Abstract Pea seed-borne mosaic virus (PSbMV) is a significant pathogen that affects the yield and quality of leguminous crops. In recent years, PSbMV has emerged as the dominant viral threat to legume cultivation in Yunnan Province, China. In this study, the complete genome of PSbMV-YN isolate (GenBank accession: PP746839) with a length of 9919 nucleotides excluding the poly(A) tail was obtained by segmented amplification combined with rapid amplification of cDNA ends (RACE). Phylogenomic analysis of full-length polyprotein-encoding nucleotide and predicted amino acid sequences revealed that PSbMV-YN clusters within the P1 pathotype clade. Genetic mapping of local pea varieties demonstrated eIF4E , a gene tightly linked to the sbm1 locus, as the susceptibility genotype, revealing the mechanistic basis for the prevalence of the P1 pathotype in Yunnan pea populations. Further analysis of the highly variable region of the P1 cistron in PSbMV-YN indicates that the P1 protein could be classified as Type A, which generally lacks RNA silencing suppressor activity. Using a GFP suppression assay, we further validated the RNA silencing suppressor activity of HC-Pro in both Nicotiana benthamiana and its natural host— Pisum sativum L. Additionally, immunoprecipitation sequencing (IP-seq) revealed that HC-Pro binds to small RNAs of variable lengths, thereby effectively inhibiting both local and systemic RNA silencing to promote viral infection. This study reports the first genomic sequence of a Chinese isolate of PSbMV and provides a comprehensive analysis of interactions between HC-Pro with endogenous small RNAs.

Pea seed lots (144) from Australian farms and research trials were assessed for pea seed-borne mosaic virus (PSbMV) seed transmission rates. High infection rates (up to 40%) were detected, particularly in the widely grown and highly PSbMV susceptible variety ‘Kaspa’, with only 12 out of 54 seed lots found to be free of the virus. PSbMV strains were isolated from 15 infected seed lots, and were pathotyped on a set of homogeneous Pisum sativum PSbMV differentials. Of the four pathotypes identified, P1 and P4 (eight and 20 isolates, respectively) were earlier reported in Australia. Pathotype P3, as yet unreported in Australia, was the most frequently identified pathotype (42 isolates). One PSbMV isolate was identified as the P2 pathotype, which has previously been isolated only from lentil seed. None of the isolated pathotypes could overcome the PSbMV resistance gene sbm1. The relevance of these findings for field pea breeding programmes and genomic studies of PSbMV are discussed.

Pulse crops, including chickpeas, lentils, faba beans, field peas etc., are vital to Australia’s agricultural economy, supporting domestic consumption, exports, and sustainable farming through nitrogen fixation. However, viral diseases pose a significant threat, causing substantial yield losses and compromising crop quality. This concise review synthesizes current knowledge on major viruses affecting Australian pulse crops, such as alfalfa mosaic virus (AMV), bean yellow mosaic virus (BYMV), cucumber mosaic virus (CMV), pea seed-borne mosaic virus (PSbMV), turnip mosaic virus (TuMV) and other common viruses. It highlights their transmission modes (e.g., aphids, seeds, mechanical), characteristic symptoms (e.g., chlorosis, stunting, leaf deformation), and economic impacts. The review also outlines common detection methods, including serological (ELISA, LFIA) and molecular techniques (RT-PCR, LAMP, HTS), and integrated disease management strategies, such as virus-free seeds, vector control, crop rotation, and resistant cultivars. By consolidating these aspects, this review provides a small look into the viruses of pulse crops in Australia.

Pea seed-borne mosaic virus (PSbMV) poses a major threat to global pulse production. This virus, transmitted through seeds, can spread within fields via insect vectors, especially pea aphids (Acyrthosiphon pisum), in a nonpersistent manner. To mitigate the risks associated with PSbMV, it is crucial to plant virus-free seeds, detect the virus at an early stage, and implement effective control measures for the vectors, given that most commercial pulse cultivars are vulnerable to the virus. This study designed and assessed multiple primers for PCR-based virus detection and demonstrated their capability to identify PSbMV isolates in infected plant tissues. The primers successfully detected PSbMV in dried plant tissues and in aphids collected from infected plants, even after being stored at room temperature for up to 3 months. Furthermore, a hydrolysis probe-based assay was developed, and its effectiveness for quantitative PCR (qPCR), digital PCR (dPCR), and droplet digital PCR (ddPCR) was evaluated. Our results showed high sensitivity and linearity of the assay, capable of detecting PSbMV at concentrations as low as 22 copies per reaction mix using digital PCRs. Our findings underscore the effectiveness of the developed primers and assay for the rapid and sensitive detection of PSbMV isolates in a variety of plant tissues, aphids, and seed samples, offering improved tools for disease monitoring and management in agricultural settings.

