Wheat Streak Research Articles

Homoeologous Recombination: A Novel and Efficient System for Broadening the Genetic Variability in Wheat

Gene transfer from wild wheat relatives to bread wheat is restricted to homologous recombination. The presence of the Pairing homoeologous 1 (Ph1) gene in the long arm of wheat chromosome 5B allows only homologous chromosomes to pair and recombine, resulting in diploid inheritance of polyploid wheat. Previously, we identified a potent homoeologous pairing promotor gene(s) (Hpp-5Mg); its carrier chromosome 5Mg derived from Aegilops geniculata and its wheat homoeologous chromosome 5D freely recombined in the presence of the Ph1 gene. In this study, we investigated the effect of Hpp-5Mg on homoeologous recombination in the absence of Ph1. In Hpp-5Mg/ph1bph1b plants, we observed a vast genome-wide increase in homoeologous recombination and multiple crossovers (CO), including CO breakpoints in proximal regions of the chromosomes where recombination is known to be suppressed. We tested the efficacy of Hpp-5Mg/ph1bph1b-induced homoeologous recombination by producing new recombinants for the wheat streak mosaic virus resistance gene, Wsm3, present in the wheat-Thinopyrum intermedium Robertsonian translocation (RobT T7BS.7S#3L). A recombination frequency of 6.5% was detected by screening the progenies double monosomic for T7BS.7S#3L and 7B by genomic in situ hybridization. This recombination frequency was about 100-fold higher compared with the recombinant frequency of 0.06% observed by using ph1b-induced homoeologous recombination alone. Our results indicate that chromosome 5Mg promotes homoeologous recombination between wheat and wild wheat relative chromosomes, which helps in the generation of pre-breeding materials thereby accelerating wheat crop improvement.

Occurrence, distribution and alternative hosts of Wheat streak mosaic virus infecting ginger in Kaduna State, Nigeria.

A field survey was conducted during the 2018 rainy season (June – October) in three major ginger producing Local Government Areas (LGAs) of Kaduna State, Nigeria to determine the occurrence and spread of wheat streak mosaic virus (WSMV) infecting ginger. Symptomatic and asymptomatic ginger leaf samples (n=180) and weed samples (n=45) were collected from the surveyed fields and indexed against WSMV using Double Antibody Sandwich Enzyme-linked Immunosorbent Assay (DAS-ELISA). The results obtained showed that WSMV occurred in all the LGAs surveyed but with significant ( P = 0.05 ) variation in distribution. Jaba had the highest virus incidence (22.67\,\%) followed by Kagarko (17.67\,\%) while Kachia had the lowest virus incidence of 10.00\,\%. To the best of our knowledge, this is the first report of WSMV infecting ginger in Nigeria. Rottboellia cochinchinensis (Lour.) Clayton and Setaria barbata (Lam.) Kunth tested positive against WSMV as alternative weed hosts of the virus. The incidence of WSMV even at lower percentage is significant as population build-up could lead to a disease outbreak. Awareness programs need to be organised for farmers on yield loss potential of WSMV on ginger crop and the role of cultural practices on the incidence and management of the virus.

Mutations in virus-derived small RNAs

RNA viruses exist as populations of genome variants. Virus-infected plants accumulate 21–24 nucleotide small interfering RNAs (siRNAs) derived from viral RNA (virus-derived siRNAs) through gene silencing. This paper describes the profile of mutations in virus-derived siRNAs for three members of the family Potyviridae: Turnip mosaic virus (TuMV), Papaya ringspot virus (PRSV) and Wheat streak mosaic virus (WSMV). For TuMV in Arabidopsis thaliana, profiles were obtained for mechanically inoculated rosette leaves and systemically infected cauline leaves and inflorescence. Results are consistent with selection pressure on the viral genome imposed by local and systemic movement. By genetically removing gene silencing in the plant and silencing suppression in the virus, our results showed that antiviral gene silencing imposes selection in viral populations. Mutations in siRNAs derived from a PRSV coat protein transgene in the absence of virus replication showed the contribution of cellular RNA-dependent RNA polymerases to the generation of mutations in virus-derived siRNAs. Collectively, results are consistent with two sources of mutations in virus-derived siRNAs: viral RNA-dependent RNA polymerases responsible for virus replication and cellular RNA-dependent RNA polymerases responsible for gene silencing amplification.

