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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 (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.

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.

During 2016 growing season, samples with leaf yellowing and mosaic like symptoms were collected from main lentil (Lens culinaris Medik L.) fields. Specific ELISA positive PSbMV samples were...

An empirical model was developed to forecast Pea seed-borne mosaic virus (PSbMV) incidence at a critical phase of the annual growing season to predict yield loss in field pea crops sown under Mediterranean-type conditions. The model uses pre-growing season rainfall to calculate an index of aphid abundance in early-August which, in combination with PSbMV infection level in seed sown, is used to forecast virus crop incidence. Using predicted PSbMV crop incidence in early-August and day of sowing, PSbMV transmission from harvested seed was also predicted, albeit less accurately. The model was developed so it provides forecasts before sowing to allow sufficient time to implement control recommendations, such as having representative seed samples tested for PSbMV transmission rate to seedlings, obtaining seed with minimal PSbMV infection or of a PSbMV-resistant cultivar, and implementation of cultural management strategies. The model provides a disease forecast risk indication, taking into account predicted percentage yield loss to PSbMV infection and economic factors involved in field pea production. This disease risk forecast delivers location-specific recommendations regarding PSbMV management to end-users. These recommendations will be delivered directly to end-users via SMS alerts with links to web support that provide information on PSbMV management options. This modelling and decision support system approach would likely be suitable for use in other world regions where field pea is grown in similar Mediterranean-type environments.

Pea seed-borne mosaic virus (PSbMV) significantly reduces yields in a broad spectra of legumes. The eukaryotic translation initiation factor has been shown to confer resistance to this pathogen, thus implying that translation and proteome dynamics play a role in resistance. This study presents the results of a proteome-wide analysis of Pisum sativum L. response to PSbMV infection. LC–MS profiling of two contrasting pea cultivars, resistant (B99) and susceptible (Raman) to PSbMV infection, detected > 2300 proteins, 116 of which responded to PSbMV ten and/or twenty days post-inoculation. These differentially abundant proteins are involved in number of processes that have previously been reported in the plant-pathogen response, including protein and amino acid metabolism, stress signaling, redox homeostasis, carbohydrate metabolism, and lipid metabolism. We complemented our proteome-wide analysis work with targeted analyses of free amino acids and selected small molecules, fatty acid profiling, and enzyme activity assays. Data from these additional experiments support our findings and validate the biological relevance of the observed proteome changes. We found surprising similarities in the resistant and susceptible cultivars, which implies that a seemingly unaffected plant, with no detectable levels of PSbMV, actively suppresses viral replication. Biological significance Plant resistance to PSbMV is connected to translation initiation factors, yet the processes involved are still poorly understood at the proteome level. To the best of our knowledge, this is the first survey of the global proteomic response to PSbMV in plants. The combination of label-free LC–MS profiling and two contrasting cultivars (resistant and susceptible) provided highly sensitive snapshots of protein abundance in response to PSbMV infection. PSbMV is a member of the largest family of plant viruses and our results are in accordance with previously characterized potyvirus-responsive proteomes. Hence, the results of this study can further extend our knowledge about these pathogens. We also show that even though no viral replication is detected in the PSbMV-resistant cultivar B99, it is still significantly affected by PSbMV inoculation.

