Viruses cause significant yield and quality losses in a wide variety of cultivated crops. Increasing international travel and the trade of plant material, as well as climate changes, enhance the risk of spreading and introducing new viruses and their vectors into production systems [1
]. Moreover, most of the viruses are multihost pathogens with a wider range of hosts, rather than the specialists (and, therefore, the host range of many viruses overlap). Hence, a single plant might be infected by different unrelated viral species [3
]. The interactions between viruses coinfecting the same host might affect their host range, transmission rate, virus accumulation and, as a consequence, the presence and intensity of symptoms [6
Moreover, plant viruses can emerge in crops from reservoir wild plant hosts in which they are often asymptomatic. Spread from the reservoirs into a new environment with the establishment of productive infections and effective between-host transmission mechanism are steps that require emergence to occur [10
]. In addition, many wild plants appear to have multiple infections, including both acute and persistent viruses [11
]. At present, novel viruses are being discovered rapidly in wild hosts in diverse natural ecosystems, and this discovery has been accelerated by metagenomics techniques that permit the sequencing of putative viral nucleic acids without a prior knowledge about the present viruses or associated host organisms [12
Most of the viruses infecting plants are RNA viruses, whose mutation rate is very high [13
]. Appropriate diagnosis is especially important in positive-sense single-stranded RNA ((+)ssRNA) viruses, which are characterised by a small genome, fast replication rate, and a lack of repair mechanisms and, therefore, a great potential for genetic differentiation [14
]. This allows the maintenance of the genetic diversity of the viral populations and the adaptation to the ever-changing environment while simultaneously disrupting precise targeted diagnostics approaches.
The use of the appropriate diagnostic method is crucial in maintaining healthy material, preventing the spread of the diseases, and carrying out phytosanitary measures. A traditional diagnostic method using transmission electron microscopy (TEM) allows only for the morphological observation of particles in leaf sap derived from infected plants [15
]. The standard diagnostic tests (ELISA assay, PCR, RT-PCR, qRT-PCR), despite their potential sensitivity and specificity, require specific primers or sera, and thus specific knowledge about the diagnosed pathogen and its group or family [16
]. A huge problem in the precise diagnosis of viruses is the appearance of the new genetic variants by mutation, reassortment or recombination that can significantly differ from the parental viral particle [20
]. Furthermore, the diagnosis of mixed infections is associated with many problems, mostly due to the presence of individual viral components in a higher or lower concentration [21
High-throughput sequencing (HTS) is a rapidly developing technique, providing novel opportunities for diagnosis and epidemiology. This technique allows for the sequencing of millions of nucleotides in a short time, which enables the detection of the most viral pathogens in the sample [22
]. Moreover, HTS does not require any prior information about pathogens before sequencing [23
]. Due to the possibility of sequencing millions of nucleotide sequences, it can deliver a global spectrum of occurring strains or species of pathogens. In combination with the bioinformatic analysis of the obtained raw data, it makes possible to detect all known pathogens and discover new ones from symptomatic or asymptomatic plants, as well as substrates, e.g. water or soil [26
]. HTS was first used in 2009 for the detection of plant viruses, and since then, it has often been used as a diagnostic tool [25
]. Therefore, it is a good alternative to other diagnostic tests, especially in the context of unexpected or unknown viruses that might be potential threats to plant health. Knowledge of the occurrence and degree of infestation of crops, weeds, trees and ornament plants allows the introduction of appropriate regulations, and thus prevents the occurrence of an epidemic, as well as allowing for the development of new strategies for plant protection.
In this study, an HTS-based approach was applied for the detection and identification of different viruses from infected plant material collected in Poland. To this end, various plants from different families were used, including cultivated and ornamental plants, weeds and trees. By following this procedure, we successfully detected new pathogens for Poland, identified mixed infections, and obtained the exact genetic characteristic of the pathogens. With the advantage of HTS methods that have been developed to look for virus-like sequences without the bias of only looking for known viruses, we were able to identify virus species that have not been detected using conventional RT-PCR. Furthermore, the phylogenetic analysis was performed in order to establish the genetic relationships of: melandrium yellow fleck virus, clover yellow mosaic virus and cucurbit aphid-borne yellows virus, identified in this study with others described to date.
In the present study, we used different diagnostic methods to detect plant viruses from crops, ornamental plants, weeds, trees and shrubs. An important issue in the disease management is the limited availability of appropriate diagnostic methods. In fact, effective detection protocols are required for maintaining healthy planting material. Standard diagnostic tests, despite potentially high sensitivity and specificity, require specific prior knowledge of the target pathogens, and thus are not suitable for detection of unknown or unexpected pathogens. Moreover, their high specificity might limit the detection of different isolates, variants or strains of particular pathogens, which can lead to false negative results [32
]. Given the magnitude of plant-infecting pathogens and the frequent occurrence of viruses in mixed infections, it is thus expected that some viruses are missed when using routine targeted diagnostic tests. Here, we used a HTS-based approach, to detect viruses in samples, which were previously tested to be negative using an array of selected targeted RT-PCR tests, but showed positive results using nonspecific TEM and bioassay-based approaches. A total of 13 viruses from different families were identified in 20 tested samples, some of which are rare or have never been found in Poland before. Some of the viruses (CMV, WMV, ZYMV) were detected using HTS, despite a previous negative result in RT-PCR. One of these viruses, CMV, using HTS has been detected in many analysed samples (in 9 of 20 samples). It may indicate the appearance of new genetic variants in nature or a low concentration of viruses in the tested samples.