Virome identification in wheat in the Czech Republic using small RNA deep sequencing

High-throughput deep-sequencing technology and bioinformatics analysis of the small RNA (sRNA) population isolated from plants allows universal virus detection and complete virome reconstruction for a given sample. In the present sRNA deep-sequencing analysis of virus-infected wheat samples in the Czech Republic, samples were firstly tested for barley yellow dwarf viruses (BYDVs), wheat streak mosaic virus (WSMV) and wheat dwarf virus (WDV) using ELISA, RT-PCR and PCR. Subsequent sRNA sequencing of these samples yielded more than ~60 million single-end 50-bp reads with high confidence for nine field samples of wheat. Overall, 16.5% of reads were virus-specific and 83.5% were mapped to the host. More 21-nt reads (~7.7E+06 reads) were found than 24-nt (~6.20E+06 reads) or 22-nt (~4.30E+06 reads) reads. De novo assembly of the high-quality contigs revealed the presence of three earlier reported viruses in the Czech Republic: BYDVs (31.48%), WSMV (24.23%) and WDV (26.66%). We also showed the presence of cereal yellow dwarf virus (14.33%; two species CYDV-RPS and CYDV-RPV (family Luteoviridae/Polerovirus) and wheat yellow dwarf virus (WYDV, 3.30%; Luteoviridae). Phylogenetic analysis showed CYDV and WYDV grouped separately from BYDVs. Furthermore, several recombination breakpoints were found among the groups of yellow dwarf viruses (BYDVs, CYDV, and WYDV). Using RNA deep sequencing, we confirmed the presence of the three known viruses (BYDVs, WSMV, and WDV) and the first record of two species of CYDV and WYDV in wheat in the Czech Republic.

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.

Wheat Streak Mosaic Virus: Good Management Can Reduce Your Risks

Wheat streak is often a sporadic disease in the Northern Great Plains. It is vectored by a very small eriophyid mite, the wheat curl mite. Symptoms include stippling or streaking of leaves, stunting, and yellowing. In the spring, symptoms can be easily confused with nutrient deficiencies. This article dicusses disease identification and management. Earn 0.5 CEUs in Integrated Pest Management by reading this article and taking the quiz at https://www.certifiedcropadviser.org/education/classroom/classes/773.

Combining multiplex PCR and high-resolution melting for the detection and discrimination of arthropod transmitted viruses of cereals

The Great Plains of the United States is a region comprised of approximately 45 million hectares of grasslands where several economically important cereal crops are grown. Arthropod-transmitted, cereal-infecting viruses vary in incidence from year-to-year and are often difficult to detect in large acreages. To facilitate the detection of economically important viruses of cereals that often exist in co-infections, a multiplex reverse transcriptase PCR (RT-PCR) platform assay was developed. This method can be used in combination with high resolution melting (HRM) to detect and allow for discrimination between three arthropod-transmitted plant viruses; Wheat streak mosaic virus (WSMV), Maize mosaic virus (MMV) and Barley yellow dwarf virus (BYDV). Multiplex PCR in combination with HRM allowed for successful detection of WSMV, MMV, and BYDV, as well as discrimination between three BYDV species, BYDV-PAS, BYDV-PAV and BYDV-MAV. All primer pairs amplified products of the predicted size. The BYDV-RT-PCR primers amplified products of identical length for all three species of BYDV. HRM was then used to discriminate between these products by determining significant differences between the melting rates for each (p < 0.05). This study demonstrates the flexibility of combining multiplex PCR with HRM to increase the specificity of plant virus diagnostics based on the needs of the diagnostician performing the assay.

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.

Transgenic Wheat Harboring an RNAi Element Confers Dual Resistance Against Synergistically Interacting Wheat Streak Mosaic Virus and Triticum Mosaic Virus.