Chickpea plants with severe yellowing and tip wilting were observed in the Cap-Bon Region of Tunisia in 2006. The viral-like symptoms resulted in yield loss of approximately 25% in some fields. A total of 110 symptomatic chickpea plants was collected from nine chickpea fields and tested at the Virology Laboratory of ICARDA, Syria for eight legume viruses using tissue-blot immunoassay (TBIA) (3). Polyclonal antisera produced at the ICARDA Virology Laboratory were used to test for Chickpea chlorotic dwarf virus (genus Mastrevirus, family Geminiviridae), Broad bean stain virus (genus Comovirus, family Secoviridae), Broad bean mottle virus (genus Bromovirus, family Bromoviridae), and Bean yellow mosaic virus and Pea seed borne mosaic virus (genus Potyvirus, family Potyviridae). Antiserum to Beet mosaic virus (BtMV; genus Potyvirus, family Potyviridae) (AS-0143) was provided by the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). In addition, three monoclonal antibodies (MAb) were used to detect Faba bean necrotic yellows virus (FBNYV; genus Nanovirus, family Nanoviridae) (MAb 3-2E9) (1), potyviruses (PVAS-769 [MAb PTY 3 Potyvirus Group] American Type Culture Collection, Manassas, VA), and luteoviruses (MAb B-2-5G4) (2). Twenty-two of the plants tested positive with MAb PTY 3 and BtMV antisera, 56 samples reacted with MAb B-2-5G4, and eight plants with the FBNYV MAb, whereas 24 plants tested negative with all antisera. Because reactions with the BtMV antiserum were unexpected, detection of BtMV was confirmed by reverse transcription-(RT)-PCR assays using BtMV-specific primers (LN26 and LN27) (4), which produced an amplicon of expected size (1,050 bp) from all plants that reacted with BtMV antiserum but not from plants that were serologically negative. Leaf tissue from a BtMV-infected plant was ground in 0.01 M potassium phosphate buffer, pH 7.2 (1:20, wt/vol), mixed with 0.5% celite, and used for mechanical inoculation of chickpea seedlings (cv. Beja 4). In addition, adults of three legume aphid species (Aphis craccivora, A. fabae, and Acyrthosiphon pisum) were starved for 1 h before feeding on BtMV-infected chickpea leaves for an acquisition access period of 5 min. Fifteen aphids of each species were placed on each chickpea plant, allowed to feed for 24 h, and then sprayed with an insecticide. Tip wilting symptoms appeared on plants 15 to 20 days after mechanical and aphid inoculations but not on plants used as negative control treatments (inoculated mechanically with healthy leaf tissue or with aphids that had fed on noninfected chickpea plants). Use of BtMV antiserum for TBIA analysis of inoculated plants revealed systemic BtMV infections in 35 of 92 plants inoculated mechanically and 15 of 75 plants inoculated with viruliferous A. fabae only. To our knowledge, this is the first record of BtMV infecting chickpea in Tunisia. References: (1) A. Franz et al. Ann. Appl. Biol. 128:255, 1996. (2) L. Katul. Characterization by serology and molecular biology of bean leaf roll virus and faba bean necrotic yellows virus. Ph.D. thesis. University of Gottingen, Germany, 1992. (3) K. M. Makkouk and A. Comeau. Eur. J. Plant Pathol. 100:71, 1994. (4) L. G. Nemchinov et al. Arch. Virol. 149:1201, 2004.

In field pea (Pisum sativum) crops worldwide, sowing seed with minimal Pea seed-borne mosaic virus (PSbMV) infection is crucial to manage its spread and the seed yield and quality losses it causes. The possibility of using seed fractionation to help decrease PSbMV infection in infected seed-stocks used for sowing crops was investigated. When six PSbMV-infected seed-lots of Australian field pea cvs Kaspa or PBA Twilight were passed through sieves of different mesh sizes to obtain different size fractions, PSbMV seed transmission rates to seedlings were significantly higher in the 6.5 mm fractions. Passing pea seed-lots through a 6.5 mm sieve can be used to: (i) provide a useful indicator warning of likely high seed infection levels, and (ii) contribute towards decreasing PSbMV infection levels below the % seed transmission risk threshold for sowing.

From 2013 to 2015, incidences of Pea seed-borne mosaic virus (PSbMV) infection were determined in semi-leafless field pea (Pisum sativum) crops and trial plots growing in the Mediterranean-type environment of southwest Australia. PSbMV was found at incidences of 2 to 51% in 9 of 13 crops, 1 to 100% in 20 of 24 cultivar plots, and 1 to 57% in 14 of 21 breeding line plots. Crops and plots of ‘PBA Gunyah’, ‘Kaspa’, and ‘PBA Twilight’ were frequently PSbMV infected but none of PSbMV resistance gene sbm1-carrying ‘PBA Wharton’ plants were infected. In 2015, 14 new PSbMV isolates obtained from these various sources were sequenced and their partial coat protein (CP) nucleotide sequences analyzed. Sequence identities and phylogenetic comparison with 39 other PSbMV partial CP nucleotide sequences from GenBank demonstrated that at least three PSbMV introductions have occurred to the region, one of which was previously unknown. When plants of ‘Greenfeast’ and PBA Gunyah pea (which both carry resistance gene sbm2) and PBA Wharton and ‘Yarrum’ (which carry sbm1) were inoculated with PSbMV pathotype P-2 isolate W1, resistance was overcome in a small proportion of plants of each cultivar, showing that resistance-breaking variants were likely to be present. An improved management effort by pea breeders, advisors, and growers is required to diminish infection of seed stocks, avoid sbm gene resistance being overcome in the field, and mitigate the impact of PSbMV on seed yield and quality. A similar management effort is likely to be needed in field pea production elsewhere in the world.

