We isolated the barley stem rust resistance genes Rpg5 and rpg4 by map-based cloning. These genes are colocalized on a 70-kb genomic region that was delimited by recombination. The Rpg5 gene consists of an unusual structure encoding three typical plant disease resistance protein domains: nucleotide-binding site, leucine-rich repeat, and serine threonine protein kinase. The predicted RPG5 protein has two putative transmembrane sites possibly involved in membrane binding. The gene is expressed at low but detectable levels. Posttranscriptional gene silencing using VIGS resulted in a compatible reaction with a normally incompatible stem rust pathogen. Allele sequencing also validated the candidate Rpg5 gene. Allele and recombinant sequencing suggested that the probable rpg4 gene encoded an actin depolymerizing factor-like protein. Involvement of actin depolymerizing factor genes in nonhost resistance has been documented, but discovery of their role in gene-for-gene interaction would be novel and needs to be further substantiated.actin depolymerizing factor ͉ barley ͉ disease resistance domains ͉ map-based cloning

Barley stripe mosaic virus (BSMV)-based virus-induced gene silencing (VIGS) is an effective strategy for rapid functional analysis of genes in wheat leaves, but its utility to transiently express genes, and silencing in other tissues including root, flower, and developing grains, has not been demonstrated in monocots. We monitored green fluorescent protein (GFP) expression to demonstrate the utility of BSMV as a transient expression vector and silenced genes in various wheat tissues to expand VIGS utility to characterize tissue-specific genes. An antisense construct designed for coronatine insensitive1 (COI1) showed an 85% decrease in COI1 transcript level in roots accompanied by a 26% reduction in root length. Similarly, silencing of seed-specific granule-bound starch synthase by antisense and hairpin constructs resulted in up to 82% reduction in amylose content of the developing grains. VIGS of meiosis-specific genes demonstrated by silencing wheat homologue of disrupted meiosis cDNA1 (DMC1) by an antisense construct resulted in a 75-80% reduction in DMC1 transcript level accompanied by an average of 37.2 univalents at metaphase I. The virus-based transient GFP expression was observed in the leaf, phloem, and root cortex at 10-17 days post-inoculation. A novel observation was made that 8-11% of the first selfed generation progeny showed VIGS inheritance and that this proportion increased to 53-72% in the second and to 90-100% in the third generations. No viral symptoms were observed in the progeny, making it possible to study agronomic traits by VIGS. VIGS inheritance is particularly useful to study genes expressing during seed germination or other stages of early plant growth.

Significance Maintaining diploid-like pairing behavior is essential for a polyploid to establish as a new species. The Pairing homeologous 1 ( Ph1 ) gene, regulating such behavior in polyploid wheat, was identified in 1958, but its molecular function remained elusive. The present communication reports identification of the candidate Ph1 ( C-Ph1 ) gene that is expressed exclusively during meiotic metaphase I, whose silencing resulted in formation of multivalents like the Ph1 gene mutations. Although the C-Ph1 gene has three homoeologous copies, the 5B copy has diverged in sequence from the other two copies. Heterologous gene silencing of the Arabidopsis homologue of the C-Ph1 gene also confirmed its function. Molecular characterization of this gene will make it possible to develop precise alien introgression strategies.