Wheat streak mosaic virus (WSMV) and triticum mosaic virus (TriMV) are economically important viruses of wheat (Triticum aestivum L.), causing significant yield losses in the Great Plains region of the United States. These two viruses are transmitted by wheat curl mites, which often leads to mixed infections with synergistic interaction in grower fields that exacerbates yield losses. Development of dual-resistant wheat lines would provide effective control of these two viruses. In this study, a genetic resistance strategy employing an RNA interference (RNAi) approach was implemented by assembling a hairpin element composed of a 202-bp (404-bp in total) stem sequence of the NIb (replicase) gene from each of WSMV and TriMV in tandem and of an intron sequence in the loop. The derived RNAi element was cloned into a binary vector and was used to transform spring wheat genotype CB037. Phenotyping of T1 lineages across eight independent transgenic events for resistance revealed that i) two of the transgenic events provided resistance to WSMV and TriMV, ii) four events provided resistance to either WSMV or TriMV, and iii) no resistance was found in two other events. T2 populations derived from the two events classified as dual-resistant were subsequently monitored for stability of the resistance phenotype through the T4 generation. The resistance phenotype in these events was temperature-dependent, with a complete dual resistance at temperatures ≥25°C and an increasingly susceptible response at temperatures below 25°C. Northern blot hybridization of total RNA from transgenic wheat revealed that virus-specific small RNAs (vsRNAs) accumulated progressively with an increase in temperature, with no detectable levels of vsRNA accumulation at 20°C. Thus, the resistance phenotype of wheat harboring an RNAi element was correlated with accumulation of vsRNAs, and the generation of vsRNAs can be used as a molecular marker for the prediction of resistant phenotypes of transgenic plants at a specific temperature.

RNAi‐Mediated Silencing of Endogenous Wheat Genes EIF(Iso)4E‐2 and EIF4G Induce Resistance to Multiple RNA Viruses in Transgenic Wheat

ABSTRACTWheat (Triticum aestivum L.) lacks sources of effective resistance to the potyviruses Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV). Potyviruses use host eukaryotic initiation factors (eIFs) to facilitate initial translation of their genomes, and many plant virus resistance genes are due to mutations in eIFs. Therefore, we hypothesized that silencing TaeIF(iso)4E and TaeIF4G would provide effective and broad‐spectrum virus resistance. Three transgenic wheat lines with an RNA interference (RNAi) hairpin construct targeting TaeIF(iso)4E and four lines with a hairpin construct targeting TaeIF4G were recovered through wheat transformation. Early generation lines were found to be resistant to WSMV and TriMV, and co‐inoculation of both. The lines were selfed to the T5 generation to insure transgene stability, as well as crossed onto the hard red winter wheat cultivar ‘Karl 92’ to test the RNAi constructs in a more adapted genetic background. Phenotypically, transgenic lines were found to be resistant to WSMV, TriMV, and mixed infections of both, were resistant to Soil‐borne wheat mosaic virus, and demonstrated a significant reduction in Barley yellow dwarf virus infection. The RNAi effectiveness on viral RNA was evaluated using reverse transcription quantitative polymerase chain reaction (PCR). An 18‐fold reduction in WSMV and TriMV viral RNA was found in the T5 transgenic lines when compared with control plants. Viral RNA reduction was also found in the F1, and BC1F1 crosses to Karl 92. These results demonstrate that a single, endogenously derived transgene in wheat can provide resistance to multiple viruses and provides a proof of concept for future gene editing.

Virulence Dynamics and Breeding for Resistance to Stripe, Stem, and Leaf Rust in Canada Since 2000.