Phylogenetic analysis, mixed infection and seed transmission of Pea seed-borne mosaic virus in Ethiopia

Plant introductions, including the movement of seeds across international borders, significantly contribute to the emergence of viral pathogens, which account for approximately half of all emerging plant diseases. Among plant virus families, the Potyviridae stand out as the largest and most economically impactful. The potyvirus genus with 201 species widespread across various crops, causes substantial crop losses globally. These viruses are primarily transmitted by aphids in a nonpersistent manner, with some also capable of transmission through seeds. Global food security is significantly impacted by the crucial part that seed transmission plays in the epidemiology and spread of different plant viruses. With transmission rates varying greatly between viruses and host plants, over 231 viruses were discovered to be capable of spreading through seeds, including 20 seed-transmitted potyviruses. The seed transmission efficiency of potyviruses such as maize dwarf mosaic virus (MDMV), cowpea aphid-borne mosaic virus (CABMV), and bean common mosaic virus (BCMV) ranges from 3% to 95%. Other notable viruses include soybean mosaic virus (SMV) (58% transmission) and pea seed-borne mosaic virus (PSbMV) (100% transmission). The diversity in seed transmission efficiency among viruses demonstrates the important role that seeds play in the propagation and dissemination of viral pathogens. In addition, seed transmission permits viruses to survive in unfavorable environments, promotes long-distance spread via international seed exchange, and acts as the main point of infection for viruses that have vertical transmission. The complex dynamics of seed transmission and the complex relationship between viruses and their host plants highlight the significance of strict phytosanitary measures in protecting global agriculture from the destructive effects of viruses transmitted through seeds. This review focuses on potyvirus seed transmission, with an emphasis on determining the significance of this mode and understanding the potential role of seed transmission in potyviruses.

With worldwide cultivation, the faba bean (Vicia faba L.) stands as one of the most vital cool-season legume crops, serving as a major component of food security. China leads global faba bean production in terms of both total planting area and yield, with major production hubs in Yunnan, Sichuan, Jiangsu, and Gansu provinces. The faba bean viruses have caused serious yield losses in these production areas, but previous researches have not comprehensively investigated this issue. In this study, we collected 287 faba bean samples over three consecutive years from eight provinces/municipalities of China. We employed small RNA sequencing, RT-PCR, DNA sequencing, and phylogenetic analysis to detect the presence of viruses and examine their incidence, distribution, and genetic diversity. We identified a total of nine distinct viruses: bean yellow mosaic virus (BYMV, Potyvirus), milk vetch dwarf virus (MDV, Nanovirus), vicia cryptic virus (VCV, Alphapartitivirus), bean common mosaic virus (BCMV, Potyvirus), beet western yellows virus (BWYV, Polerovirus), broad bean wilt virus (BBWV, Fabavirus), soybean mosaic virus (SMV, Potyvirus), pea seed-borne mosaic virus (PSbMV, Potyvirus), and cucumber mosaic virus (CMV, Cucumovirus). BYMV was the predominant virus found during our sampling, followed by MDV and VCV. This study marks the first reported detection of BCMV in Chinese faba bean fields. Except for several isolates from Gansu and Yunnan provinces, our sequence analysis revealed that the majority of BYMV isolates contain highly conserved nucleotide sequences of coat protein (CP). Amino acid sequence alignment indicates that there is a conserved NAG motif at the N-terminal region of BYMV CP, which is considered important for aphid transmission. Our findings not only highlight the presence and diversity of pathogenic viruses in Chinese faba bean production, but also provide target pathogens for future antiviral resource screening and a basis for antiviral breeding.