Pea seed-borne mosaic virus (PSbMV) stability in sap and its contact transmission between field pea plants were investigated in glasshouse experiments. When infective leaf sap was kept at room temperature and inoculated to plants in the absence of abrasive, it was still highly infective after 6 h and low levels of infectivity remained after 30 h. PSbMV was transmitted from infected to healthy plants by direct contact when leaves were rubbed against each other. It was also transmitted when intertwining healthy and PSbMV-infected plants were blown by a fan to simulate wind. When air was blown on plants kept at 14 to 20°C, contact transmission of PSbMV occurred consistently and the extent of transmission was enhanced when plants were dusted with diatomaceous earth prior to blowing. In contrast, when plants were kept at 20 to 30°C, blowing rarely resulted in transmission. No passive contact transmission occurred when healthy and infected plants were allowed to intertwine together. This study demonstrates that PSbMV has the potential to be transmitted by contact when wind-mediated wounding occurs in the field. This may play an important role in the epidemiology of the virus in field pea crops, especially in situations where contact transmission expands initial crop infection foci before aphid arrival.

Drivers of Pea seed-borne mosaic virus (PSbMV) epidemics in rainfed field pea crops were examined under autumn to spring growing conditions in a Mediterranean-type environment. To collect aphid occurrence and PSbMV epidemic data under a diverse range of conditions, 23 field pea data collection blocks were set up over a 6-year period (2010 to 2015) at five locations in the southwest Australian grain-growing region. PSbMV infection levels in seed sown (0.1 to 13%), time of sowing (22 May to 22 June), and cultivar (Kaspa or PBA Twilight) varied with location and year. Throughout each growing season, rainfall data were collected, leaf and seed samples were tested to monitor PSbMV incidence in the crop and transmission from harvested seed, and sticky traps were used to monitor flying aphid numbers. Winged migrant Acyrthosiphon kondoi, Lipaphis erysimi, Myzus persicae, and Rhopalosiphum padi were identified in green tile traps in 2014 and 2015. However, no aphid colonization of field pea plants ever occurred in the blocks. The deductions made from collection block data illustrated how the magnitude of PSbMV spread prior to flowering is determined by two primary epidemic drivers: (i) PSbMV infection incidence in the seed sown, which defines the magnitude of virus inoculum source for within-crop spread by aphids, and (ii) presowing rainfall that promotes background vegetation growth which, in turn, drives early-season aphid populations and the time of first arrival of their winged migrants to field pea crops. Likely secondary epidemic drivers included wind-mediated PSbMV plant-to-plant contact transmission and time of sowing. PSbMV incidence at flowering time strongly influenced transmission rate from harvested seed to seedlings. The data collected are well suited for development and validation of a forecasting model that informs a Decision Support System for PSbMV control in field pea crops.

Legume crop cultivation is widespread in Greece and contributes significantly to the agricultural economy of the country. In contrast to pea, Pea seed-borne mosaic virus (PSbMV) detection and pathotype characterization in lentil is limited. The aim of this study was the molecular characterization of the first reported lentil isolate of PSbMV in Greece. Specific primers against three genes of PSbMV, namely the coat protein (CP) and the P1 and HC-Pro proteases have been designed and tested in RT-PCR detection and quantitation of the virus in lentil. Phylogenetic and recombination analysis of the Greek CP and HC-Pro amplicons was performed with the aim of detecting sequence variability indicative of pathotype classification. Moreover, the identified point mutations of the Greek HC-Pro sequence were further exploited in RT-RFLP protocols that together with the phylogenetic analysis lead to the classification of the Greek PSbMV isolate into pathogroup 1.

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.

Resistance to a newly recognized pathotype of pea seed-borne mosaic virus, PSbMV-P4, was found in PI 347492, an accession of Pisum sativum from India. In cross and backcross populations between PI 347492 and the susceptible cultivars Bonneville, Ranger, and PI 269816, resistance was determined to be monogenically recessive. The symbol sbm-4 is proposed for the gene conferring resistance to this pathotype of PSbMV