Wheat (Triticum spp.) is a major field crop in Canada in terms of acreage, annual production, and export market value. There are nine classes of Canadian wheat based on growth habit (winter or spring), kernel hardness (hard or soft), seed coat color (red or white), and quality factors (grain protein content and gluten strength). Wheat was described by Newman in 1928 as "the economic fairy to the industrial and commercial life of Canada, having built practically the whole economic structure of the Prairie Provinces." Wheat production in Canada is affected by several biotic and abiotic stresses. The major abiotic stresses are frost damage, drought, and heat stress. Among biotic stresses, diseases caused by fungal pathogens are the most important although wheat streak mosaic virus (WSMV) has caused some localized outbreaks in some years. In context of cultivar registration in Canada, there are certain diseases that breeders have to take into account while developing resistant cultivars. The Prairie Recommending Committee for Wheat, Rye, and Triticale (PRCWRT) classify wheat diseases into priority one, priority two, and priority three depending on prevalence and potential damage they can cause. However, priority one diseases are more of a concern and a minimum level of resistance in commercial cultivars is recommended for those.

Differences in Aceria tosichella population responses to wheat resistance genes and wheat virus transmission

Severe winter wheat yield losses due to infestations of wheat curl mite, Aceria tosichella Keifer, and mite-transmitted viruses occur in wheat production areas of the United States and Canada. Mite infestation alone causes stunted, chlorotic plants in susceptible wheat varieties, and mites transmit Wheat Streak Mosaic (WSMV), High Plains Wheat Mosaic (HPWMoV), and Triticum Mosaic Virus (TriMV). Wheat curl mites were collected from 25 sites in Kansas, Missouri, Nebraska, Texas, North Dakota, and South Dakota in 2014 and 2015. At each site, mite virulence was determined to wheat plants harboring the Cmc2-, Cmc3-, or Cmc4 mite resistance gene; or Cmc4 plus the Wsm2 WSMV resistance gene. Mites collected from 92%, 36%, and 24% of sites were virulent to susceptible Jagger wheat plants (no Cmc), Cmc2, and Cmc3, respectively. The mega-population consisting of all 25 mite sub-populations was avirulent to 80% of plants containing Cmc4 + Wsm2 or Cmc4. WSMV, HPWMoV, or TriMV was present in mites at 76%, 16%, and 8% of the 25 sites, respectively. Our results will enable breeders to increase the efficiency of wheat production by releasing wheat varieties containing wheat curl mite resistance genes that reduce wheat yield losses.

Incidence of Mite-Vectored Viruses of Wheat in the Texas High Plains and Interactions With Their Host and Vector

Mite-vectored virus diseases of wheat are common throughout the Great Plains and cause significant economic losses to growers each year. These diseases are caused by Wheat streak mosaic virus (WSMV), Triticum mosaic virus (TriMV), and Wheat mosaic virus (WMoV), all of which are transmitted by the wheat curl mite (WCM), Aceria tosichella Keifer. New wheat cultivars with tolerance or resistance to WSMV have been released recently, but their widespread cultivation and potential impact on mite-transmitted virus incidence in the Texas Panhandle was unknown. A total of 648 symptomatic wheat samples were collected from 26 counties, predominately in the Texas Panhandle, and tested by enzyme-linked immunosorbent assay (ELISA) for WSMV, TriMV, and WMoV. Samples that tested negative by ELISA were subsequently tested by real-time quantitative PCR (qPCR) for each virus. Approximately 93% of the samples tested by ELISA were positive for WSMV, 43% were positive for TriMV, and 7% were positive for WMoV. Eleven samples tested positive only for TriMV, but none were positive only for WMoV. When samples that tested negative for the different viruses by ELISA were retested by real-time qPCR, detection of each virus was significantly increased. When results of the ELISA test and qPCR were combined, 100% of the 648 samples tested positive for WSMV, approximately 94% were positive for TriMV, and 23% were positive for WMoV. This demonstrated that the incidence of TriMV in the Texas High Plains is much greater than previously reported. The fact that real-time qPCR revealed over a 2-fold increase in the incidence of TriMV and a 3-fold increase in WMoV demonstrated that the ELISA test, which is commonly used by diagnostic laboratories in the Great Plains, should not be used for studies requiring a high degree of sensitivity and accuracy in virus detection. After initial virus infection status was determined, samples that tested positive for WSMV and TriMV were further observed for WCM infestation. A total of 292 samples were inspected and a total of 101 mites were collected from 40 tillers. Individual mites and the tillers from which they were recovered were tested by real-time qPCR to determine how copy numbers of WSMV and TriMV in mites and host tissue compared, and whether the WSMV/TriMV copy number ratio in individual mites was similar to that of the host tissue from which they were collected. In all mites and tillers tested, the WSMV copy number was always higher than that of TriMV and copy numbers of both viruses were always higher in plant tissue than in mites. Although there was a significant correlation between the WSMV/TriMV copy number ratio in plant tissue and in associated mites, the correlation coefficient was very low (r = 0.31, P = 0.0248). In the majority of comparisons, the WSMV/TriMV ratio was higher in individual mites than in the tiller from which they were recovered. The reason for this increase is unknown but indicates that mites may preferentially acquire WSMV from tillers coinfected with WSMV and TriMV, a finding that could have significant implications for virus transmission and disease epidemiology.