During a project investigating pea viruses in the United Kingdom and developing a workflow for high-throughput sequencing in crop surveillance, pea necrotic yellow dwarf virus (PNYDV) was identified at one site, along with pea enation mosaic virus-1, pea enation mosaic virus-2, pea enation mosaic virus satellite RNA, pea seedborne mosaic virus, and turnip yellows virus. Following an initial finding of small PNYDV contigs by an RNA sequencing protocol on MiSeq (Illumina), confirmation and prevalence testing by PCR and Sanger sequencing determined the average PNYDV prevalence to be 3%. This is the first report of PNYDV, and a nanovirus, in the United Kingdom. Initial characterization of the octopartite genome by PCR revealed that component DNA N had a truncated sequence that differed from those previously reported. This led to confirmation testing using rolling circle amplification (RCA) into MiSeq and then MinION (Oxford Nanopore). Sequences were obtained by both RCA-MiSeq and RCA-MinION, with MinION producing more complete genomes than RCA-MiSeq. Data obtained from sequencing were used to provide an initial estimation of the genomic formula of PNYDV. Previous work has looked into comparing each method; however, this study shows how these methods can be used together. The truncated sequence for DNA N was also found by RCA-MiSeq and RCA-MinION and confirmed by specific primers, but further work is required to understand the impact of this truncation. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Incidence, Distribution and Molecular Characterization of Pea Seed-borne Mosaic Virus (PSbMV) in Egypt

Pea seed-borne mosaic virus (PSbMV), a nonpersistently aphid-transmitted potyvirus, has been reported in field pea (Pisum sativum L.)-growing regions worldwide. In 2014, PSbMV was first identified in field peas in North Dakota, U.S.A. Susceptibility and yield losses attributed to PSbMV infection are influenced by viral pathotype and host genotype. Isolate ND14-1, recovered from North Dakota infected seed and identified as pathotype 4 (P4), was mechanically inoculated onto 20 field pea cultivars under greenhouse conditions. PSbMV susceptibility, number of seeds and pods per plant, yield, symptom expression, and PSbMV seed transmission rates were assessed by cultivar. A risk assessment was developed based on cultivar susceptibility, yield reduction, and PSbMV seed transmission. Risk factors were weighted based on perceived importance to commercial field pea producers. Three cultivars were classified as low risk, seven cultivars were classified as intermediate risk, and 10 cultivars were classified as high risk. Two of the low-risk cultivars, Aragorn and Cruiser, were confirmed to be resistant to this isolate of PSbMV. Cultivar Arcadia was susceptible to PSbMV infection with mild expression of symptoms, but was classified as low risk based on a low seed transmission rate and diminished yield losses. This risk assessment could prove a useful tool for growers in field pea cultivar selection where PSbMV is prevalent.

Pea seed-borne mosaic virus

There is only limited knowledge of the presence and incidence of viruses in peas within the United Kingdom, therefore high-throughput sequencing (HTS) in combination with a bulk sampling strategy and targeted testing was used to determine the virome in cultivated pea crops. Bulks of 120 leaves collected from twenty fields from around the UK were initially tested by HTS, and presence and incidence of virus was then determined using specific real-time reverse-transcription PCR assays by testing smaller mixed-bulk size samples. This study presents the first finding of turnip yellows virus (TuYV) in peas in the UK and the first finding of soybean dwarf virus (SbDV) in the UK. While TuYV was not previously known to be present in UK peas, it was found in 13 of the 20 sites tested and was present at incidences up to 100%. Pea enation mosaic virus-1, pea enation mosaic virus-2, pea seed-borne mosaic virus, bean yellow mosaic virus, pea enation mosaic virus satellite RNA and turnip yellows virus associated RNA were also identified by HTS. Additionally, a subset of bulked samples were re-sequenced at greater depth to ascertain whether the relatively low depth of sequencing had missed any infections. In each case the same viruses were identified as had been identified using the lower sequencing depth. Sequencing of an isolate of pea seed-borne mosaic virus from 2007 also revealed the presence of TuYV and SbDV, showing that both viruses have been present in the UK for at least a decade, and represents the earliest whole genome of SbDV from Europe. This study demonstrates the potential of HTS to be used as a surveillance tool, or for crop-specific field survey, using a bulk sampling strategy combined with HTS and targeted diagnostics to indicate both presence and incidence of viruses in a crop.

Pea seed-borne mosaic virus (PSbMV) is both seedborne and aphid-transmitted and can cause economic losses for pea (Pisum sativum L.) production by reducing yield through decreased seed weight and number. The P1 pathotype is especially virulent, affecting this important vegetable crop across the United States and internationally in regions of West Asia, North Africa, Europe, and Australia. Previously, two kompetitive allele-specific PCR (KASP) genotyping markers (eIF4E resistant 1 and 2) were developed and validated on P. sativum accessions identifying two PSbMV pathotype P1 resistance alleles in the eukaryotic translation initiation factor gene, eIF4E. The current study utilized these novel markers to rapidly evaluate 318 genetic resource accessions maintained as part of the United States Department of Agriculture National Plant Germplasm System's Pea Single Plant Collection (PSPC). The evaluations also included 58 commercial and other plant introduction (PI) lines that were assessed for the two eIF4E resistance alleles. All genotyping results were validated in greenhouse assays by confirmation of observable disease symptoms after inoculations and by enzyme-linked immunosorbent assays. The eIF4E resistant 1 and 2 alleles were found in 18 accessions from the PSPC, five commercial lines, and 14 other PI accessions. A single PSPC accession showed resistance to PSbMV pathotype P1 that is believed to be a novel source of resistance based on sequencing analysis of eIF4E. Sources of resistance were identified in the PSPC and in commercial cultivars that can be introgressed into breeding lines using traditional techniques to reduce the time and cost required to generate germplasm with superior disease-resistant traits.

