In total, sequencing reads from 27 viruses were found collectively across all alfalfa seed germplasm sources (Table 2 and Additional File 2). Each of the seed samples averaged hits from 10 different viruses (Fig. 1). Based on the bioinformatic analysis, the viruses belong to no less than 15 genera representing 10 different families. Most prevalent among them were known seedborne and seed transmitted species such as alfalfa mosaic virus (AMV), Medicago sativa amalgavirus 1, and partitiviruses (Fig. 1). Their respective assembled contigs covered complete or near-complete genomes (Additional File 2). The identified members of the family Partitiviridae included unclassified viruses Panax cryptic virus 3 (46.1% protein identity), Dichroa partitivirus 1 (48.3%), and Polygonatum partitivirus 1 (69.5%) that have not been reported in alfalfa previously.

Distribution of viral communities in alfalfa (Medicago sativa L.) seed samples

Reads of bean leafroll virus (BLRV) and pea streak virus (PeSV), commonly infecting alfalfa, were also detected in several of the germplasm seed sources (Table 2). In some of them (germplasm sources 5 and 10), the assembled or overlapped contigs of the PeSV covered a near-complete viral genome (Additional File 2). As of today, both viruses are not considered seedborne although they have been previously found in alfalfa seeds [11]. While very few sequencing reads mapped to BLRV (~ 100 reads in all samples), nearly 100,000 of them aligned to the reference genome of PeSV (Additional File 2), indicating a likely seedborne nature of the virus, whether it is transmitted to offspring or merely localizes in the seed.

Alfalfa virus S (AVS), a member of the genus Allexivirus, was detected in the seeds of three different germplasm sources, including the two commercial cultivars. A near-complete AVS genome (~ 98.6% of GenBank ID: NC_034622.1) was recovered from the germplasm source 5 (Table 1, Additional File 2). We proposed earlier a potential role of seed transmission in the distribution of AVS [11]. The recently discovered Snake River alfalfa virus (SRAV) was also detected in the seeds of nine of the ten alfalfa germplasm samples. Some of them contained complete or nearly complete viral genome (germplasm sources 1, 2, 4, 5, 6, 7, 8, 9, and 10; Additional File 2). SRAV was proposed to belong to a flavi-like lineage [12] but later suggested to be a persistent, vertically transmitted virus distantly related to endornaviruses [13].

Several mitoviruses, for which the natural host is fungi, were most likely associated with fungal infections that can be carried internally in alfalfa seeds [14]. The exact fungal hosts of these mitoviruses are unclear, although suggestions can be made, contingent on the similarity scores with known viruses. The seed-infecting fungi would likely include economically important Alternaria spp., Botrytis spp., as well as Peronospora spp. in the Oomycota phylum.

Soybean chlorotic mottle virus (SbCMV) was recently reported to be an endogenous virus integrated into the alfalfa genome [15] and thus the presence of its genomic segments in the seed virome is not incidental. It is conceivable that the endogenous SbCMV-like elements are stable constituents of the host genome and have functional roles in alfalfa’s development. Whether they also represent a source of exogenous infection is currently unknown but cannot be excluded.

Scattered reads of two recently reported rhabdoviruses, alfalfa cytorhabdovirus (ACRV) and alfalfa nucleorhabdovirus (ANRV) [6] were found in the seeds of seven different germplasm sources. Larger genomic portions of these viruses may not have been recovered due to the possible limitations of RNA-seg depth. Rhabdoviruses are recognized as a cause of serious economic losses in plant crop species. A rhabdovirus infecting alfalfa in Argentina was associated with diseased plants displaying shortened internodes, a bushy appearance, deformations, puckering, epinasty of leaflet blades, vein enations, and varying sized papillae on the adaxial leaflet surfaces [16].

Several potyviral genomic fragments were recovered from commercial cultivar source 1. The longest (3.9 kb) translated contig had 26.6% identity with the polyprotein of sugarcane streak mosaic virus (SCSMV), covering the HC-Pro, P3, 6K1, and C1-encoding regions of the genome (PSI BLAST query cover = 79%; E-value = 3e-86), (Additional File 2). The second longest contig (3.1 kb) was 31.4% identical to the polyprotein of Passiflora edulis symptomless virus (PaeSV), covering 6K2, NIa-VPg, NIa-Pro, Peptidase C4, NIb and RdRp- encoding regions of the genome (PSI BLAST query cover = 99%; E-value = 3e-134). The third translated contig (1.3 kb) was 31.68% identical to sweet potato mild mottle virus (SPMMV), covering nearly complete coat protein of the virus (PSI BLAST query cover = 99%; E-value = 4e-55), respectively. It is thus possible that all these fragments represent a genome of one novel potyvirus, which we tentatively named alfalfa-associated potyvirus (AaPV1).

