Tomato (Solanum lycopersicum) plants from a commercial glasshouse were identified with symptoms compatible with a tomato brown rugose fruit virus (ToBRFV) infection. Reverse transcription-PCR and quantitative PCR confirmed the presence of ToBRFV. Subsequently, the same RNA sample and a second from tomato plants infected with a similar tobamovirus, tomato mottle mosaic virus (ToMMV), were extracted and processed for high-throughput sequencing with the Oxford Nanopore Technology (ONT). For the targeted detection of ToBRFV, the two libraries were synthesized by using six ToBRFV sequence-specific primers in the reverse transcription step. This innovative target enrichment technology enabled deep coverage sequencing of ToBRFV, with 30% of the total reads mapping to the target virus genome and 57% mapping to the host genome. The same set of primers applied to the ToMMV library generated 5% of the total reads mapping to the latter virus, indicating that sequencing of similar, non-target viral sequences was also allowed. Further, the complete genome of pepino mosaic virus (PepMV) was also sequenced from the ToBRFV library, thus suggesting that, even using multiple sequence-specific primers, a low rate of off-target sequencing can usefully provide additional information on unexpected viral species coinfecting the same samples in an individual assay. These results demonstrate that targeted nanopore sequencing can specifically identify viral agents and has sufficient sensitivity towards non-target organisms to provide evidence of mixed virus infections.

The discovery of a plum tree isolate of plum pox virus (PPV, Potyvirus plumpoxi), done in Eastern Albania in 2011 in the frame of an EU-funded survey, which represents a divergent strain named PPV-An, proved to be original and informative for the unraveling of PPV evolutionary history. Maximum likelihood and Bayesian phylogenetic methods applied on full-length genomes or selected regions analyzed the affinities of the PPV-An with other PPV strains. Potential recombination events were also evaluated. A refined timeline of PPV evolutionary history integrating recombination events, strains migration and ancestral host state is proposed. Altogether, the analyses confirm previous hypotheses that PPV-An corresponds to an ancestral, non-recombinant PPV strain. PPV-An likely served as the origin of the PPV-M and T strains through recombination with isolate(s) of the D strain. Molecular clock analyses dated the most recent common ancestor (TMRCA) of PPV at 4546 years ago and phylogeny separated the main PPV strains from the cherry-adapted strains around 3100 years ago. Meanwhile, the recombination events that gave rise to the M and T strains are estimated to have occurred in the early sixteenth century of the common era (CE). The characterization of the PPV-An strain enabled a comprehensive phylogenetic analysis of PPV. PPV-An is confirmed to be the previously unidentified progenitor, which, together with PPV-D led through recombination to the emergence of the currently prevalent and evolutionary successful recombinant strains of European origin (e.g., M, Rec, and T). The low representation of PPV-An in current PPV populations is likely the consequence of a population replacement phenomenon possibly linked to a higher fitness of the recombinant strains deriving from it. These results highlight the PPV-An strain as a key player in PPV evolutionary history and consolidate PPV as one of the promising models to study host-adaptive evolution processes and phylogeography among the most damaging viruses of agricultural systems.

Xylella fastidiosa (Xf) is a quarantine pathogen heavily affecting economically important crops worldwide. Different sequence types (STs) belonging to Xf subspecies are present in various areas of Spain, including the Balearic Islands, and cause the almond leaf scorch disease (ALSD) in Prunus spp. The increased demand for rapid tests for early detection of the pathogen should enforce strict containment measures. Molecular detection through isothermal amplification reactions enables simplified instrumentation and the use of raw nucleic acid extracts. Colorimetric loop-mediated isothermal amplification (cLAMP) was applied to rapidly detect Xf in naturally infected almonds on Mallorca Island (Spain), using a quick crude sap extraction without DNA purification. Following tissue homogenization, an alkaline treatment for target DNA extraction was conducted before the cLAMP test. The cLAMP assay was able to detect up to 100 CFU/mL of the Xf bacterial suspension diluted in healthy almond sap. The same crude extracts used in the cLAMP test were also tested by qPCR. An overall positive agreement of about 47% was observed between the results of the two techniques, while a decrease in cLAMP sensitivity was evident as the bacterial titer declined in infected plants over Cq > 26–27. This study shows the potential of the cLAMP application as a rapid and low-cost point-of-care diagnostic method for the timely monitoring of Xf directly in the field.

Climatic changes are heavily affecting the sustainability of vegetable crops crucial for food supply worldwide, mainly in subtropical countries. One of the main threats to food security is the spread of diseases caused by plant viruses, favored by irregular rains and extreme temperatures, which reduce crop yield and quality. During a preliminary field survey carried out in two provinces of Angola in 2024, a few symptomatic plants of tomato, habanero pepper, common bean and a wild weed were sampled. These plants generally showed dwarfing, yellowing and leaf curl and were submitted to high-throughput sequencing to detect any viral agent. The evidence of mixed infections of several polyphagous viruses with RNA or DNA genomes, variously affecting the selected plants, was assessed from the sequence analysis and further confirmed for most samples by molecular tests, like (RT)-PCR or qPCR. Emerging polero-, begomo and tobamoviruses were denoted as infecting these plants. A novel, previously unknown carlavirus was also described in a wild weed. Most of those viruses are efficiently mechanically transmitted or airborne vehiculated by insect vectors. Although based on a limited number of samples, this study provides a first insight into the diversity of viruses infecting vegetable crops in Angola. It also highlights the pressing need for a broader monitoring to better understand virus distribution and epidemiology, and suggests the use of virus-free seeds to reduce the potential risk to crop production.