First report of infections with isolates of the Asian genotype of Cucumber green mottle mosaic virus in cucumber crops in Belgium.

In July 2019, four rows of cucumber plants (Cucumis sativus) in a commercial glasshouse in the north of Belgium showed severe mosaic, blistering and distortion of the leaves, with symptoms resembling those caused by Cucumber green mottle mosaic virus (CGMMV). CGMMV is a Tobamovirus that mainly affects cucurbit crops worldwide (Dombrovsky et al., 2017). Phylogenetic analyses in previous studies have shown two major clades, one including isolates that were initially identified in Europe and Russia (European genotype) and the second one with isolates initially identified in Asia and Israel (Asian genotype) (Dombrovsky et al., 2017; Pitman et al., 2022; Mackie et al., 2023). A symptomatic leaf sample was collected and total RNA was isolated from 100 mg of leaf tissue (Spectrum™ Plant Total RNA kit, Sigma-Aldrich). CGMMV was detected using a one-step TaqMan RT-qPCR (Hongyun et al., 2008). High-throughput sequencing (HTS) confirmed the presence of CGMMV. The sample was prepared using the Novel enrichment technique of viromes protocol (NETOVIR protocol, Conceição-Neto et al., 2015). The leaf material was homogenized, enriched for virus-like particles and the RNA was extracted (QIAamp Viral RNA mini kit, QIAGEN). The extract was randomly amplified (Whole Transcriptome Amplification kit, Sigma Aldrich), used for library preparation (Nextera XT DNA library preparation kit, Illumina) and sequenced on a NovaSeq platform. HTS data analysis was performed using Geneious Prime software (Biomatters, Auckland, New Zealand, version 2023.2). After quality filtering and trimming, 26.7M reads were obtained (132 nt mean length). In total, 20.6M reads were mapped to two genomes KP772568 and GQ411361 (considered as reference for the Asian and European genotypes respectively) with Geneious. This revealed 100% coverage of the full sequences (6422 nt) with 99.4% and 90% nucleotide identities to the reference genomes of Asian and European genotypes, respectively. Phylogenetic analyses confirmed that isolate 2019-26A-BE, with GenBank ID OR724740, relates to the Asian genotype. The HTS data were additionally processed using the ViPER pipeline (De Coninck, 2021). The raw reads were quality filtered and trimmed, (Trimmomatic) and then used to perform de novo assembly (metaSPAdes). The produced contigs were classified using DIAMOND and visualized with KronaTools. The results showed that no other virus was detected in the sample. Finally, cucumber seedlings were inoculated using the original symptomatic sample and were grown in a research glasshouse. After 3 weeks, severe CGMMV symptoms, similar to the original symptoms observed in the commercial glasshouse, were observed in the inoculated plants. Infection with CGMMV was verified via RT-qPCR, and the isolate present in the inoculated plants was confirmed to belong to the Asian genotype via RT-PCR-RFLP (Crespo et al., 2017). Later samplings of symptomatic leaves confirmed the presence of isolates belonging to the Asian genotype of CGMMV at four other commercial glasshouse locations, specializing in cucumber crop, in Belgium in 2020, 2021 and 2023 by RT-PCR-RFLP. While the Asian genotype was previously found elsewhere in Europe (Pitman et al., 2022), to our knowledge, this is the first report of infections with isolates of this genotype in glasshouse cucumber crops in Belgium. Further investigation is required to determine the spread and impact of infections with isolates of the Asian genotype in cucumber crops in Belgium.

High population densities of Macrolophus pygmaeus on tomato plants can cause economic fruit damage: interaction with Pepino mosaic virus?

BACKGROUND: The zoophytophagous predator Macrolophus pygmaeus Rambur (Hemiptera: Miridae) is a successful biocontrol agent against several pest species in protected tomato crops. This predator is considered to be harmless for the crop. However, in recent years, Heteroptera feeding punctures on tomato fruit in Belgian and Dutch greenhouses have been misinterpreted as Pepino mosaic virus (PepMV) symptoms. In this study, three hypotheses were tested: (1) M. pygmaeus causes fruit damage that increases with population density and surpasses economic thresholds; (2) the presence of prey or alternative prey reduces the damage; (3) an infection of the tomato plants by PepMV triggers or aggravates M. pygmaeus fruit damage. RESULTS: At increasing M. pygmaeus densities, the severity of fruit damage increased from a few dimples towards yellowish discoloration and deformed fruits. A correlation with an infection with PepMV was found. The severity of the symptoms was independent of the presence of prey. A minimum economic density threshold was estimated at 0.32 M. pygmaeus per leaf. CONCLUSION: M. pygmaeus can cause economic damage to tomato fruits at densities common in practice. An infection of the plants with PepMV enhances fruit symptoms significantly. Interacting plant defence responses are most likely the key to explaining this, although confirmation is required. © 2015 Society of Chemical Industry.

Ratio of mutated versus wild-type coat protein sequences in Pepino mosaic virus determines the nature and severity of yellowing symptoms on tomato plants.

Recently, Pepino mosaic virus (PepMV) infections causing severe yellowing symptoms in tomato plants have been reported in glasshouse tomato crops. When studying this phenomenon in commercial glasshouses, two different types of yellowing symptoms, occurring in adjacent plants, were distinguished: interveinal leaf yellowing and yellow mosaics. After several weeks, the interveinal leaf yellowing symptoms gradually disappeared and the plant heads became green again, with yellow mosaic patterns on the leaves as an intermediate stage. The sequencing of multiple isolates causing interveinal leaf yellowing identified two point mutations, occurring in positions 155 and 166 of the coat protein (CP), as unique to the yellowing pathotype. Site-directed mutagenesis of infectious clones confirmed that both CP mutations are determinants of the interveinal leaf yellowing symptoms. Sequencing of CP clones from plants or plant parts with the yellow mosaic symptoms resulted in a mixture of wild-type and mutated sequences, whereas sequencing of CP clones from the green heads of recovered plants resulted in only wild-type sequences. Yellow mosaic symptoms could be reproduced by inoculation of an artificial 1:1 mixture of RNA transcripts from the wild-type and mutated infectious clones. These results show that the ratio of mutated versus wild-type sequences can determine the nature and severity of symptom development. The gradual recovery of the plants, which coincides with the disappearance of the yellowing mutations, suggests that selection pressure acts to the advantage of the wild-type virus. Experiments with wild-type and mutated infectious clones showed that reverse mutation events from mutant to wild-type occur and that the wild-type virus does not have a replicative advantage over the mutant. These results suggest that reverse mutation events occur, with subsequent selection pressure acting in favour of the wild-type virus in the growing plant parts, possibly related to a lower long-distance movement efficiency of the mutant.