Seed transmission of potyviruses, which are among the most agriculturally significant plant viral pathogens, is not unusual, although its mechanism has not been completely described [17]. Research previously reported that pathogenic maize dwarf mosaic potyvirus (MDMV) was present in male and female floral organs at all organogenesis stages and was subsequently detected in mature pollen grains of the infected maize plants and all parts of the maturing seeds [3]. This suggests a systemic invasion of germ line by potyviruses via mother plant tissues. To our knowledge, no potyviruses have been detected or reported in alfalfa seed prior to this study. Traces of a virus distantly resembling PNG bee virus 10 [18] are likely incidental unless introduced by infected bees through pollen grains.

In order to randomly confirm the presence of HTS-identified viruses and to exclude the possibilities of cross-contamination from other samples impacting the data, we performed RT-PCR with primers specific for several identified viruses: ACRV, AVS, BLRV, PeSV, SRAV, PaeSV, SPMMV, and SCSMV. Primers were designed based on the obtained HTS contigs (Additional File 1). The RT-PCR led to the amplification of the correct products from all these viruses (Fig. 2). This experiment validated the HTS findings. Nevertheless, as is always the case with HTS, the potential effect of contaminating sequences cannot be underestimated or ignored.

Reverse transcription-polymerase chain reaction to validate the presence of viral sequences in alfalfa (Medicago sativa L.) seeds. M, 1 kb Plus DNA ladder (Thermo Fisher Scientific Inc., Waltham, MA USA). Lane 1: amplification with primers LN1052/53 (BLRV, 473 bp), germplasm source №7. Lane 2: primers LN1052/53, control reaction. Lane 3: primers LN1054/55 (PeSV, 340 bp), germplasm source №5. Lane 4: primers LN1054/55, control reaction. Lane 5: primers LN1056/57 (ACRV, 480 bp), germplasm source № 5. Lane 6: primers LN1056/57, control reaction. Lane 7: primers LN1058/59 (AVS, 338 bp), germplasm source № 9. Lane 8: primers LN1058/59, control reaction. Lane 9: primers LN1060/61 (SRAV, 320 bp), germplasm source № 10. Lane 10: primers LN1060/61, control reaction. Lane 11: primers LN1062/63 (SCSMV, 603 bp), germplasm source № 9. Lane 12: primers LN1062/53, control reaction. Lane 13: primers LN1064/65 (PaeSV, 694 bp), germplasm source № 9. Lane 14: primers LN1064/65, control reaction. Lane 15: primers LN1066/67 (SPMMV, 213 bp), germplasm source № 9. Lane 16: primers LN1066/67, control reaction

We next attempted to learn if the presence of the detected viruses in alfalfa seeds may lead to their actual seed transmission. For this purpose, we germinated surface-sterilized seeds of germplasm sources 5, 7, 8, 9, and 10 (Table 1) in Petri dishes. After one week, total RNA was extracted from seedlings and used for RT-PCR with the same sets of primers. The only amplicons produced were from ACRV and SRAV (Fig. 3). This experiment once again confirmed our previous suggestion of the persistent nature of SRAV in alfalfa [13]. The amplification of the ACRV sequence from germinated seedlings is of particular interest, since rhabdoviruses are not known to infect seeds of any plant species and mainly depend on transmission by phytophagous insects [19].

Reverse transcription-polymerase chain reaction validating seed transmission of the selected viruses in alfalfa (Medicago sativa L.). M, 1 kb Plus DNA ladder (Thermo Fisher Scientific Inc., Waltham, MA USA). Lane 1: amplification with primers LN1052/53 (BLRV), germplasm source №7. Lane 2: primers LN1052/53, control reaction. Lane 3: primers LN1054/55 (PeSV), germplasm source №5. Lane 4: primers LN1054/55, control reaction. Lane 5: primers LN1056/57 (ACRV, 480 bp), germplasm source № 5. Lane 6: primers LN1056/57, control reaction. Lane 7: primers LN1058/59 (AVS), germplasm source № 9. Lane 8: primers LN1058/59, control reaction. Lane 9: primers LN1060/61 (SRAV, 320 bp), germplasm source № 9. Lane 10: primers LN1060/61, control reaction. Lane 11: primers LN1062/63 (SCSMV), germplasm source № 9. Lane 12: primers LN1062/63, control reaction. Lane 13: primers LN1064/65 (PaeSV), germplasm source № 9. Lane 14: primers LN1064/65, control reaction. Lane 15: primers LN1066/67 (SPMMV), germplasm source № 10. Lane 16: primers LN1066/67, control reaction.

The remaining known and novel candidate viruses found in mature seeds by HTS and RT-PCR (AVS, BLRV, PeSV, PaeSV, SPMMV, and SCSMV) were likely unstable and inactivated in the embryo and thus did not retain their infectivity or were unable to replicate [2]. While seed transmission was not supported by this testing, it cannot be completely ruled out. It is also important to emphasize that all seeds used in this study, except for the commercial cultivars, have been maintained as accessions of the NPGS for a long period of time, some for as long as ~ 30 years, which could significantly affect virus transmissibility. From this perspective, it is remarkable that viral sequences were still detected in the seeds and some of them apparently retained infectivity.