Pea seed-borne mosaic virus (PSbMV) infection causes a serious disease of field pea (Pisum sativum) crops worldwide. The PSbMV transmission efficiencies of five aphid species previously found landing in south-west Australian pea crops in which PSbMV was spreading were studied. With plants of susceptible pea cv. Kaspa, the transmission efficiencies of Aphis craccivora, Myzus persicae, Acyrthosiphon kondoi and Rhopalosiphum padi were 27%, 26%, 6% and 3%, respectively. Lipaphis erysimi did not transmit PSbMV in these experiments. The transmission efficiencies found for M. persicae and A. craccivora resembled earlier findings, but PSbMV vector transmission efficiency data were unavailable for A. kondoi, R. padi and L. erysimi. With plants of partially PSbMV resistant pea cv. PBA Twilight, transmission efficiencies of M. persicae, A. craccivora and R. padi were 16%, 12% and 1%, respectively, reflecting putative partial resistance to aphid inoculation. To examine aphid alighting preferences over time, free-choice assays were conducted with two aphid species representing efficient (M. persicae) and inefficient (R. padi) vector species. For this, alatae were set free on multiple occasions (10-15 repetitions each) amongst PSbMV-infected and mock-inoculated pea or faba bean (Vicia faba) plants. Following release, non-viruliferous R. padi alatae exhibited a general preference for PSbMV-infected pea and faba bean plants after 30min-4h, but preferred mock-inoculated plants after 24h. In contrast, non-viruliferous M. persicae alatae alighted on mock-inoculated pea plants preferentially for up to 48h following their release. With faba bean, M. persicae preferred infected plants at the front of assay cages, but mock-inoculated ones their backs, apparently due to increased levels of natural light there. When preliminary analyses were performed to detect PSbMV-induced changes in the volatile organic compound profiles of pea and faba bean plants, higher numbers of volatiles representing a range of compound groups (such as aldehydes, ketones and esters) were found in the headspaces of PSbMV-infected than of mock-inoculated pea or faba bean plants. This indicates PSbMV induces physiological changes in these hosts which manifest as altered volatile emissions. These alterations could be responsible for the differences in alighting preferences. Information from this study enhances understanding of virus-vector relationships in the PSbMV-pea and faba bean pathosystems.

The effect of direct application of artificial dsRNA molecules derived from the viral coat protein gene and the possibility of RNA interference induction leading to protection against Pea seed borne mosaic virus (PSbMV) have been investigated. Effectivity of virus transmission was not afected by the dsRNA treatment. Simultaneous application of dsRNA and virus inoculation as well as dsRNA application one day before PSbMV inoculation led to a significant short-term decrease in the PSbMV concentration in experimental pea plants. Either the curative effect of the artificial dsRNA biolistic application was not noticed.

Tasmanian vegetable crops (broccoli, carrots, cauliflower, common bean, and pea) were tested for virus infection with Potyviruses (all crops), members of the Luteoviridae (all except carrot), Alfalfa mosaic virus (all except brassicas), Cucumber mosaic virus (all except carrot and cauliflower), Subterranean clover stunt virus (legumes only), Tomato spotted wilt virus (legumes only) and Cauliflower mosaic virus (brassicas only) over three seasons (2007–2010). Low (and sporadic) incidence of each virus (or virus group) was found in pea crops. Only Luteovirid and SCSV infections were found in bean crops at low frequency and incidence. Virus infection was not detected in carrot crops. Broccoli crops recorded generally low incidence of all four viruses or groups tested for, whilst cabbage crops recorded very low incidence of CaMV only. In the pea and broccoli crops members of the Luteoviridae were the most prevalent viruses detected. Samples that tested positive for generic Potyvirus and Luteoviridae tests were retested using virus specific antiserum and/or RT-PCR. Potyvirus detections (from pea crops in the first year of testing) were due to infection with Pea seed-borne mosaic virus (PSbMV). These samples were all sourced from a single imported seed line. Subsequent testing over the next two seasons failed to show evidence of PSbMV which may have been eliminated with destruction of the seed source. Molecular analyses of a selection of Luteovirid isolates revealed most infections were due to Turnip yellows virus with five isolates from pea belonging to a putative novel Polerovirus species with closest homology to Cucurbit aphid-borne yellows virus.

A survey was conducted to identify viruses affecting chickpea and lentil crops in the major production areas of Azerbaijan during June 2007 and May 2008. The survey covered randomly selected 16 farmers’ fields (13 chickpea and three lentil) and one lentil field in research station during 2007, and 14 fields (nine chickpea and five lentil fields) during 2008. Infection incidences were determined for 11 legume viruses by tissue blot immunoassays (TBIA) on 50–200 randomly samples collected from each field. The TBIA and confirmatory nucleic acid analyses indicated that Luteoviridae species [Beet western yellows virus (BWYV), Bean leafroll virus (BLRV) and Chickpea chlorotic stunt virus (CpCSV)] were collectively the most common viruses in chickpea and lentil fields followed by Faba bean necrotic yellows virus (FBNYV). Pea seed-borne mosaic virus (PSbMV) was not detected in chickpea but reached high incidences in lentil fields. This is the first survey of legume viruses in Azerbaijan and first record of FBNYV, BWYV, BLRV and CpCSV naturally infecting chickpea and lentil crops, and PSbMV infecting lentil crop in Azerbaijan.

