Spatial and temporal differences in viral populations

The spatial and temporal compositions of the viromes in the different regions over a period of three growing seasons show, perhaps not unsurprisingly, similarities as well as fundamental differences. For example, PEMV1, PEMV2 and PNYDV were the viruses that could be located in all regions (but not in every season; Figure 8). The polerovirus TuYV was detected in all regions except for the Landkreis Rostock region (Figure 8). This location is close to the Baltic Sea with winds blown inland from the seaside; therefore, aphids as virus vectors are unlikely to carry viruses into the crops.

Interestingly, some viruses could only be located in very few regions. For example, WCMV was detected in four regions i.e., Salzlandkreis-2, Münster, Kreis Stormarn and Landkreis Rostock whereas several other viruses were only present in two different regions. BYMV was detected in two regions i.e., Salzlandkreis-1 and Salzlandkreis-2.

BGCV2 was in Salzlandkreis-1 and Landkreis Rostock. ClYVV was in Salzlandkreis-2 and Münster. PSbMV was detected Salzlandkreis-2 and Kreis Stormarn. RCEV 1 and RCUV were detected in two regions i.e., Münster and Landkreis Meißen. SbDV and WCCV2 were detected Münster and Kreis Stormarn. Furthermore, BLRV and PaEV were detected only in one region (Salzlandkreis-2 and Landkreis Meißen, respectively).

Chap te r 3: Pe a v irome

Figure 8: List of viruses and their associated nucleic acids detected in each of the six German pea growing regions sampled over three seasons 2016, 2017 and 2018. The seasons in which the viruses were detected are mentioned in brackets. The names of the viruses and their associated nucleic acids 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 associatted emaravirus, PEMV1: pea enation mosaic virus 1, PEMV2: pea enation mosaic virus 2, PEMVsatRNA: pea enation mosaic virus satellite RNA, PNYDVαSat1: pea necrotic yellow dwarf alphasatellite 1, PNYDVαSat3: pea necrotic yellow dwarf alphasatellite 3, PNYDVαSat4: pea necrotic yellow dwarf alphasatellite 4, PNYDVαSat5: pea necrotic yellow dwarf alphasatellite 5, PNYDVαSat6: pea necrotic yellow dwarf

Chap te r 3: Pe a v irome

alphasatellite 6, PNYDVαSat7: 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.

Other viruses were found in only one season in one region i.e., BVG, BCMV, CaTV1, CLRV, CMV, HLV, RCCV1, RCNVA, SMV, TVCV, WHIV21 and all mitoviruses. So far, many of these viruses have not yet been included in standard monitoring programs of pea diseases, therefore the incidence and impact of these viruses on pea and other legume crops is currently unknown.

PEMV1, PEMV2 and their satellites were not detected in 2017 in Landkreis Rostock and in 2018 in Kreis Stormarn. In 2018, PEMV1 was not detected in Landkreis Rostock, however PEMV2 and the satellite RNA were present. TuYV was detected in all three seasons in Salzlandkreis-1, Münster and Landkreis Meißen, however after its detection in Kreis Stormarn in 2016, it could not be detected in 2017 and 2018. Meanwhile, TuYV could not be identified in Salzlandkreis-2 in 2017.

PNYDV, an emerging nanovirus in Germany, was detected in all the regions in 2016 (Figure 8). PNYDV was detected during in all three seasons in Salzlandkreis-1 and Landkreis Meißen, consistent with previous monitoring. PNYDV was not detected in the other four regions in 2017; however, in 2018, it was again detected in Salzlandkreis-2. In 2016 and 2017, WClMV could be found in Salzlandkreis-2 and Kreis Stormarn while in 2018 it was detected again in Kreis Stormarn with additional confirmations in Münster and Landkreis Rostock. PSbMV was detected in all seasons in Salzlandkreis-2 and Kreis Stormarn. Although BLRV in one region, it was detected for two successive season 2016 and 2017 but could not be found in 2018. PaEV was also detected for two seasons in 2017 and 2018.

Taken together, when we look at the spatial distribution of viruses, we can only observe minor differences in the viral community compositions. No major differences could be observed between the regions as e.g., a total of 36 virus incidences were detected in Salzlandkreis-2 followed by Münster with 33 virus incidences (over the three seasons). A total of 28 virus incidences were reported from Salzlandkreis-1, 25 for both Kreis Stormarn and Landkreis Meißen and the lowest rate for Landkreis Rostock (20).

The temporal virus incidences appear to be relatively stable over the successive seasons 2016, 2017 and 2018 with 53, 56 and 54 virus incidences, respectively (Supplementary Table S4).

Chap te r 3: Pe a v irome

3.5. Discussion

This is the first HTS-based study designed to describe the pea virome in Germany.

In addition to the focus on viruses infecting pea crops, we also explored spatio-temporal aspects across six different production regions in Germany over three years. We also examined as potential virus reservoirs non-pea plants surrounding the pea crops, including those related to peas (belonging to the Fabaceae) and plants that were completely unrelated. This distinguishes our study from many metagenomics studies that focused on either just one crop plant, one production area, one season or combination thereof. We believe that this study demonstrates the importance of spatio-temporal aspects into metagenomics studies in order to draw a more complete picture of all the viruses present.

To discover viruses with different genomes and prevent bias we used a ribosomal RNA depleted total RNA (Pecman et al., 2017). As demonstrated, this method detected RNA viruses with both a plus and negative sense genome, and viruses with a DNA genome, namely PNYDV and its associated satellites. We discovered a surprisingly high number of viruses in the different pools with some present in high abundance, i.e., CMV, PEMV1, PEMV2, PEMVSatRNA, TuYV, TuYVaRNA1 and TuYVaRNA2. The recovered reads were pool-dependent as well as dependent on the viral genome, virus titre and incidence within the pool (Supplementary Table S3, Figure 3). All these viruses are positive single-stranded viruses. Interestingly, dsRNA viruses, i.e., partitivirids, and the new negative sense RNA emaravirus were also detected using the ribo-depletion method, despite a low number of reads.