborne wheat mosaic disease in Brazil

Soilborne wheat mosaic disease (SBWMD), originally attributed to infections by Soilborne wheat mosaic virus (SBWMV) and Wheat spindle streak mosaic virus (WSSMV), is one of the most frequent virus diseases and causes economic losses in wheat in southern Brazil. This study aimed to characterize molecularly the viral species associated with wheat plants showing mosaic symptoms in Brazil. Wheat leaves and stems displaying mosaic symptoms were collected from different wheat cultivars in Passo Fundo municipality, Rio Grande do Sul State, southern Brazil. Double‐stranded RNA was extracted and submitted to cDNA library synthesis and next‐generation sequencing. No sequences of SBWMV and WSSMV were detected but the complete genome sequence of a putative new member of the family Benyviridae was determined, for which the name wheat stripe mosaic virus (WhSMV) is proposed. WhSMV has a bipartite genome with RNA 1 and RNA 2 organization similar to that of viruses belonging to Benyviridae. WhSMV RNA 1 has a single open reading frame (ORF) encoding a polyprotein with putative viral replicase function. WhSMV RNA 2 has six ORFs encoding the coat protein, the major protein (read‐through), triple gene block movement proteins (TGB 1, 2 and 3) and ORF 6 (hypothetical protein). In addition to the genomic organization and nucleotide and amino acid sequence identities, phylogenetic analyses also corroborated that WhSMV is a virus species of the Benyviridae. However, isolates of WhSMV formed a clade distinct from members of the genus Benyvirus. It was also demonstrated that the plasmodiophorid Polymyxa graminis is associated with wheat roots showing SBWMD symptoms and infected by WhSMV.

Genome‐wide association analysis of resistance to wheat spindle streak mosaic virus in bread wheat

Wheat spindle streak mosaic virus (WSSMV) is a major concern for cereal crops in Europe and North America. A strong increase in the occurrence of WSSMV has been observed in each French region where susceptible cultivars are cultivated. Most European bread wheat cultivars are resistant, but assessing the status of newly registered cultivars or breeding lines regarding WSSMV resistance is of importance. This paper describes a genome‐wide association study carried out on a panel of 163 cultivars and tested for their resistance to WSSMV. Two regions on chromosomes 5B and 7D showed minor effects on WSSMV resistance. More importantly, a large genomic region on chromosome 2D explained most of the resistance to WSSMV. More than 99% of the cultivars carrying the AA genotype at the most associated marker (Excalibur_c15426_661) were resistant to WSSMV, while 100% of the cultivars showing the GG genotype were susceptible. This large genomic region of 45.8 Mb was found distal to the centromere and showed very high linkage disequilibrium. It is hypothesized that this region may be an alien introgression originating from a wild related species. This region contains a total of 2605 predicted genes based on the Chinese Spring IWGSC RefSeq v. 1.0 including genes potentially involved in plant disease resistance. A kompetitive allele‐specific PCR (KASP) single‐nucleotide polymorphism (SNP) marker was designed in order to identify breeding lines or registered cultivars resistant to WSSMV.

First Report of Association of Candidatus Phytoplasma cynodontis (16SrXI-B Group) With Streak, Yellowing, and Stunting Disease in Durum and Bread Wheat Genotypes From Central India

Since 2011, chlorosis with defined chlorotic streak symptoms, yellowing, stunting, and drying of infected clumps of 155 durum (Triticum durum L.) and two bread wheat (T. aestivum L.) genotypes (30- to 45-day-old seedlings) were regularly observed at Regional Station of ICAR - Indian Agricultural Research Institute, Indore (Madya Pradesh), India. The disease incidence was greater in durum (1.7 to 20%) compared with bread wheat (1.3 to 10.4%). Ten randomly selected symptomatic leaf samples were analyzed for the presence of virus particles using leaf dip electron microscopy, PCR, and rolling circle amplification, but no virus particles viz. Wheat streak mosaic virus, Barley yellow dwarf virus, Wheat spindle streak mosaic virus, and Soilborne wheat mosaic virus were detected, nor positive serological relationships established with the antibodies of these viruses. To detect the association of phytoplasma with symptomatic wheat samples, genomic DNA from three representative durum wheat genotypes (Is-1: H18638; Is-2: PDW245; Is-3: MACS3063) and one bread wheat genotype (Is-271: HW2004) were assayed for amplification of 16S rRNA gene using primers P1 and P7 followed by nested PCR using primers R16F2n and R16R2 (Gundersen and Lee 1996). The 16S rRNA gene of phytoplasma was detected in all symptomatic samples, and no amplification was observed in asymptomatic wheat samples. The obtained PCR fragments of 1.25 kb were sequenced directly using primers 343R, 704F, 907R, and 1028F. The 1,245-kb sequences were deposited in GenBank (LT594113–16), which showed 100% sequence identity with each other and 98.16% identity with reference phytoplasma strain BGWL-C1 (Candidatus Phytoplasma cynodontis, AJ550984) when analyzed using EzTaxon database. The MEGA6 constructed neighbor joining phylogenetic analysis and virtual RFLP analysis using 17 restriction enzymes of 16SrRNA gene sequences allowed affiliating the wheat phytoplasma strains with 16SrXI-B subgroup strain with a similarity coefficient of 1 (Zhao et al. 2009). The association with the 16SrXI-B group was further established by amplifying