Traditional plant breeding relies upon crosses and subsequent selection of genotypes to meet desirable traits. The incorporation of marker-assisted selection into breeding strategies would result in a reduction in the number of offspring to be propagated, selected and tested. In the case of pea (Pisum sativum L.), the testing of resistance to viral pathogens such as pea seed-borne mosaic virus (PSbMV) is included in the breeding process. Resistance to the common strains of PSbMV is conferred by a single recessive gene (eIF4E), localized on LG VI (sbm-1 locus). We have analyzed for variation in the eIF4E genomic sequences from 43 pea varieties and breeding lines, reported as donors of resistance. This enabled a comprehensive investigation of the eIF4E gene structure and mutations responsible for PSbMV resistance were identified. Subsequently, PCR-based and gene-specific single nucleotide polymorphism and co-dominant amplicon length polymorphism markers were developed. All together 60 accessions were analyzed using sequence data and/or allele specific DNA markers. Developed allele specific markers were reproducibly amplified across a broad spectra of pea varieties and breeding lines. These were found to be 100% accurate in detecting the presence of the respective alleles when compared to symptomology and ELISA, testing (74% reliable). Hence, these molecular markers will substantially speed-up PSbMV diagnosis and resistance breeding processes in pea.

The molecular diversity of 14 isolates of Pea seed-borne mosaic virus (PSbMV) from southern Australia, 13 previously described isolates from Pakistan, and a reference isolate from the United States have been studied to determine whether a relatively simple molecular diagnostic assay and classification scheme could be developed for this virus. The Australian isolates were placed into either pathotype P1 or pathotype P4 by bioassay on differential genotypes of Pisum sativum. The Pakistani isolates represented pathotypes P1, P4, U1, and U2, and an undetermined pathotype. The reference US isolate was pathotype P1. A reverse transcription-polymerase chain reaction (RT-PCR) assay based on an amplicon from the variable HC-Pro coding region of potyviruses was shown to distinguish PSbMV from seven other legume infecting potyviruses. Restriction fragment length polymorphisms (RFLPs) generated from the HC-Pro RT-PCR products of all 28 isolates using seven restriction endonucleases placed them into eight groups. A phylogenetic tree based on a Bray-Curtis similarity comparison placed the groups into three clusters. The groups and clusters had no clear association with either pathotype or geographic source. It is concluded that within the range of viruses and isolates tested, the RT-PCR-RFLP method will both specifically identify PSbMV and provide a simple, qualitative, and rapid means for placing PSbMV isolates into groups. Applications could include mapping and tracking isolates in space and time.

The phenotypic expression and heritability of quantitative traits vary due to genotypic differences and environmental influences. Maximum heritability was found in number of pods/plant (99.43%) followed by seed yield/plant (99.06%) and number of primary branches/plant (98.74%). The presence of considerable degree of genotypic variances among tested genotypes under different environments suggests that success in lentil breeding could possibly be achieved through direct phenotypic selection. Seed yield showed highly significant positive correlation with number of pods/plant and pod length. Maximum direct effect on seed yield was observed for pod length. There was a wide range of variability in disease reaction among the genotypes. Pea seed borne mosaic virus is basically a disease of pea has been found to be transmitted to lentil. It is suggested that these two crops should not be planted near each other. Therefore, number of pods/plant, pod length, days to maturity and number of seeds/pod was emphasized as primary seed yield.

Pea seed-borne mosaic virus (PSbMV) is not considered a problem in Australian lentils as no seed transmission in commercial lentil seed lots has so far been reported. PSbMV lentil-seed transmissible strains were found in accessions from the Australian germplasm collection that were imported from the USDA germplasm collection and were previously reported to contain PSbMV. Strategies to clean up infected entries and to avoid the spread of these strains to commercial fields are discussed.

Two virus resistance loci on linkage groups II and VI have provided the only sources of natural resistance against Pea seed-borne mosaic virus (PSbMV, Potyviridae) in the important crop plant Pisum sativum L. A combination of parallel approaches was used to collate linked markers, particularly for sbm-1 resistance on linkage group VI. We have identified sequences derived from the genes for the eukaryotic translation initiation factors eIF4E and eIF(iso)4E as being very tightly linked to the resistance gene clusters on linkage groups VI and II, respectively. In particular, no recombinants between sbm-1 and eIF4E were found amongst 500 individuals of an F2 cross between the BC4 resistant line (JI1405) and its recurrent susceptible parent 'Scout'. In a different mapping population, the gene eIF(iso)4E was also shown to be linked to sbm-2 on linkage group II. A parallel cDNA-AFLP comparison of pairs of resistant and susceptible lines also identified an expressed tag marker just 0.7 cM from sbm-1. eIF4E and eIF(iso)4E have been associated with resistance to related viruses in other hosts. This correlation strengthens the use of our markers as valuable tools to assist in breeding multiple virus resistances into peas, and identifies potential targets for resistance gene identification in pea.