Globally, high-throughput sequencing (HTS) has been used for virus detection in germplasm certification programs. However, sequencing costs have impeded its implementation as a routine diagnostic certification tool. In this study, the targeted genome sequencing (TG-Seq) approach was developed to simultaneously detect multiple (four) viral species of; Pea early browning virus (PEBV), Cucumber mosaic virus (CMV), Bean yellow mosaic virus (BYMV) and Pea seedborne mosaic virus (PSbMV). TG-Seq detected all the expected viral amplicons within multiplex PCR (mPCR) reactions. In contrast, the expected PCR amplicons were not detected by gel electrophoresis (GE). For example, for CMV, GE only detected RNA1 and RNA2 while TG-Seq detected all the three RNA components of CMV. In an mPCR to amplify all four viruses, TG-Seq readily detected each virus with more than 732,277 sequence reads mapping to each amplicon. In addition, TG-Seq also detected all four amplicons within a 10−8 serial dilution that were not detectable by GE. Our current findings reveal that the TG-Seq approach offers significant potential and is a highly sensitive targeted approach for detecting multiple plant viruses within a given biological sample. This is the first study describing direct HTS of plant virus mPCR products. These findings have major implications for grain germplasm healthy certification programs and biosecurity management in relation to pathogen entry into Australia and elsewhere.

One of the most important diseases of pea is caused by Pea seed-borne mosaic virus (PSbMV), which has a relatively wide host range. Since there are few varieties with resistance against the virus, and spraying insecticides is not very effective, the determination of the disease and the pathogen in the seeds is very important. Inoculum prepared from pea seeds showing typical virus symptoms caused very mild symptoms on Chenopodium amaranticolor and C. quinoa, but several chlorotic/necrotic lesions on bean (Phaseolus vulgaris) cv. Scarlet, and systemic symptoms with mosaic and curling of top leaves on bean cv. Maxidor. The detection of the virus was carried out by PCR using universal primers and virus sequence analysis. According to the phylogenetic analysis the PSbMV isolate identified in Hungary belongs to the pathotype P1 and associated with the cluster 2 isolates.

Pea (Pisum sativum L.) is an economically important legume crop that is commonly used as dry beans, fresh peas, pods and shoots (Guo et al. 2009). Pea enation mosaic is an important virus disease of pea caused by two viruses in an obligate symbiosis, pea enation mosaic virus 1 (PEMV-1, Enamovirus, Luteoviridae) and pea enation mosaic virus 2 (PEMV-2, Umbravirus, Tombusviridae) (Hema et al. 2014). In November 2019, foliar yellow mosaic and vein enations symptoms were observed from pea plants in five fields of Honghe autonomous prefecture, Yunnan province, China. Incidence of symptomatic plants ranged from 20 to 40% and was distributed in both small and large fields. Leaves with typical virus-like symptoms were collected from five symptomatic pea plants in two fields and used for total RNA extraction. The five extracts of equimolar quantities were pooled into a sample and subjected to High Throughput Sequencing (HTS) by Illumina HiSeq system. Analyses of raw RNA reads were performed using CLC Genomics Workbench 12 (Qiagen). A total of 60,009,746 RNA reads were obtained from the sample, and de novo assembly of the reads using the CLC Genomics generated 88,105 contigs. BLASTN searches revealed the presence of contigs with high similarities to PEMV-1, PEMV-2, Pea seed-borne mosaic virus, and Bean yellow mosaic virus. To confirm the presence of PEMV-1 and PEMV-2 in the samples, two virus-specific primer pairs were designed based on the contig sequences obtained by HTS in this study. Primer pairs PEMV-1F/PEMV-1R (5'-ATGCCGACTAGATCGAAATC-3'/5'-TCAGAGGGAGGCATTCATTA-3') that flank the cp gene of PEMV-1 and PEMV-2F/PEMV-2R (5'-ATGACGATAATCATTAATG-3'/5'-TCACCCGTAGTGAGAGGCA-3') that target the ORF3 region of PEMV-2 were used to amplify the two viruses in RT-PCR. DNA fragments of the expected sizes (PEMV-1, 570 bp; PEMV-2, 693 bp) were amplified from all five samples. The RT-PCR products were cloned and sequenced. Sequence analysis showed that the 570-bp amplicon (MT481989) shared the highest nucleotide sequence identity of 98.95% with PEMV-1 (Z48507), while the 693-bp fragment (MT481990) had the highest nucleotide sequence identity of 97.4% with PEMV-2 isolate JKI (MK948534). One gram of the symptomatic leaves from each of the five plants was homogenized with 5 mL of 0.01 M phosphate-buffered saline (PBS buffer), pH 7.0. Each of the resulted saps was used to inoculate onto five healthy pea seedlings. A total of 25 healthy pea seedlings were inoculated, and 16 inoculated plants developed yellowing and mottling at 10 days post inoculation (dpi); no symptoms were observed on control plants inoculated only with PBS buffer. The formation of the typical enation was observed along the veins of lower side of the symptomatic leaves of the inoculated plants at 30 dpi. PEMV-1 and PEMV-2 infection were confirmed by RT-PCR assays using the specific primer pairs described above. Although the presence of the pea enation mosaic virus complex was suspected in China based on symptomatology (Brunt et al. 1997), to our knowledge, this is the first molecular confirmation of PEMV-1 and PEMV-2 occurrence in China. The co-infection of PEMV-1 and PEMV-2 usually cause severe yield losses; therefore, integration of detection and control measures is important in pea production regions where the two viruses occurred.

