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High-throughput sequencing (HTS), more specifically RNA sequencing of plant tissues, has become an indispensable tool for plant virologists to detect and identify plant viruses. During the data analysis step, plant virologists typically compare the obtained sequences to reference virus databases. In this way, they are neglecting sequences without homologies to viruses, which usually represent the majority of sequencing reads. We hypothesized that traces of other pathogens might be detected in this unused sequence data. In the present study, our goal was to investigate whether total RNA-seq data, as generated for plant virus detection, is also suitable for the detection of other plant pathogens and pests. As proof of concept, we first analyzed RNA-seq datasets of plant materials with confirmed infections by cellular pathogens in order to check whether these non-viral pathogens could be easily detected in the data. Next, we set up a community effort to re-analyze existing Illumina RNA-seq datasets used for virus detection to check for the potential presence of non-viral pathogens or pests. In total, 101 datasets from 15 participants derived from 51 different plant species were re-analyzed, of which 37 were selected for subsequent in-depth analyses. In 29 of the 37 selected samples (78%), we found convincing traces of non-viral plant pathogens or pests. The organisms most frequently detected in this way were fungi (15/37 datasets), followed by insects (13/37) and mites (9/37). The presence of some of the detected pathogens was confirmed by independent (q)PCRs analyses. After communicating the results, 6 out of the 15 participants indicated that they were unaware of the possible presence of these pathogens in their sample(s). All participants indicated that they would broaden the scope of their bioinformatic analyses in future studies and thus check for the presence of non-viral pathogens. In conclusion, we show that it is possible to detect non-viral pathogens or pests from total RNA-seq datasets, in this case primarily fungi, insects, and mites. With this study, we hope to raise awareness among plant virologists that their data might be useful for fellow plant pathologists in other disciplines (mycology, entomology, bacteriology) as well.

High-throughput sequencing (HTS) technologies have become indispensable tools assisting plant virus diagnostics and research thanks to their ability to detect any plant virus in a sample without prior knowledge. As HTS technologies are heavily relying on bioinformatics analysis of the huge amount of generated sequences, it is of utmost importance that researchers can rely on efficient and reliable bioinformatic tools and can understand the principles, advantages, and disadvantages of the tools used. Here, we present a critical overview of the steps involved in HTS as employed for plant virus detection and virome characterization. We start from sample preparation and nucleic acid extraction as appropriate to the chosen HTS strategy, which is followed by basic data analysis requirements, an extensive overview of the in-depth data processing options, and taxonomic classification of viral sequences detected. By presenting the bioinformatic tools and a detailed overview of the consecutive steps that can be used to implement a well-structured HTS data analysis in an easy and accessible way, this paper is targeted at both beginners and expert scientists engaging in HTS plant virome projects.

Grapevine leafroll disease is one of the most severe viral diseases of grapevine caused by Grapevine leafroll-associated viruses (GLRaVs). Physiological processes were monitored on grapevines with single (GLRaV-1) and mixed (GLRaV-1 and GLRaV -3) viral infection under greenhouse conditions from June to September, in vegetation period 2014. In the mid of the season (July) negative effects of the virus infections on physiological processes were more severe in mixed than in single infection. The net-photosynthesis (Pn) of the leaves infected with GLRaV-1 and GLRaV-3 reached only a half of the Pn in GLRaV-1 infected grapevines. Similar reduction was found for stomatal conductance, transpiration and parameters related to photochemical efficiency (electron transport rate).

Agrobacterium vitis causes common grape vine (Vitis vinifera L.) crown gall disease that destroyed a lot of Slovenian vineyards more than a decade ago. Eighty isolates of Agrobacterium spp. collected during monitoring in 2006 were identified as A. vitis and A. tumefacies by pehA and multiplex PCR method. Tumor-inducing capacity of these strains was assessed on test plants and with PCR methods for detection of the Ti plasmid responsible for tumor induction. With VCF3/VCR3 primer pair six false negatives and no false positives were detected. The high genetic diversity of pathogenic Agrobacterium spp. strains affects the performance of molecular methods, thus biological test should be performed where results from molecular methods are doubtful.

Sensitivity of field tests (AgriStrip and Immunochromato), DAS-ELISA, two step RT-PCR and real-time RT-PCR for Plum pox virus (PPV) detection was tested in various tissues of apricot, peach, plum and damson plum trees infected with isolates belonging to PPV-D, PPV-M or PPV-Rec, the three strains present in Slovenia. Flowers of apricot and plum in full bloom proved to be a very good source for detection of PPV. PPV could be detected with all tested techniques in symptomatic parts of leaves in May and with one exception even in the beginning of August, but it was not detected in asymptomatic leaves using field tests, DAS-ELISA and partly also molecular techniques. PPV was detected only in some of the samples of asymptomatic parts of the leaves with symptoms and of stalks by field tests and DAS-ELISA. Infections were not detected in buds in August using field tests or DAS-ELISA. Field tests are useful for confirmation of the PPV infection in symptomatic leaves, but in tissues without symptoms DAS-ELISA should be combined or replaced by molecular techniques.

Five isolates of a new member of the family Closteroviridae, tentatively named blackcurrant leafroll-associated virus 1 (BcLRaV-1), were identified in the currant. The 17-kb-long genome codes for 10 putative proteins. The replication-associated polyprotein has several functional domains, including papain-like proteases, methyltransferase, Zemlya, helicase, and RNA-dependent RNA polymerase. Additional open reading frames code for a small protein predicted to integrate into the host cell wall, a heat-shock protein 70 homolog, a heat-shock protein 90 homolog, two coat proteins, and three proteins of unknown functions. Phylogenetic analysis showed that BcLRaV-1 is related to members of the genus Closterovirus, whereas recombination analysis provided evidence of intraspecies recombination.