SUMMARY Transgenic peas containing a copy of the Pea seed-borne mosaic virus (PSbMV) isolate DPD1 NIb sequence develop a 'recovery' phenotype when challenged with either the homologous (DPD1) or a heterologous (NY) PSbMV isolate. However, the specificity of the subsequent resistance differs with respect to the initiation by and targeting of different virus isolates. Analysis of tissue in which recovery had been induced by either of the isolates, revealed the presence of low molecular RNA molecules (siRNA) derived from degradation of the NIb transgene mRNA. When non-transgenic scions were grafted on to transgenic stocks in which recovery had been induced, all the scions became infected, indicating that the virus can be exported from recovered tissue. Experiments in which recovered scions were grafted on to non-transgenic stocks revealed that the recovery phenotype could be maintained in the apparent absence of a source of virus. However in a number of cases, side-shoots which developed on the non-transgenic stock became infected. These results indicate that recovered tissue contains extremely low levels of infectious virus with the potential, directly or indirectly, to confer the observed resistance specificity. Indirectly, the viral genome could act as a source of specific siRNA molecules, which are present in infected tissues but are below the level of detection in recovered tissues. These could act in conjunction with siRNAs derived from the transgene mRNA to maintain a level of PTGS (post-transcriptional gene silencing) activity which is effective in preventing further accumulation of homologous or related viruses. We suggest a model to explain the differential specificity.

Resistance to pea seed-borne mosaic virus (PSbMV) pathotype P-1 in peas is conferred by sbm1 with recessive inheritance. PSbMV is an economically important pathogen with world-wide distribution that causes significant losses in pea yield and reduces seed and produce quality. The sbm1 gene was previously mapped to linkage group VI on molecular linkage maps of the pea genome. To improve plant breeders' ability to develop varieties resistant to PSbMV, two random amplified polymorphic DNA markers (G05―2537 and L01―910) and one restriction fragment length polymorphism (P446) linked to sbm1 have been identified. The genomic sequences for these markers have been characterized and the information used to develop three simple polymerase chain reaction-based STS (sequence tagged site) assays. Linkage analysis in two F 2 populations showed that the most tightly linked of these three STS loci (sG05―2537) is approximately 4 cM from sbm1. Characterization of a collection of resistant and susceptible germplasm demonstrated a strong correlation between STS alleles and sbm1 alleles, indicating the utility of these markers for marker-assisted selection in breeding programmes using a range of germplasm sources.

The mechanism of Pisum sativum pathotype-specific resistance to pea seed-borne mosaic potyvirus (PSbMV) was investigated and the coding region determinant of PSbMV virulence was defined. Homozygous recessive sbm-1 peas are unable to support replication of PSbMV pathotype 1 (P-1), whereas biochemically and serologically related pathotype 4 (P-4) is fully infectious in the sbm-1/sbm-1 genotype. We were unable to detect viral coat protein or RNA with double antibody sandwich-enzyme-linked im-munosorbent assay and reverse transcription-polymerase chain reaction in sbm-1/sbm-1 P-1-inoculated protoplasts and plants. Lack of viral coat protein or RNA in P-1 trans-fected sbm-1/sbm-1 protoplasts suggests that sbm-1 resistance is occurring at the cellular level and that inhibition of cell-to-cell virus movement is not the operating form of resistance. In addition, because virus products were not detected at any time post-inoculation, resistance must either be constitutive or expressed very early in the virus infection process. P-1-resistant peas challenged with full-length, infectious P-1/P-4 recombinant clones demonstrated that a specific P-4 coding region, the 21-kDa, genome-linked protein (VPg), was capable of overcoming sbm-1 resistance, whereas clones containing the P-1 VPg coding region were noninfectious to sbm-1/sbm-1 peas. VPg is believed to be involved in potyvirus replication and its identification as the PSbMV determinant of infectivity in sbm-1/sbm-1 peas is consistent with disruption of an early P-1 replication event.

