Investigating the Pea Virome in Germany-Old Friends and New Players in the Field(s)

Abstract

Peas are an important legume for human and animal consumption and are also being used as green manure or intermediate crops to sustain and improve soil condition. Pea production faces constraints from fungal, bacterial, and viral diseases. We investigated the virome of German pea crops over the course of three successive seasons in different regions of pea production to gain an overview of the existing viruses. Pools from 540 plants, randomly selected from symptomatic and asymptomatic peas, and non-crop plants surrounding the pea fields were used for ribosomal RNA-depleted total RNA extraction followed by high-throughput sequencing (HTS) and RT-PCR confirmation. Thirty-five different viruses were detected in addition to nine associated nucleic acids. From these viruses, 25 are classified as either new viruses, novel strains or viruses that have not been reported previously from Germany. Pea enation mosaic virus 1 and 2 were the most prevalent viruses detected in the pea crops, followed by pea necrotic yellow dwarf virus (PNYDV) and turnip yellows virus which was also found also in the surrounding non-legume weeds. Moreover, a new emaravirus was detected in symptomatic peas in one region for two successive seasons. Most of the identified viruses are known to be aphid transmissible. The results revealed a high virodiversity in the German pea fields that poses new challenges to diagnosticians, researchers, risk assessors and policy makers, as the impact of the new findings are currently unknown.

Keywords: PEMV; PNYDV; Pisum sativum; TuYV; aphid transmitted viruses; emaravirus; high throughput sequencing.

FIGURE 1

Map of the six different pea crop sampling regions in Germany and the sample pooling strategy used for each of three successive seasons 2016, 2017, and 2018.

FIGURE 2

The virus species found in the four sampling categories symptomatic peas (SP), asymptomatic peas (aSP), surrounding legumes (sL), and surrounding non-legumes (snL). Samples were collected in six pea-growing regions (Salzlandkreis-1, Salzlandkreis-2, Münster, Kreis Stormarn, Landkreis Rostock, and Landkreis Meißen) in Germany over three successive seasons 2016, 2017, and 2018. A network illustration of virus species of each category and shared viruses among them. Background colors of virus acronyms correspond to the background colors of the respective virus family. The virus names are: BVG, barley virus G; BCMV, bean common mosaic virus; BLRV, bean leafroll virus; BYMV, bean yellow mosaic virus; BGCV2, black grass cryptic virus 2; CaTV1, carrot torradovirus 1; CLRV, cherry leaf roll virus; ClYVV, clover yellow vein virus; CMV, cucumber mosaic virus; HLV, Heracleum latent virus; PaMV1, pea associated mitovirus 1; PaMV2, pea associated mitovirus 2; PaMV3, pea associated mitovirus 3; PaEV, pea associated emaravirus; PEMV1, pea enation mosaic virus 1; PEMV2, pea enation mosaic virus 2; PNYDV, pea necrotic yellow dwarf virus; PSbMV, pea seed-borne mosaic virus; RCCV1, red clover carlavirus 1; RCEV1, red clover enamovirus 1; RCNVA, red clover nepovirus a; RCUV, red clover umbravirus; SsMV4, Sclerotinia sclerotiorum mitovirus 4; sLaIV, sL associated ilarvirus; snLaCV, snL associated chordovirus; snLaIV, snL associated ilarvirus; snLaSV, snL associated secoviridae; snLaWV, snL associated waikavirus; SbDV, soybean dwarf virus; SMV, soybean mosaic virus; TVCV, turnip vein-clearing virus; TuYV, turnip yellows virus; WCCV2, white clover cryptic virus 2; WClMV, white clover mosaic virus; WHIV21, Wuhan insect virus 21.

FIGURE 3

A summary of virus species (in red) detected in and around sampled pea crops from six different German pea-growing regions (Salzlandkreis-1 [1], Salzlandkreis-2 [2], Münster [3], Kreis Stormarn [4], Landkreis Rostock [5], and Landkreis Meißen [6]) over three seasons 2016, 2017, and 2018. Virus acronyms, virus names, and their associated nucleic acids: BVG, barley virus G; BCMV, bean common mosaic virus; BLRV, bean leafroll virus; BYMV, bean yellow mosaic virus; BGCV2, black grass cryptic virus 2; CaTV1, carrot torradovirus 1; CLRV, cherry leaf roll virus; ClYVV, clover yellow vein virus; CMV, cucumber mosaic virus; HLV, Heracleum latent virus; PaMV1, pea associated mitovirus 1; PaMV2, pea associated mitovirus 2; PaMV3, pea associated mitovirus 3; PaEV, pea associated emaravirus; PEMV1, pea enation mosaic virus 1; PEMV2, pea enation mosaic virus 2; PEMVsatRNA, pea enation mosaic virus satellite RNA; PNYDVaSat1, pea necrotic yellow dwarf alphasatellite 1; PNYDVaSat3, pea necrotic yellow dwarf alphasatellite 3; PNYDVaSat4, pea necrotic yellow dwarf alphasatellite 4; PNYDVaSat5, pea necrotic yellow dwarf alphasatellite 5; PNYDVaSat6, pea necrotic yellow dwarf alphasatellite 6; PNYDVaSat7, pea necrotic yellow dwarf alphasatellite 7; PNYDV, pea necrotic yellow dwarf virus; PSbMV, pea seed-borne mosaic virus; RCCV1, red clover carlavirus 1; RCEV1, red clover enamovirus 1; RCNVA, red clover nepovirus a; RCUV, red clover umbravirus; SsMV4, Sclerotinia sclerotiorum mitovirus 4; sLaIV, sL associated ilarvirus; snLaCV, snL associated chordovirus; snLaIV, snL associated ilarvirus; snLaSV, snL associated secoviridae; snLaWV, snL associated waikavirus; SbDV, soybean dwarf virus; SMV, soybean mosaic virus; TVCV, turnip vein-clearing virus; TuYV, turnip yellows virus; TuYVaRNA, turnip yellows virus associated RNA; TuYVaRNA2, turnip yellows virus associated RNA 2; WCCV2, white clover cryptic virus 2; WClMV, white clover mosaic virus; WHIV21, Wuhan insect virus 21.

FIGURE 4

Neighbor joining tree (NJ) of the nucleocapsid protein (NP) of the two isolates of pea associated emaravirus (PaEV) detected in Germany and representative emaraviruses. The phylogenetic trees are based on alignments of the amino acid sequences. The sequences were aligned with Clustal W and NJ trees constructed with MEGA X. The percentage of the bootstrap values above 50% (1,000 replications) are shown at the nodes. Virus acronyms and names are as follows: AcCRaV, Actinidia chlorotic ringspot-associated emaravirus; EMARaV, European mountain ash ringspot-associated emaravirus; FMV, fig mosaic emaravirus; HPWMEV, high plains wheat mosaic emaravirus; PaEV, pea associated emaravirus; PPSMV1, pigeonpea sterility mosaic emaravirus 1; PPSMV2, pigeonpea sterility mosaic emaravirus 2; RLBV, raspberry leaf blotch emaravirus; RRV, rose rosette emaravirus; RSV, rice stripe virus (Tenuivirus; Phenuiviridae) and RYRSaV, redbud yellow ringspot-associated emaravirus. The GenBank accession numbers are in brackets. The scale bar indicates the genetic distance.