Epistatic determinism of durum wheat resistance to the wheat spindle streak mosaic virus

Key messageThe resistance of durum wheat to the Wheat spindle streak mosaic virus (WSSMV) is controlled by two main QTLs on chromosomes 7A and 7B, with a huge epistatic effect.Wheat spindle streak mosaic virus (WSSMV) is a major disease of durum wheat in Europe and North America. Breeding WSSMV-resistant cultivars is currently the only way to control the virus since no treatment is available. This paper reports studies of the inheritance of WSSMV resistance using two related durum wheat populations obtained by crossing two elite cultivars with a WSSMV-resistant emmer cultivar. In 2012 and 2015, 354 recombinant inbred lines (RIL) were phenotyped using visual notations, ELISA and qPCR and genotyped using locus targeted capture and sequencing. This allowed us to build a consensus genetic map of 8568 markers and identify three chromosomal regions involved in WSSMV resistance. Two major regions (located on chromosomes 7A and 7B) jointly explain, on the basis of epistatic interactions, up to 43% of the phenotypic variation. Flanking sequences of our genetic markers are provided to facilitate future marker-assisted selection of WSSMV-resistant cultivars.

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.

A new sequence variant of wheat spindle streak mosaic virus in Germany

Wheat spindle streak mosaic virus is a Bymovirus transmitted by the plasmodiophorid Polymyxa graminis that infects small grains such as wheat, rye and triticale. Nucleotide sequences of WSSMV isolates from several locations in Germany used for virus resistance trials differ from the sequences of the isolates found in France, Denmark, and the USA by a nona-nucleotide insertion in the 14K protein region.

Occurrence of Viruses and Associated Grain Yields of Paired Symptomatic and Nonsymptomatic Tillers in Kansas Winter Wheat Fields.

Vector-borne virus diseases of wheat are recurrent in nature and pose significant threats to crop production worldwide. In the spring of 2011 and 2012, a state-wide sampling survey of multiple commercial field sites and university-managed Kansas Agricultural Experiment Station variety performance trial locations spanning all nine crop-reporting regions of the state was conducted to determine the occurrence of Barley yellow dwarf virus-PAV (BYDV-PAV), Cereal yellow dwarf virus-RPV, Wheat streak mosaic virus (WSMV), High plains virus, Soilborne wheat mosaic virus, and Wheat spindle streak mosaic virus using enzyme-linked immunosorbent assays (ELISA). As a means of directly coupling tiller infection status with tiller grain yield, multiple pairs of symptomatic and nonsymptomatic plants were selected and individual tillers were tagged for virus species and grain yield determination at the variety performance trial locations. BYDV-PAV and WSMV were the two most prevalent species across the state, often co-occurring within location. Of those BYDV-PAV- or WSMV-positive tillers, 22% and 19%, respectively, were nonsymptomatic, a finding that underscores the importance of sampling criteria to more accurately assess virus occurrence in winter wheat fields. Symptomatic tillers that tested positive for BYDV-PAV produced significantly lower grain yields compared with ELISA-negative tillers in both seasons, as did WSMV-positive tillers in 2012. Nonsymptomatic tillers that tested positive for either of the two viruses in 2011 produced significantly lower grain yields than tillers from nonsymptomatic, ELISA-negative plants, an indication that these tillers were physiologically compromised in the absence of virus-associated symptoms. Overall, the virus survey and tagged paired-tiller sampling strategy revealed effects of virus infection on grain yield of individual tillers of plants grown under field conditions and may provide a complementary approach toward future estimates of the impact of virus incidence on crop health in Kansas.