In this study, we identified two new viruses which originated from weeds: ClYMV and MYFV. The first one, ClYMV infects a broad bean, pea, alfalfa, fat hen, chickweed, and tulips [34
]. Moreover, it causes an important disease of clovers in the United States and Canada [36
]. In Europe, it does not pose a significant threat to crops, although it has been identified in the United Kingdom [35
] and the Czech Republic [37
]. ClYMV belongs to Potexvirus
genus within Alphaflexiviridae family. Its genome consists of single-stranded plus RNA about 7000 nt in length. The phylogenetic analysis revealed that the Polish isolate is closely related to the isolate from the United Kingdom. The second one—MYFV—belongs to the Bromovirus
genus of the Bromoviridae
family. Bromoviruses have three positive-sense RNAs as their genome, designated RNA1, RNA2, and RNA3 [38
]. MYFV occurs sporadically and there is only one sequence deposited in the GenBank. The nucleotide sequence identity between the Polish isolate and Hungarian originated from M. album
ranged between 91.1–94.8% for the genomic RNA1-3. ClYMV and MYFV were detected in weeds that can serve as alternative hosts and potential sources of virus infection. In addition, many wild plants are often infected with more than one viral species at the same time [11
]. Surprisingly, when analysing weed plants using HTS, we only detected the presence of single viral infections. Perhaps this is the result of collecting the samples in urban locations, where the presence of potential vectors and the possibility of mechanical viral transmission is more limited.
We have shown that plants from the Cucurbitaceae
family sampled in Poland are infected with a large spectrum of viruses in mixed infections, including CMV, ZYMV, WMV, CLSV and CABYV. Cucurbits are the major vegetables cultivated worldwide and are affected by more than 70-well-characterised viruses belonging to the main plant virus groups [39
]. Most are transmitted by aphids, which in combination with global warming, can promote the faster spread of viruses and the appearance of more severe infections, as well as the horizontal transmission of the viruses to new hosts by their vectors [40
]. In 4 of the 11 cucurbit samples, we identified the presence of CABYV. The virus was detected in Poland for the first time in 2018 on zucchini crops in the Wielkopolska region [31
]. This was the first report of the occurrence of CABYV in Poland, and the virus has not been previously detected using conventional RT-PCR. CABYV infects cucumber, melon, squash and watermelon, and has also been detected in many weed species, which may be efficient reservoirs [39
]. The virus is efficiently transmitted in a persistent, circulative manner by a few aphid species (Aphis gossypii, M. persicae
and Macrosiphum euphorbiae
). The abundance of CABYV reservoirs found around cultivated fields and the large populations of A. gossypii
and M. persicae
vectors indicated the potential for virus spread, and represents a serious threat to cucurbit production in Poland. It has been shown that CABYV is one of the most common cucurbit viruses in open-field crops, distributed worldwide [41
]. Increasing international travel and the trade of plant material enhances the risk of introducing new viruses and their vectors into production systems. It is very likely that, due to the similarity of symptoms induced by CABYV with those induced by other viruses, the presence of the CABYV in Poland remained unnoticed for some years. Our phylogenetic analysis showed geographical and host divergence of CABYV isolates. The Polish isolates of CABYV clustered with isolates from Spain or Brazil, as well as with isolates originating from Asian countries. No clear division by host plant on phylogenetic tree may suggest that CABYV isolates can easily adapt to ever-changing environmental conditions and different hosts.
We have also identified the presence of CLSV in complex with other viruses on zucchini plants. This is the second study reporting the presence of this pathogen in our country [42
]. The virus is rarely observed in crops, but it can cause symptoms in the form of chlorotic spots with necrotic centers, slight stunting and the delay of flowering [42
]. Mixed infections were also identified in the case of tomato crops, where the complex of PepMV and CMV or PepMV and TYRV (tomato sample that gave an unusual pattern of symptoms on infected plants) was observed in tested samples. PepMV is one of the most dangerous viruses that currently infects tomato plants in many European countries, North and South America and China [44
]. In Poland, it has been widely distributed since 2005. PepMV causes a wide spectrum of symptoms on infected plants, leading to a lower quality and quantity of crops [49
]. There are various pathotypes of the virus, including necrotic ones, which can lead to plant death. In addition, the presence of the virus in mixed infections with other viruses can lead to higher crop losses. In recent years, the severe damages caused by mixed infections of the viruses belonging to the Tospovirus
genus: tomato spotted wilt virus and TYRV were observed in tomato crops in Poland [57
]. The virus can cause brown necrotic spots on leaves and stems, as well as necrotic spots on fruits. TYRV is relatively rare in Europe. CMV is a pathogen with a very wide host range. On tomatoes, the virus causes the inhibition of growth and stunting of plants, leading to a reduced crop yield. The presence of TYRV and CMV in a mixed infection with PepMV, especially due to their easy mechanical transmission, may pose a serious threat to tomato crops. The presence of mixed virus infections can modulate the symptoms, transmission and pathogenicity [6
]. The simultaneous occurrence of different viruses on infected plants can stimulate the emergence of new genetic variants, and thus affect the degree of genetic diversity of their populations [58
]. Such occurrence of mixed infections in plants can affect the evolutionary dynamics of the virus or virus population, change the population structure and, therefore, can contribute to stronger and more frequent infections.
In summary, we detected a wide spectrum of pathogens using the HTS-based approach. We have shown that HTS technology is appropriate for the detection of viruses in plants in the absence of prior information on the type of pathogen and its genome structure, or in the case of unclear symptoms of infection. Moreover, we confirmed the potential of HTS for use in the identification of all viruses in mixed infections, including those that could escape detection by RT-PCR. The use of HTS for virus detection will improve the current diagnostic methods, by thoroughly investigating the genomic sequence and the variability of known pathogens, and by designing new diagnostic tools for new viruses.