Pepino mosaic virus: an endemic pathogen of tomato crops

Owing to their large population size and short generation time, viruses generally have a huge potential to evolve and adapt under natural selection pressure. Despite tremendous efforts in human, animal and plant health management, viral diseases remain difficult to control and eradicate. Moreover, existing control strategies are compromised by the continuous emergence of new viruses. In Chapter 1 emerging viruses of tomato crops are reviewed. This includes Pepino mosaic virus (PepMV), a Potexvirus with a single stranded RNA genome, a rapidly emerging virus which has become one of the most important viral diseases in tomato production worldwide over the recent years. Infection by PepMV can cause a broad range of symptoms on tomato plants, of which especially the typical fruit marbling can lead to significant economical losses. Presently, five PepMV genotypes (EU, LP, CH2, US1 and US2) have been described worldwide, three of which (EU, LP and US2) have previously been reported to occur in Europe. As nature and severity of PepMV symptoms are highly variable, economical damage caused by PepMV is difficult to assess and the identification of factors contributing to symptom severity is warranted. In Chapter 2 the genetic diversity of the PepMV population in Belgian greenhouses is studied and related to the symptom development in tomato crops. Previously, no correlation has been found between different PepMV genotypes and the symptomatology of infected plants. A novel assay based on restriction fragment length polymorphism (RFLP) was developed to discriminate the different PepMV genotypes. Both RFLP and sequence analysis revealed the occurrence of two genotypes, the EU genotype as well as the CH2 genotype, within the tomato production in Belgium. Surprisingly, a clear dominance of the CH2 genotype in the Belgian PepMV population was found, although this genotype has previously not been found in commercial tomato production. Whereas no differences were observed in symptom expression between plants infected by one of the two genotypes, co-infection with both genotypes resulted in more severe PepMV symptoms. Furthermore, our study revealed that PepMV recombinants frequently occur in such mixed infections. So far, it remained unclear whether different PepMV isolates can cause differential symptom severity. Therefore, PepMV symptomatology of different isolates was studied in Chapter 3. Based on the survey described in Chapter 2, four isolates that differed in symptom expression in the crop of origin were selected for greenhouse trials. The selected isolates were inoculated onto tomato plants grown in separate plastic tunnels. PepMV symptom development was assessed regularly and extensive sampling followed by ELISA analyses, genotyping and nucleotide sequencing was performed to study viral presence and variation in PepMV sequences throughout the trial period. Two isolates (EU mild and CH2 mild) that were selected based on mild symptom expression in the crop of origin caused only mild symptoms in the trial, while two other isolates (CH2 aggressive and EU+CH2) that were selected for severe symptom display, caused considerably more severe symptoms. Sequence identity between the mild and the aggressive CH2 isolates was as high as 99.4%. Results of this study show that differential symptom expression can, at least partially, be attributed to the PepMV isolate, which may be related to minor differences at the nucleotide level between isolates. In Chapter 4, seed transmission of PepMV in tomato is demonstrated. Fruit was harvested from the greenhouse trials described in Chapter 3 and more than 100,000 seeds were extracted and cleaned using an enzymatic treatment without disinfection. Infection assays using indicator plants confirmed the presence of infectious virus particles on the seeds. In the framework of a European project, seeds were distributed to 10 different laboratories in three separate batches, and germinated for seedling analyses by ELISA. In total over 87,000 plants were tested, and 23 PepMV-infected plants were detected, indicating an overall transmission rate of 0.026 %. Our results clearly show that PepMV can be transmitted from contaminated seeds to seedlings, highlighting the risk of using seeds from PepMV-infected plants, and revealed the potential for seed transmission to contribute to spread of PepMV. In Chapter 5, the potential of three mild PepMV isolates, belonging to the CH2, EU and LP genotypes, to protect a tomato crop against the aggressive CH2 isolate (Chapter 3) as the challenge isolate, was assessed in greenhouse trials. After challenge infection, enhanced symptom display was recorded in plants that were pre-inoculated with a protector isolate that belonged to a different genotype (EU, LP) than the challenge isolate. A quantitative genotype-specific TaqMan assay revealed that in these plants, the accumulation of the challenge isolate only temporarily slowed down. By contrast, efficient cross-protection was obtained using the mild isolate of the CH2 genotype, and in this case the challenge isolate was barely detectable in the pre-inoculated plants. These results suggest that the interaction between PepMV isolates largely depends on RNA sequence homology and that post-transcriptional gene silencing plays an important role in cross-protection. As plant viruses are obligate intracellular parasites that hijack host cellular functions and resources for their replication and movement, they generally induce a wide variety of alterations in host gene expression and cell physiology. In Chapter 6, we used a custom-designed Affymetrix tomato GeneChip array that contains probe sets to interrogate over 22,000 tomato transcripts to study transcriptional changes in response to inoculation with the highly similar (99.4% nucleotide sequence identity) mild and aggressive CH2 isolates that are characterized in Chapter 3. Interestingly, our results show that both isolates induce differential transcriptomic responses in the tomato host despite accumulation to similar viral titers. PepMV inoculation resulted in an extensive transient repression of host genes which clearly affected primary metabolism. Especially the defense response intensity was higher upon inoculation with the aggressive isolate, and defense was mediated by salicylic acid signaling rather than by jasmonate signaling. Our results furthermore show that PepMV differentially regulates the RNA silencing pathway, suggesting a role for PepMV-encoded silencing suppressors, and the ubiquitination pathway. In addition, perturbation of pigment biosynthesis, as monitored by differential regulation of the flavonoid/anthocyanin and lycopene biosynthesis pathways, was monitored, which can be associated with the typical PepMV-induced marbling of tomato fruit. Finally, Chapter 7, the general discussion, is a PepMV pathogen profile in which the results obtained in this work are discussed and integrated into a review on the current knowledge on this highly successful pathogen of tomato crops