‘TAM 204’ Wheat, Adapted to Grazing, Grain, and Graze‐out Production Systems in the Southern High Plains

‘TAM 204’ (Reg. no. CV‐1155, PI 686859), a hard red winter wheat (Triticum aestivum L.) cultivar with the experimental designation of TX06V7266, was developed and released by Texas A&amp;M AgriLife Research in 2014. TAM 204 is an F4–derived line from the cross ‘TAM 112’/TX01M5009 made at Vernon, TX, in 2001. TAM 204 is an apically awnletted, medium‐maturing, semidwarf wheat with red glumes. It was released primarily as a grain, dual‐purpose, and graze‐out wheat with high grain and forage yield potential. It has good acid soil tolerance, is resistant to Soil‐borne wheat mosaic virus, Wheat streak mosaic virus and its vector wheat curl mite, greenbug, stem rust and stripe rust, and it is moderately susceptible to leaf rust. TAM 204 has a high level of resistance to Hessian fly biotypes GP and vH9 and a moderate level of resistance to biotype vH13. Compared with ‘TAM 111’, which is currently the most widely grown cultivars in Texas, TAM 204 has significantly higher grain yield in the Texas High Plains under both irrigated and dryland environments. Compared with ‘TAM 401’ and ‘Weathermaster 135’, which are currently the two most popular apically awnletted cultivars in Texas, TAM 204 has similar forage yield but higher grain yield across a wide range of environments. TAM 204 could be a better option for wheat growers and cattle owners under grain‐only, dual‐purpose (grazing‐plus‐grain), and graze‐out production system in the southern US Great Plains.

Role of chelated fertilizers` and nano-particles application for formation of elements of productivity аt artificial infected with phytoplasmas and virus

Aim. Investigation of the role of chelated fertilizer and nanoparticles in the formation of elements of productivity for artificial infection of phytoplasmas and wheat streak mosaic virus. Methods. Microbiological, serological, biophysical, biometric, statistical. Results. In the field conditions, the effect of leaf treatment of wheat plants with chelated fertilizers and nanoparticles of copper and silver for artificial infection of phytoplasmas and WSMV on the magnitude of the quantum efficiency of photosystem II for controlling replication of the virus by the method of immunoassay analysis and the study of the influence of nanoparticles on the development of phytoplasmas, as well as the weight of 1000 grains of wheat was investigated. Conclusions. Foliar treatment with chelated fertilizer can activate the plant’s phytoimmunity of wheat plants, thus preventing the active replication of the virus in the plant organism, thereby reducing somewhat the loss of productivity for the damage to the WSMV. The use of foliar treatment of wheat plants with 1% solution nanoparticles of silver and copper citrates can stop the development of phytoplasma infection, which allows maintaining a level of productivity close to the intact plants.&#x0D; Keywords: Triticum aestivum L., WSMV, phytoplasma, chlorophyll a fluorescence induction, productivity.

Wheat Virus Identification Within Infected Tissue Using Nanopore Sequencing Technology.