Two isolates of pea seed-borne mosaic potyvirus, DPD1 and NY, were identified as pathotypes P-1 and P-4, respectively, by their infectivity on Pisum sativum L. lines homozygous for the recessive resistance genes sbm-1 and sbm-4. The two isolates differed in several biological characteristics. DPD1 induced transient vein clearing, downward rolling of leaflets and internode shortening on P. sativum, whereas NY only caused a slight growth reduction. DPD1 moved systemically in Chenopodium quinoa whereas NY was restricted to inoculated leaves. DPD1 was frequently transmitted by seeds whereas NY was rarely seed-transmitted: 24% and 0.3%, respectively, in P. sativum '549'. Both DPD1 and NY were transmitted by aphids (Myzus persicae), though a DAG triplet was not present in the N terminus of the coat protein. The nucleotide sequence and deduced amino acid sequence of NY were determined and compared to the corresponding sequences of DPD1.

Nucleotide sequences of complete or partial coat protein (CP) genes were determined for 11 isolates of pea seed-borne mosaic virus (PSbMV) from Australia and one from China, and compared with known sequences of 20 other isolates. On phylogenetic analysis, the isolates from Australia and China grouped into 2 of 3 clades. Clade A contained three sub-clades (Ai, Aii and Aiii), Australian isolates were in Ai or Aiii, and the Chinese isolate in Aii. Clade A contained isolates in pathotypes P-1, P-2 and U-2; clade B, one isolate in P-2; and clade C, only isolates in P-4.

In a survey for viruses of cultivated legumes in West Asia and North Africa, pea seed-borne mosaic virus (PSbMV) was found in faba bean, lentil and pea. Using ELISA, it was detected in 107 out of 1554 faba bean samples and 40 out of 496 lentil samples with virus-like symptoms collected in Algeria, Egypt, Ethiopia, Jordan, Lebanon., Libya, Morocco, Sudan, Tunisia and Turkey.

Pea, lentil, dwarf and broad bean crops were each monitored over two seasons for virus infection using sample surveys, ELISA, and indicator hosts to estimate disease incidence. The incidence of individual viruses in most crops was usually < 11%, exceptions include cucumber mosaic virus (CMV) in lentils and alfalfa mosaic virus (AMV) in broad beans. Multiple virus infections were detected in 55–100% of pea, lentil, and broad bean crops. Soybean dwarf virus (SDV), beet western yellows virus (BWYV), pea seed‐borne mosaic (PSbMV), and CMV were the most common aphid‐borne or seed‐transmitted viruses detected. AMV, CMV, BWYV, bean yellow mosaic virus (BYMV), and PSbMV were recorded from lentil and CMV and PSbMV from broad beans for the first time in New Zealand. Survey results are presented and their implications are discussed.

The reaction of 25 UK pea cultivars to pea seedborne mosaic virus (PSbMV) was studied in the glasshouse. All cultivars proved to be susceptible. When leaf or tendril samples from inoculated plants were analysed, it was found that most combining cultivars had a lower virus content, as measured by A-405 values in ELISA, than garden and vining cultivars, and that garden and vining cultivars had a similar virus content. Seed transmission of PSbMV in 20 cultivars ranged from 0% in cvs Maro, Princess and Progreta to 74% in cv. Vedette. There was no obvious relationship between virus content and the efficiency of seed transmission in different cultivars. Wide variation in percentage seed transmission between individuals of single cultivars was observed; for seven cvs (ST 2–49%) tested, all showed some plants escaping seed transmission altogether Seedlings infected through the seed showed symptoms similar to those in the infected parent plants. Virus particles were readily detectable by immunosorbent electron microscopy in infected seedlings, and in elongated infected embryonic shoots produced 3 days after seed germination, by ELISA. An ELISA test of seed samples from cv. Wavercx showed that virus was as readily detected 3 days after germination as after growth to symptomatic seedlings (4 weeks), and probably provides the most sensitive, accurate and time-saving assay for seed transmission of PSbMV.

