https://doi.org/10.36253/phyto-15934

Accepted: April 16, 2025; Published: May 15, 2025

Short Notes

Pea seed-borne mosaic virus pathotypes isolated from Australian pea (Pisum sativum) seed

Joop van Leur^1,* , Mohammad Aftab² , Angela Freeman²

¹ New South Wales Department of Primary Industries and Regional Development, Tamworth Agricultural Institute, Tamworth, Australia

² Agriculture Victoria, The Grains Innovation Park, Horsham, Australia

*Corresponding author. E-mail: joop.vanleur@dpi.nsw.gov.au

Summary. 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.

Keywords. PSbMV, BYMV, virus resistance, pathogenicity test.

Contents

Introduction

Material and methods

Results and Discussion

Acknowledgements

Literature cited

Introduction

Pea seed-borne mosaic virus (PSbMV, Potyvirus pisumsemenportati) is present in field pea (Pisum sativum) in many countries, probably because the virus is seed transmitted at high rates. Distribution of this virus, seed transmission, host and vector range, and symptomatology have been reviewed extensively (Khetarpal and Maury, 1987; Congdon, 2017). Depending on the pea genotype, PSbMV symptoms can be difficult to identify in the field, and serological or molecular tests are required to determine its presence in host plants.

Cultivation of field pea, and of winter pulses in general, is recent in Australia (Siddique and Sykes, 1997), and it was not until 1991 that PSbMV was reported in commercial pea crops (Ligat et al., 1991). Later surveys showed that the virus is widespread in Australia’s pea crops, both in the western (Latham and Jones, 2001a, Congdon et al., 2016) and eastern (Freeman et al., 2013) regions of the country.

PSbMV causes considerable yield losses in Austra-lian field peas, despite the generally mild symptoms it causes (Coutts et al., 2009), and the level of PSbMV infection in a pea crop is closely related to the level of seed-transmission in the seed stock used (Coutts et al., 2009; Freeman et al., 2013). Use of virus-free seed would therefore provide adequate control of PSbMV-induced yield losses. However, the virus can be introduced from neighbouring infested fields, and Australian pea growers generally save their own seed and rarely test the seed stocks for virus presence. Host resistance will provide a lasting control option, and the Australian field pea breeding programme has made the incorporation of PSbMV resistance in new varieties a priority (Rosewarne, 2016).

Breeding peas for PSbMV resistance is facilitated by the availability of single recessive genes in the host that provide immunity, and these can be identified in seedling tests using mechanical inoculations. Provvidenti and Alconero (1988) proposed four independent recessive resistance genes, sbm1, sbm2, sbm3 and sbm4, to explain the differential reactions to the three PSbMV pathotypes, P1, P4 and P2, reported at the time (Alconero et al., 1986). The gene sbm1 conferred resistance to P1, sbm2 and sbm3 conferred resistance to P2, and sbm4 gave resistance to P4. These authors also reported strong linkages between gene sbm2 and the mo resistance gene for bean yellow mosaic virus (BYMV) and between sbm1, sbm3 and sbm4 resistance genes. Johan-sen et al. (2001) showed that the differences between the three PSbMV pathotypes could be explained by the properties of two viral cistrons, and predicted the existence of a fourth pathotype, P3. The P3 pathotype was subsequently isolated from seed of a faba bean gene-bank accession originating from Nepal (Hjulsager et al., 2002). Gao et al. (2004) showed that only two recessive resistance genes were operating in the pea/PSbMV pathosystem. The sbm1 gene (present in a range of germplasm of Indian and Ethiopian origin), confers resistance to all four PSbMV pathotypes (P1, P2, P3, P4), while a different allele of the sbm1 gene, sbm1¹ (present in the germplasm accessions PI 269774 and PI 269818), gives resistance only to the P1 and P2 pathotypes, and the sbm2 gene (in ‘Dark Skin Perfection’ and a large number of commercial pea lines) only provides resistance to pathotypes P2 and P3. The use of two differentials, one with the sbm1¹ gene and one with the sbm2 gene, allows the classification of PSbMV to one of the four pathotypes (Table 1).

Within Australia, limited attention has been given to pathotyping of PSbMV strains. Ligat and Randles (1993) pathotyped three Australian isolates as P1 (one isolate) or P4 (two isolates), using a differential set of six pea genotypes. Torok and Randles (2007) pathotyped 14 Australian PSbMV isolates, and classified ten as P4 and four as the P1 pathotype.

For pea breeding programmes to successfully incorporate PSbMV resistance, knowledge is required of the composition of the local PSbMV strain population. The present study isolated PSbMV strains from pea seed originating from geographically distinct locations in Australia, and pathotyped the isolates using a differential set of homogeneous pea genotypes.

Material and methods

Tissue blot immunoassays (TBIA) were used for all virus diagnostics, as these provide a reliable, rapid, and cost-efficient methodology for processing large numbers of plant samples (Freeman et al., 2013; van Leur et al., 2013a). Samples were each blotted onto nitrocellulose membranes (Schleiger & Schuell Protran, 0.45 µm pore size), and were processed using a polyclonal PSbMV antibody (DSMZ, AS-0129), following the procedures described by Kumari et al. (2022).