Viral diseases are a limiting factor to wheat production. Viruses are difficult to diagnose in the early stages of disease development and are often confused with nutrient deficiencies or other abiotic problems. Immunological methods are useful to identify viruses, but specific antibodies may not be available or require high virus titer for detection. In 2015 and 2017, wheat plants containing Wheat streak mosaic virus (WSMV) resistance gene, Wsm2, were found to have symptoms characteristic of WSMV. Serologically, WSMV was detected in all four samples. Additionally, High Plains wheat mosaic virus (HPWMoV) was also detected in one of the samples. Barley yellow dwarf virus (BYDV) was not detected, and a detection kit was not readily available for Triticum mosaic virus (TriMV). Initially, cDNA cloning and Sanger sequencing were used to determine the presence of WSMV; however, the process was time-consuming and expensive. Subsequently, cDNA from infected wheat tissue was sequenced with single-strand, Oxford Nanopore sequencing technology (ONT). ONT was able to confirm the presence of WSMV. Additionally, TriMV was found in all of the samples and BYDV in three of the samples. Deep coverage sequencing of full-length, single-strand WSMV revealed variation compared with the WSMV Sidney-81 reference strain and may represent new variants which overcome Wsm2. These results demonstrate that ONT can more accurately identify causal virus agents and has sufficient resolution to provide evidence of causal variants.

Development of Single Nucleotide Polymorphism Markers for the Wheat Curl Mite Resistance Gene Cmc4

ABSTRACTWheat curl mite (Aceria tosichella Keifer) is an important wheat (Triticum aestivum L. em. Thell.) pest in many wheat‐growing regions worldwide. Mite feeding damage not only directly affects wheat yield, but A. tosichella also transmits Wheat streak mosaic virus (WSMV). Wheat resistance to A. tosichella, therefore, helps control WSMV. OK05312 (PI 670019) is an advanced breeding line released from Oklahoma that shows a high level of A. tosichella resistance. To map the gene(s) conditioning wheat resistance to A. tosichella in OK05312, a genetic linkage map was constructed using single nucleotide polymorphism (SNP) markers derived from genotyping‐by‐sequencing (GBS) and a population of 186 recombinant inbred lines (RILs) from the cross ‘Jerry’ (PI 632433)/OK05312. Seedlings of both parents and the RIL population were infested by A. tosichella Biotype 1 in greenhouse experiments. One major quantitative trait locus was identified on the short arm of chromosome 6D, which corresponds to the previously reported gene Cmc4 for A. tosichella resistance. This gene explained up to 71% of the phenotypic variation and was delimited in a 1.7‐Mb (∼3.3‐cM) region by SNPs 370SNP7523 and 370SNP1639. We successfully converted 12 GBS‐SNPs into Kompetitive allele specific polymerase chain reaction (KASP) markers. Two of them tightly linked to Cmc4 were validated to be highly diagnostic in a US winter wheat population and can be used for marker‐assisted breeding for incorporation of Cmc4 into new wheat cultivars.