Summary Four isolates of pea seed-borne mosaic virus (PSbMV) representing pathotypes P1 (isolates US and Q) and P4 (isolates S4 and S6), and groups III (US and Q) and V (S4 and S6) have been used in a study of the survival and partitioning of PSbMV under conditions of continuous seed transmission in the commercial pea cultivar Dundale. Assays suitable for detecting virus in small tissue samples were developed, and included dot-immunobinding assay with antisera to both PSbMV and cytoplasmic inclusion body (CIB) protein, and dot hybridisation assay (DHA) with cDNA transcribed from virus RNA. Under the conditions of our experiments, seed transmission occurred at rates exceeding 90% for all virus isolates. Virus was detectable by serology and symptoms in inoculated plants, and in all vegetative tissue of second generation plants raised from seed of the inoculated plants. However, in the third, fourth and fifth sequential generations raised from seed, all plants were symptomless. Neither virus nor CIB were detectable in leaf, stem or roots by serology, but both were readily detectable in some floral parts, and in immature and mature seed. Mature seed contained virus and CIB antigen in the testa, cotyledon and embryo. Inoculum prepared from whole seeds was infectious. The testa was shown not to be involved in transmission between generations, thus implicating the embryo alone in vertical transmission. Virus antigen could not be detected in the emerging cotyledons of germinating seed and all true leaves by serology, but the leaves contained PSbMV RNA detectable by DHA. These results show that PSbMV infection can be transferred through the vegetative phase at a subliminal level, and reaches relatively high concentrations in floral parts and seeds. Thus PSbMV may be maintained at a high level of infection in seed in the absence of any apparent symptoms in the plant, and without a requirement for horizontal transmission between plants by vectors. Such a mechanism may explain the high levels of infection commonly reported in pea breeding lines.

The location of sbm-1 on the Pisum sativum genetic map was determined by linkage analysis with eight syntenic molecular markers. Analysis of the progeny of two crosses confirmed that sbm-1 is on chromosome 6 and permitted a more detailed map of this chromosome to be constructed. The inclusion of Fed-1 and Prx-3 among the markers facilitated the comparison of our map with the classical genetic map of pea. The sbm-1 gene is most closely linked to RFLP marker GS185, being separated by a distance of about 8 cM. To determine the practical value of GS185 as a marker for sbm-1 in plant breeding programs, the GS185 hybridization pattern and virus-resistance phenotype were compared in of a collection of breeding lines and cultivars. Three GS185 hybridization patterns were discerned among the lines. A strong association was found between one of these patterns and resistance to PSbMV.

Seed transmission of an isolate of pea seed-borne mosaic virus (PSbMV) in several pea genotypes has been studied. Cross-pollination experiments showed that pollen transmission of PSbMV did not occur and accordingly, virus was not detected in pollen grains by ELISA or electron microscopy. Comparative studies between two pea cultivars, one with a high incidence of seed transmission and one with none, showed that PSbMV infected the floral tissues (sepals, petals, anther and carpel) of both cultivars, but was not detected in ovules prior to fertilization. Virus was detected equally well in seed coats of the progeny in both cultivars. Analysis of virus incidence and concentration in pea seeds of different developmental stages demonstrated that in the cultivar with a high incidence of seed transmission, PSbMV directly invaded immature embryos, multiplied in the embryonic tissues and persisted during seed maturation. In contrast, the cultivar without seed transmission did not show invasion of immature embryos by the virus; there was no evidence for virus multiplication or persistence during embryo development and seed maturation. Hence seed transmission of PSbMV resulted from direct invasion of immature pea embryos by the virus and the block to seed transmission in the non-permissive cultivar probably occurred at this step.

Field experiments examined the effects of sowing field pea seed with different amounts of infection with Pea seed-borne mosaic virus (PSbMV) on virus spread, seed yield, and infection levels in harvested seed. Plots were sown with seed with actual or simulated seed transmission rates of 0.3 to 6.5% (2005) or 0.1 to 8% (2006), and spread was by naturally occurring migrant aphids. Plants with symptoms and incidence increased with the amount of primary inoculum present. When final incidence reached 97 to 98% (2005) and 36% (2006) in plots sown with 6.5 to 8% infected seed, yield losses of 18 to 25% (2005) and 13% (2006) resulted. When incidence reached 48 to 76% in plots sown with 1.1-2 to 2% initial infection, seed yield losses were 15 to 21% (2005). Diminished seed weight and seed number both contributed to the yield losses. When the 2005 data for the relationships between percent incidence and yield or yield gaps were plotted, 81 to 84% of the variation was explained by final incidence and, for each 1% increase, there was a yield decline of 7.7 to 8.2 kg/ha. Seed transmission rates in harvested seed were mostly greater than those in the seed sown when climatic conditions favored early virus spread (1 to 17% in 2005) but smaller when they did not (0.2 to 2% in 2006). In 2007, sowing infected seed at high seeding rate with straw mulch and regular insecticide application resulted in slower spread and smaller seed infection than sowing at standard seeding rate without straw mulch or insecticide. When data for the relationship between final percent incidence and seed transmission in harvested seed were plotted (all experiments), 95 to 99% of the variation was explained by PSbMV incidence. A threshold value of