PSbMV seed-to-plant transmission (PSbMV-SPT) rates were determined from 144 seed lots (92 harvested from farmer fields and 52 from trial fields), submitted to the Tamworth laboratory for PSbMV seed testing during 2006-2010. ‘Kaspa’ was the predominant cultivar in this evaluation, with 54 seed lots, followed by ‘Excell’ (18 seed lots), ‘Morgan’ (12), and ‘Parafield’ (nine).

Seeds were incubated on wet filter paper at 22°C in the dark for 7-10 d, after which plumules or radicles of the germinated seeds were tested for PSbMV presence by TBIA. Seed lots were first tested in batches of 30 seeds. A sequential sampling approach was later taken, with further tests made on seed lots that had low infection levels. For most seed lots, more than 90 seeds were tested. The PSbMV-SPT rate was calculated as:

100 × (total number of PSbMV positive germlings / total number of germinated seeds).

To isolate PSbMV strains, 20 seed lots were selected. This selection was based on PSbMV-SPT rates, but also included a range of varieties and seed sources, from three Australian states New South Wales (NSW), Western Australia (WA) and Victoria. For each selected seed lot, 20 small pots were sown with three seeds/pot, using a commercial potting mix, and the pots were then held in an aphid-proof, temperature-controlled (18–24°C) greenhouse. Two to 3 weeks after sowing, all emerged plants were tested by TBIA, and PSbMV positives were selected. The PSbMV strains were isolated by inoculating 2- to 3-week-old plants of the faba bean variety ‘Fiesta’ with individual PSbMV positive seedlings, using the inoculation methods described below. PSbMV presence in the faba bean plants was confirmed with TBIA prior to pathogenicity tests.

Three PSbMV pea lines with reported single resistance genes (Hjulsager et al., 2002; Gao et al., 2004), including PI 269774 (sbm1¹ gene), Dark Skin Perfection (sbm2) and PI 193835 (sbm1) were obtained from the Australian Grains Genebank (Horsham, Australia). To ensure host plant homogeneity, single plant selections were made from these lines prior to testing. For pathogenicity tests, four to five plants each of the three differentials, and a universally susceptible pea line (‘Kaspa’) were inoculated 10 to 14 d after sowing, by dusting first and second leaves of each plant with carborundum powder (silicon carbide # 400), and rubbing into the leaves a virus suspension from young virus-infected faba bean leaves. The suspension was prepared by homogenisation of the faba bean leaves in a cold 0.1 M sodium phosphate buffer (pH = 7.0). Each inoculation was repeated after one week on the third leaves of the plants. Two weeks after the second inoculation, the plants were TBIA-tested for virus presence. Virus isolates that gave variable results, or failed to infect the susceptible ‘Kaspa’ plants, were retested.

Results and Discussion

PSbMV seed infections were common in the 144 submitted seed lots assessed, with 52 seed lots PSbMV-free and 47 having PSbMV-SPT rates greater than 5% (Table 2). ‘Kaspa’ and ‘Excell’ were found to be particularly susceptible to PSbMV seed infection, with only 12 (22%) ‘Kaspa’ seed lots PSbMV-free and 25 (46%) with infection levels greater than 5%. For ‘Excell’, five (27%) seed lots were PSbMV-free and six (33%) had infection levels greater than 5%. In contrast, ten out of 12 seed lots of ‘Morgan’ were PSbMV-free, although this variety does not contain any of the sbm genes for PSbMV resistance (van Leur et al., 2013a). The greatest PSbMV-SPT rate, 40%, was found in an ‘Excell’ seed lot from a trial plot at the Wagga Wagga Agricultural Institute, NSW. The greatest ‘Kaspa’ infection, 25%, was from a trial plot at the Plant Breeding Institute at Narrabri, NSW. Greater infection levels were found in seed originating from research stations as compared to farmer fields; out of nine ‘Kaspa’ seed lots harvested from trial fields, four (44%) had PSbMV-SPT levels >10%, while nine out of 45 ‘Kaspa’ seed lots (20%) harvested in farmer fields had similar infection levels. Similarly, for all seed lots combined, 37 of 52 (71%) seed lots harvested from trial fields, and ten of 92 (11%) from farmer fields, had PSbMV-SPT levels >10%. These differences are probably a result of research stations growing a wide range of germplasm, including highly susceptible genotypes that can be inoculum reservoirs for viruses and virus vectors.