First Report of Barley virus G in Australia

Wheat (Triticum aestivum), barley (Hordeum vulgare), and oats (Avena sativa) are some of the most economically important cereals in Australia and worldwide. Several cereal viruses are present in southeastern Australia with varied incidence and prevalence depending on year, location, and climatic conditions. Barley and cereal yellow dwarf viruses (family Luteoviridae) are increasingly prevalent in southeastern Australia (Nancarrow et al. 2018). They are transmitted by aphid vectors, mainly the bird cherry-oat aphid (Rhopalosiphum padi) and the corn leaf aphid (Rhopalosiphum maidis). High Plains wheat mosaic virus (HPWMoV) (family Fimoviridae) has been reported in Western Australia (Coutts et al. 2014), and there have been unconfirmed reports of its presence in Victoria; wheat streak mosaic virus (WSMV) (family Potyviridae) was reported in Victoria and other Australian states in 2003 (Ellis et al. 2003). Both HPWMoV and WSMV are transmitted by the wheat curl mite (Aceria tosichella) (Coutts et al. 2014). In late April 2018, a high number of R. maidis was observed on volunteer cereal plants consisting of wheat, barley, and wild oats (Avena fatua) in fields near Horsham, Victoria, Australia, together with yellowing and streaking discoloration on some plants. No evidence of wheat curl mite infestation was observed. Initially, 148 samples were collected from symptomatic and nonsymptomatic plants and tested for WSMV (DSMZ-AS0544), luteoviruses (DSMZ, AS-0227/1), and the yellow dwarf viruses BYDV-PAV, BYDV-MAV, and CYDV-RPV by tissue blot immunoassay (TBIA) (Nancarrow et al. 2018); 16 samples were positive for WSMV, 34 were positive for luteoviruses, and of the 34 luteovirus-positive samples, six were positive for at least one of the yellow dwarf viruses typically found in the Horsham region. The remaining 28 luteovirus-positive samples required further identification. In early May, a further 68 samples, prioritizing plants with streaking discoloration, were collected from the same area and tested for WSMV, luteoviruses, and HPWMoV (Agdia, SRA17200) by TBIA; 26 samples were positive for WSMV, three were positive for luteoviruses, and two were positive for HPWMoV. Two of the 216 samples tested by TBIA had mixed infections. Virus was detected in symptomatic and nonsymptomatic samples, and a number of symptomatic plants tested negative. A subset of 23 samples requiring either further identification or confirmation was additionally tested by reverse transcription polymerase chain reaction (RT-PCR) for WSMV (French and Robertson 1994), luteoviruses (Robertson et al. 1991), and HPWMoV (Lebas et al. 2005). Amplicons of the expected sizes were obtained with 11 samples positive for WSMV, 12 positive for luteoviruses, and two positive for HPWMoV. Amplicons were directly sequenced. Nucleotide sequences of the WSMV (GenBank accession MK103385) and HPWMoV (accession MK103386) amplicons were identical to accessions AY327865 (WSMV) and KT988872 (HPWMoV), respectively, confirming the presence of both viruses and hence the presence of HPWMoV in Victoria. The nucleotide sequence of the luteovirus amplicon showed 99% identity to barley virus G (BVG) (accession NC_029906). The 12 samples that tested positive for luteoviruses by TBIA and RT-PCR were further tested using two sets of BVG-specific primers (Jo et al. 2018; Park et al. 2017), each targeting a distinct region of the BVG genome; all samples were positive. An amplicon from each BVG-specific RT-PCR test was directly sequenced (accessions MK411425 and MK103388), with each showing 96% nucleotide identity to BVG (accession NC_029906), confirming the presence of BVG in these samples. Some BVG-positive samples showed yellow discoloration of the leaf tips. This is the first reported detection of BVG in Australia. BVG was reported for the first time in South Korea in 2016 (Zhao et al. 2016). Epidemiology and yield losses associated with BVG are not known in Australia or elsewhere.

P7 and P8 proteins of High Plains wheat mosaic virus, a negative-strand RNA virus, employ distinct mechanisms of RNA silencing suppression

High Plains wheat mosaic virus (genus Emaravirus), an octapartite negative-sense RNA virus, encodes two RNA silencing suppressors, P7 and P8. In this study, we found that P7 and P8 efficiently delayed the onset of dsRNA-induced transitive pathway of RNA silencing. Electrophoretic mobility shift assays (EMSA) revealed that only P7 protected long dsRNAs from dicing in vitro and bound weakly to 21- and 24-nt PTGS-like ds-siRNAs. In contrast, P8 bound strongly and relatively weakly to 21- and 24-nt ds-siRNAs, respectively, suggesting size-specific binding. In EMSA, neither protein bound to 180-nt and 21-nt ssRNAs at detectable levels. Sequence analysis revealed that P7 contains a conserved GW motif. Mutational disruption of this motif resulted in loss of suppression of RNA silencing and pathogenicity enhancement, and failure to complement the silencing suppression-deficient wheat streak mosaic virus. Collectively, these data suggest that P7 and P8 proteins utilize distinct mechanisms to overcome host RNA silencing for successful establishment of systemic infection in planta.