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High Plains wheat mosaic virus (HPWMoV) is a monocistronic octapartite single-stranded negative-sense RNA virus in the genus Emaravirus, family Fimoviridae (ICTV 2018). It was first reported in 1993 in several High Plains states of the USA (Jensen et al. 1996). It infects a number of cereal crops including wheat (Triticum aestivum L.), maize (Zea mays L.), barley (Hordeum vulgare L.), and some weeds (Seifers et al. 1998). Symptoms induced by HPWMoV are similar to those caused by wheat streak mosaic virus (WSMV) with leaf veins showing yellow flecks and streaks, and severity varying from mild to high. HPWMoV is transmitted by the wheat curl mite Aceria tosichella Keifer (Seifers et al. 2009). It can co-infect wheat with some cereal viruses including wheat streak mosaic virus (WSMV), triticum mosaic virus, barley yellow dwarf virus PAV, and cereal yellow dwarf virus RPV (Burrows et al. 2009). In aA field survey conducted in Southern southern Alberta, Canada, in late June 2017, we sampledevaluated 34 and 37 plants of wheat and grassy weed species, respectively, for the presence of cereal viruses. The sampled wheat plants showed yellow flecks and streaks, with some heavily chlorotic leaves showing necrotic patches along necrosis at leaf margins. Some leaves evenLeaves with extreme symptoms were became completely scorched and/or desiccated. Grassy weed species were collected nearby symptomatic wheat, but did not show any typical symptoms. The incidence of symptoms varied among the sampled wheat fields, with some fields showing more than 75% crop impactedranging from trace levels to 75% infected. The samples were subjected to RT-PCR test for HPWMoVwas performed using primers WMoV-F (Byamukama et al. 2016) and WMoV-RR targeting the RNA 3B region of HPWMoV, after total nucleic acid extraction using in-house procedure (SOP L036). All primer sequences, their location in the genome, and amplicon sizes are given in supplementary table S1. Four wheat plants (1 plant of cv. AAC Elevate, and 3 plants of unknown cultivar), and one plant of foxtail barley plant (Hordeum jubatum L.), from Lethbridge County tested positive for HPWMoV. One wheat plant (cv. Glenn) from the Medicine Hat area also tested positive for HPWMoVthe virus. The assay wasResults were confirmed by two independent RT-PCRs with primers targeting RNA-6 segment (WmoV-F1 and WmoV-R1) and RNA-2 segment (WmoV-F2 and WmoV-R2), respectively. BLASTn analysis of a representative of each of the two fragments, which have been submitted to NCBI under accession numbers MT124696 and MT124697, showed that they share more than 99 % sequence identity with respective RNA species of somethe HPWMoV isolates from the USANebraska and Kansas (KT988861, KJ939624, KJ939629, and KT988866). All but one of the HPWMoV infected wheat plants were also positive for WSMV. WSMV infection was confirmed using DAS-ELISA (Agdia, IN, USA) and RT-PCR with primers targeting the WSMV coat protein (WSMV-CP2 and WSMV-CP4, Dwyer et al. 2007). NAs a second confirmatory method, next-generation sequencing analysis of one RT-PCR positive sample yielded 10,012,849 clean reads. The dsRNA extraction method, library preparation, sequencing and bioinformatics analyses were done according to Rott et al. (2017). The obtained clean reads were searched for viral sequences using Virtool software (https://www.virtool.ca). This analysis revealinged 31 HPWMoV specific contigs. Sixteen contigs, ranging in length from 252 to 1728 bp, covered 72.92% of HPWMoV KS7 isolate’s genome (KT988860 – KT988868) and shared 95-100% identity with it. This virus could be present in, or spread into, other parts of the Canadian prairies where complex co-infections with the other wheat curl mite-transmissible viruses could occur. Co-infections of WSMV and HPWMoV have been reported to correlate with increased symptom severity (Burrows et al. 2009). Primer sequences, their location in genome and amplicon sizes are given in supplementary table S1. WSMV infections have led to wheat yield losses of 7-18% in the Texas panhandle, and the North American prairies, including Alberta, Canada (Hadi et al. 2011). The appearance of HPWMoV in Canada is concerning because co-infections with WSMV could increase severity and yield loss.

Hemp (Cannabis sativa L.) is a herbaceous annual grown mainly for its blast fiber and seed oil. In 1999, Health Canada issued licenses to plant 12,145 ha of hemp in Canada. Of these, 730 ha were in Alberta. During the last week of August, hemp plants (cv. Fasamo) in a central Alberta field showed the following symptoms and signs: wilting foliage turning light brown; dry tan to gray lesions on stems; shredding and breaking of stems at the lesion; presence of white mycelium in the lesion; and black round, irregular, or oblong sclerotia (up to 5 mm diameter and 2 to 11 mm long) present externally at the lesion on the stem and inside the pith cavity. Lesions were found at the crown, near the inflorescence, and along the entire stem length. Disease incidence in a survey of six commercial fields (40 ha) ranged from 1 to 8%. The organism isolated from lesions on potato dextrose agar produced white aerial mycelia and large numbers of sclerotia characteristic of Sclerotinia sclerotiorum. Pathogenicity was confirmed by inoculating 23-day-old greenhouse-grown hemp plants (cv. Fasamo) with autoclaved wheat grains colonized for 14 days with a S. sclerotiorum culture previously isolated from an infected hemp plant. The grains were placed on soilless growing medium near the plant and covered very lightly. One week after inoculation, grayish lesions appeared on the stems, white mycelia appeared on lesions, and plants wilted. The pathogen was reisolated from the lesions. This is the first report of S. sclerotiorum on hemp in Alberta, Canada. The disease known as hemp canker has been reported to cause severe losses under cool wet conditions in the Netherlands (1). Reference: (1) J. M. McPartland. J. Int. Hemp Assoc. 3:19, 1996.

Rubus stunt, caused by ‘Candidatus Phytoplasma rubi’ (Rubus stunt phytoplasma; RSP), is an economically important disease of Rubus spp. . This disease occurs in wild and cultivated Rubus in Europe but has not been reported from North America; however, its major leafhopper vector is well established in Western Canada and the United States. RSP has the potential to impact the cane-fruit industry by significantly compromising yields and impacting export potential for Rubus propagation material. To mitigate the risk of this disease entering or establishing, import and export testing of propagation material is a phytosanitary requirement in Canada, the U.S and other countries regulating RSP. In the absence of a specific test for RSP, the current testing scheme involves the use of a generic test to screen for phytoplasmas followed by additional time-consuming procedures to confirm the phytoplasma species. In this study, a real-time PCR assay, targeting 154 bp region of tuf gene, was developed for sensitive and specific detection of RSP in Rubus spp. The developed assay detected a minimum of 5 target copies, and no cross-reactivity was observed even with the ‘Ca. P. rubi’-related strain associated with blackberry witches’ broom which differs from RSP only by a single nucleotide polymorphism in the target region. Repeatability of the developed assay was checked on two real-time PCR platforms with acceptable results. In conclusion, this real-time PCR assay provides a sensitive and specific detection of RSP for mitigating the introduction and spread of Rubus stunt disease in Rubus spp..

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