Out of the 20 selected seed lots, five did not yield PSbMV. From the remaining 15 seed lots, 88 PSbMV strains were isolated of which 71 were pathotyped (Table 3). None of the isolates infected PI 193835, confirming the effectiveness of the sbm1 gene against all known PSbMV pathotypes. Both the P1 pathotype (with eight isolates identified), and the P4 pathotype (20 isolates identified), were previously reported in Australia (Ligat and Randles, 1993; Torok and Randles, 2007). The most frequently found pathotype was P3, with 42 isolates able to infect PI 269774 (containing sbm1¹), but not ‘Dark Skin Perfection’ (containing sbm2). The pathotype P3 has not been recorded in Australia previously, but previous studies (Ligat and Randles, 1993; Torok and Randles, 2007) may have missed this pathotype because they did not use a reliable sbm2 differential. Ligat et al. (1991) compared symptom development of four Australian PSbMV isolates with an American isolate on a range of pea varieties, and noted that the Australian isolates did not infect the BYMV resistant pea variety ‘Greenfeast’. This could indicate that the Australian isolates were P3 pathotypes.

Identification of a single P2 pathotype strain (isolate Ps11-13/19) from a ‘Dundale’ seed lot originating from the Wagga Wagga Agricultural Institute (NSW) field trial was unexpected. To date, P2 pathotypes have only been isolated from lentil seed (Hampton, 1982; Alconero et al., 1986; van Leur et al., 2013b). Wylie et al. (2011) reported a P2 pathotype (isolate W1) obtained from a pea plant grown at the Medina Research Station near Perth, WA (Latham and Jones, 2001a). Latham and Jones (2001b) noted severe PSbMV symptoms in germplasm plots at this station that could have been caused by exotic virus strains, and the W1 strain could have originated from imported lentil germplasm. Ashby et al. (1986) pathotyped a PSbMV strain isolated from peas in New Zealand using sbm1, sbm1¹, and sbm2 differentials, and found that the strain reacted as a P2 pathotype. PSbMV is common in New Zealand lentil crops (Fletcher, 1993), and cross-infection of pea crops from lentil seed-borne PSbMV could have occurred. While isolate Ps11-13/19 originated from a pea seed, the seed was harvested at a research station where a wide range of legume germplasm, including lentils, is grown. The infection of the mother plant could have originated from a seed-infected lentil plant.

Identification of P3 and P2 pathotypes depends on their inability to infect sbm2 differentials. Alconero et al. (1986) noted that their P4 strain had delayed and erratic infection in pea lines that had the mo/sbm2 gene. The present study showed that infection of the ‘Dark Skin Perfection’ differential was occasionally not detected on all inoculated plants, despite the use of homogenous differentials derived from single seed progenies. However, the identities of the P2 and P3 pathotypes were confirmed with repeated inoculations of ‘Dark Skin Perfection’ and pea varieties such as ‘Greenfeast’ and ‘Bundi’, that showed BYMV resistance in previous trials (van Leur et al., 2013a).

Pathotyping of PSbMV strains is required for pea breeding programmes, but these biological tests are time consuming. Developments in the gathering and analyses of genetic information using molecular methods may enable rapid PSbMV pathotype identification. To date, phylogenetic analyses of PSbMV strains have been achieved for limited numbers of virus isolates. Safarova et al. (2008) examined eight pathotyped PSbMV isolates from the Czech Republic, and showed distinct grouping of P1 and P4 pathotypes. Wylie et al. (2011) analysed six pathotyped Australian PSbMV isolates; their P1 and P4 isolates clustered in two separate groups together with the Czech strains of the same pathotype, but their P2 isolate did not group with the published P2 pathotype strain (PSbMV-L1 isolate, GenBank accession code: AJ252242). Further development of pathotyping techniques would require analyses of large numbers of PSbMV strains for each pathotype, and the use of strains from diverse geographic backgrounds. Until that is achieved, pathotype identification should be based on biological indexing, and care must be taken not to propose pathotypes based solely on phylogenetic analyses.

The outcome of the present study is particularly relevant to pea breeding programmes. Firstly, the study confirmed the effectiveness of the sbm1 gene against all four PSbMV pathotypes. This gene is widely used in the Australian pea breeding programme, and is present in currently grown varieties such as ‘Yarrum’ and ‘PBA Wharton’, although none of the commercial varieties are homogeneous for resistance (van Leur et al., 2013a). Pea varieties that are heterogeneous for PSbMV resistance could provide enough protection for commercial purposes, but these varieties will require purification before being used as parents in breeding programmes. Secondly, given the prevalence of P3 pathotypes, varieties carrying the mo/sbm2 gene may appear to be resistant to PSbMV. However, cultivation of these cultivars will result in selection for the P1 and P4 pathotypes, as is demonstrated by the absence of P3 pathotypes in the two tested seed lots harvested from the BYMV resistant varieties ‘Moonlight’ and ‘Soupa’.

The present study results have shown how quickly PSbMV can spread. They also provide clear indications for differences in PSbMV seed transmission rates among host varieties that lack sbm resistance genes. While highly susceptible varieties (e.g. ‘Kaspa’ and ‘Excell’) are being replaced with new varieties, some carrying the sbm1 gene, it remains important to monitor commercial seed lots for PSbMV transmission to identify varieties with high seed transmission rates for PSbMV.

Acknowledgements

This study was financially supported by the Grains Research and Development Corporation, Australia.

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