A survey of Potato spindle tuber viroid was initiated in Slovenia in 2006. Until the end of 2010, 100 samples of ornamental plants were found to be infected. Analysis of 96 complete master sequences of Slovene samples revealed new genetic variants. Our sequence variants showed considerable variation in their predicted secondary structure. The variability was observed in the pathogenic, central and variable domains. In several cases even single mutations were sufficient for the change in predicted secondary structure.

V Sloveniji se na vinski trti pojavljajo kaparji Neopulvinaria innumerabilis, Pulvinaria vitis, Parthenolecanium corni in Planococcus ficus. Te žuželke sesajo rastlinski sok in povzročajo prezgodnje odpadanje listov in bolezenske okužbe grozdov, kar vpliva na kakovost pridelka. Kaparji so tudi pomembni prenašalci rastlinskih virusov. Vrsti P. vitis in P. corni sta razširjena po vsej Sloveniji, vrsta N. innumerabilis v vinogradih na Primorskem, vrsto P. ficus pa največkrat najdemo v zavarovanih prostorih. Običajno kaparje zatiramo z uporabo insekticidov, vendar so sodobne strategije varstva rastlin usmerjene v naravi prijaznejše načine, kot sta mehansko in biotično zatiranje škodljivcev. Pri nas biotično zatiranje kaparjev še ni dobro raziskano, čeprav se je v srednji Evropi že izkazalo za uspešno.

Successful total RNA (totRNA) extraction is a prerequisite for a successful real-time PCR. In the present work we compared one manual and one automated totRNA extraction method for detection of Plum pox potyvirus (PPV) in leaves of different Prunus sp. using developed real-time RT-PCR assay. Advantages and disadvantages of compared methods are described in the view of sensitivity, reproducibility and in terms of laboratory use. The results suggest that the column based totRNA extraction method is more effective for apricot, plum and peach. In the case of damson the differences in real-time PCR results between both extraction methods were negligible. In case of negative results obtained with automated method, manual column based extraction method should be used additionally.

To introduce testing of Grapevine leafroll-associated virus-2 (GLRaV-2) and Grapevine virus B (GVB) in sanitary selection of grapevine, commercially available antibodies were evaluated and conditions for routine ELISA testing were optimized. Extraction procedure with Granex 91 - special machine, which is used in routine testing in Slovenia, was compared with grinding samples in mortar. Three different extraction buffers were applied in order to overcome the inconvenience of using more than one extraction procedure when testing grapevine material for several viruses in a routine large-scale testing scheme. Results were verified with Western blot and immuno-electron microscopy. The best results were obtained using extraction buffer with unknown composition (pH 9.0) from BIOREBA kit for GLRaV-2. Other extraction buffers gave less positive samples and they are not convenient for routine testing where extraction with Granex is done. Both viruses, GLRaV-2 and GVB were found in Slovenia, but they couldn't be correlated with rougose wood disease that appears on indigenous cultivar Refošk grafted on 'SO4' from collection vineyard in Komen.

Chrysanthemum stem necrosis virus (CSNV), an RNA virus, belongs to the genus Tospovirus and family Bunyaviridae. The disease symptoms on host plants can not be distinguished from those caused by closely related viruses of the same genus, such as Tomato spotted wilt virus (TSWV). The disease symptoms may vary between host plants and can be quite severe. Diagnostics of tospoviruses is of great importance in prevention of greater economic and environmental damage. Viruses within the genus show serological similarity and therefore make the detection of the viruses by serological methods unreliable. To avoid false positive results other methods can be used for detection of RNA viruses such as RT-PCR or RT real-time PCR. CSNV was first found in Slovenia in 2001. Different methods were used to confirm the identity of the isolate. Development of disease symptoms was observed on 15 different test plants and was compared to the symptoms caused by closely related TSWV. Since serological cross-reac- tivity was observed, molecular tests were developed (RT-PCR and real-time PCR) that further confirmed the identity of the isolate and also increased sensitivity of the assays. CSNV can now be reliably detected and distinguished from related tospoviruses.

High throughput sequencing of total RNA isolated from symptomatic leaves of a sweet cherry tree (Prunus avium cv. 0900 Ziraat) from Turkey identified a new member of the genus Robigovirus designated cherry virus Turkey (CVTR). The presence of the virus was confirmed by electron microscopy and overlapping RT-PCR for sequencing its whole-genome. The virus has a ssRNA genome of 8464 nucleotides which encodes five open reading frames (ORFs) and comprises two non-coding regions, 5′ UTR and 3′ UTR of 97 and 296 nt, respectively. Compared to the five most closely related robigoviruses, RdRp, TGB1, TGB2, TGB3 and CP share amino acid identities ranging from 43−53%, 44−60%, 39−43%, 38−44% and 45−50%, respectively. Unlike the four cherry robigoviruses, CVTR lacks ORFs 2a and 5a. Its genome organization is therefore more similar to African oil palm ringspot virus (AOPRV). Using specific primers, the presence of CVTR was confirmed in 15 sweet cherries and two sour cherries out of 156 tested samples collected from three regions in Turkey. Among them, five samples were showing slight chlorotic symptoms on the leaves. It seems that CVTR infects cherry trees with or without eliciting obvious symptoms, but these data should be confirmed by bioassays in woody and possible herbaceous hosts in future studies.