Registration of OK05312, a High‐Yielding Hard Winter Wheat Donor of Cmc4 for Wheat Curl Mite Resistance

Wheat streak mosaic (WSM) is a devastating disease in the High Plains region that is advancing eastward toward lower elevations. Its control is best achieved by combining known disease resistance genes with resistance to the insect vector. A hard red winter (HRW) wheat (Triticum aestivum L.) germplasm line, OK05312 (Reg. No. GP‐995, PI 670019), was co‐developed by Oklahoma State University, Kansas State University, the Wheat Genetics Resource Center, and USDA‐ARS and released in 2014. It possesses gene Cmc4 and confers strong resistance to the wheat curl mite (WCM, Aceria tosichella Keifer). OK05312 is an F4–derived line from the double cross TX93V5919/KS96WGRC40//OK94P549/KS96WGRC34 and is expected to provide an improved source of Cmc4 over KS96WGRC40. OK94P549 was the parent line from which HRW cultivar Endurance was selected. OK05312 was released for its high grain yield potential in a late‐maturing background with resistance to the with resistance to the wheat soil‐borne mosaic–wheat spindle streak mosaic complex and acceptable milling and baking quality. Hence, OK05312 provides wheat breeders an alternative source of WCM resistance and a critical component to mitigate yield losses to WSM and other WCM‐transmitted viruses.

First Report of Wheat spindle streak mosaic virus on Wheat in Croatia

Wheat spindle streak mosaic (WSSM ; genus Bymovirus, family Potyviridae), transmitted by plasmodiophorid Polymyxa graminis Led., is one of the most important wheat viruses that cause significant yield losses (Deb and Anderson 2008). In March 2013, irregularly distributed, light green to yellow, circular patches in a wheat crop characteristic of soilborne viruses were observed in one local winter wheat (Triticum aestivum L.) cultivar Super Žitarka in the Karanac locality (Baranja Country, Croatia). Initial symptoms included light green to yellow dashes, mottling, and chlorotic, spindle-shaped streaks with green center on the leaves, which became yellow and eventually necrotic. Affected plants were stunted, with dark brown, slightly swollen, and enlarged roots. The long period of wet and cool spring weather most likely favored disease development and severe symptoms were visible by the end of vegetation with estimated incidence of 40%. The roots of infected plants were stained in lactophenol cotton blue and cystosori of P. graminis were observed in all assayed wheat roots by light microscope. Because of that, soil samples were collected from this locality and used in bait plant test. Wheat bait plants showed chlorosis and mild mosaic 6 weeks after sowing, and cystosori were detected in their roots. Using double antibody sandwich (DAS)-ELISA test (Loewe Biochemica, Sauerlach, Germany), WSSM was detected serologically in all 15 collected wheat samples as well as in five wheat bait plants. For further confirmation, total RNA was extracted from leaves of all naturally infected and wheat bait plants using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and served as a template in reverse transcription (RT)-PCR. RT-PCR was carried out with One-Step RT-PCR Kit (Qiagen) using degenerate primers, WMVCPF and WMVCPR (Clover and Henry 1999), yielding an 879- to 882-bp fragment corresponding to the coat protein (CP) gene of both WSSMV and Wheat yellow mosaic virus (WYMV). Total RNAs extracted from healthy wheat leaves as well as RNase-free water were included as negative controls in RT-PCR analysis. Amplicons of the expected size were obtained from all 15 naturally infected and five bait wheat plants, while no amplification products were observed in the healthy controls. After the purification with QIAquick PCR Purification Kit (Qiagen), the RT-PCR product obtained from one selected isolate 361-13 was sequenced directly in both directions using the same primer pair as in RT-PCR (GenBank Accession No. KP257576). Pairwise comparison of the 361-13 isolate CP sequence with other homologous sequences available in GenBank, conducted using MEGA5 software (Tamura et al. 2011), revealed that wheat isolate from Croatia showed the highest nucleotide identity of 99.2% (100% amino acid identity) with the WSSMV isolate (AJ237926) originating from the United States. To our knowledge, this is the first report of WSSMV occurrence on wheat in Croatia. Wheat is the most important field crop in Croatia and the presence of this harmful virus could represent a major threat to its production. Further investigation toward establishing distribution of WSSMV and P. graminis as its vector will be conducted, followed by testing the resistance of local cultivars.