As pesticides have become heavily relied on for management of insect pests vectoring economically important pathogens of vegetable crops, development of pathogen-resistant germplasm remains a promising alternative to reduce or eliminate costly and timely chemical inputs. Molecular markers can be used to rapidly identify resistant genotypes to aid breeders in advancing germplasm. This study developed two kompetitive allele-specific PCR (KASP) genotyping markers for rapid screening of Pisum sativum genotypes for resistance to Pea seedborne mosaic virus pathotype P1 (PSbMV-P1), the most economically devastating strain worldwide. The KASP markers differentiate two eIF4E PSbMV-P1-resistant allelic variants from susceptible eIF4E variants. A single nucleotide polymorphism (Resistant 1) and a 3-basepair deletion (Resistant 2) present in either of the two resistant alleles were used for marker design. Forty-four P. sativum lines previously characterized for resistance to PSbMV were inoculated with PSbMV-P1 in a greenhouse, observed for visual symptoms, assayed for virus susceptibility by enzyme-linked immunosorbent assay (ELISA), and genotyped by KASP marker analysis. The KASP markers were 100% accurate in characterizing PSbMV-P1-susceptible and PSbMV-P1-resistant genotypes when correlated with the ELISA results. The Resistant 1 marker also correlated with resistance to PSbMV pathotypes P2 and P4 completely, making this marker a new advanced tool for P. sativum breeding programs.

Whole genome sequences of three Czech Pea seed-borne mosaic virus isolates belonging to P1 pathotype and causing different symptom intensity were obtained. Using RDP4 analysis the natural recombinant isolate PSB204CZ bearing two breakpoints in nucleotide positions 4053 and 6080 was identified. The isolate was composed of fragment 2028 nt in length partially covering CI and 6K2 regions of the minor parent (PSB262CZ) incorporated into the major parent (PSB194CZ). The results suggest that the observed recombination in CI-6K2 region is responsible for severity of developed symptoms. This observation detected for the first time natural recombination within PSbMV isolates of an important pathogen of leguminous plants.

Occurrence of <i>Pea seed-borne mosaic virus</i> in Field Peas in North Dakota

For the first time, Cucumber mosaic virus (CMV), Pea seed-borne mosaic virus (PSbMV) and Turnip yellows virus (TuYV) were detected in a naturally infected fenugreek (Trigonella foenum-graecum) crop in Australia. The level of infection reached an alarming 90%, 75% and 20% respectively for CMV, TuYV and PSbMV, resulting in total crop loss. Both CMV and TuYV were detected in non-symptomatic fenugreek plants while PSbMV was not. Seed lots from the crop in question were established, planted in the glasshouse and tested for a range of seed-borne viruses, none of which were detected. Additionally, fenugreek seedlings grown from the seeds in the glasshouse were found to be readily infected with a TuYV isolate from canola when inoculated using viruliferous Myzus persicae.