ABSTRACT
      
Nowadays Pepino mosaic virus (PepMV) is an important tomato-infecting virus. Ten years after be discovered in the Netherlands, PepMV is widely and rapidly spread throughout the tomato crops worldwide, where sometimes produces important economical losses. The high variability in symptom expression and large genetic diversity of this virus complicates the diagnosis, but the effective mechanical transmission of PepMV between plants with contaminated tools, hands, clothing, and by direct plant-to-plant contact, complicates the control and eradication in the affected areas. Contact between healthy and infected plants as a result of routine handling during cultivation of the crop constitutes the main cause for transmission within a plot. However, the mechanism for long-distance dissemination of this virus may be the contaminated seeds, or fruits.
      Due to the high risk of the seed transmission of PepMV for the world tomato trade, we considered in first place to check the possibility of seed transmission. Rates of seed transmission for PepMV were estimated in seedlings grown from seeds obtained from symptomatic tomato fruits of plants naturally infected with the virus. The proportion of seeds infected with PepMV was at least 25% as estimated from enzyme-linked immunosorbent assay (ELISA) analysis of grouped seeds. The seeds from symptomatic fruits were planted, and seedlings at the cotyledon and transplant stage were assayed for PepMV by ELISA. Three of 168 seedlings grown from infected seeds were PepMV-positive, corresponding to a seed-to-seedling transmission rate of 1.84%. Various tomato seed treatments were evaluated for their ability to prevent seed transmission of PepMV. This virus was largely eradicated by immersing the seeds in 10% trisodium phosphate for 3 h. Although heat treatments of 24 h at 80°C and 48 h at 74°C eliminated PepMV in seedlings, these treatments did not eradicate the virus in whole seeds. The three treatments did not adversely affect seed germination. The results suggest that trisodium phosphate can be used to eradicate PepMV in tomato seed without hindering germination.
      On the other hand, the population structure of PepMV was not determined in any country where the virus was present. We analyzed the genetic diversity of the Spanish population of PepMV, which has caused severe epidemics in tomato in Spain since 2000. Isolates were characterized by the nucleotide sequence of the triple gene block (TGB) and coat protein gene (CP) and, for a subset of isolates, a part of the RNA-dependent RNA polymerase (RdRp) gene. The full-length sequence of the genomic RNA of a Solanum muricatum isolate from Peru also was determined. In spite of high symptom diversity, the Spanish population of PepMV mostly comprised highly similar isolates belonging to the strain reported in Europe (European tomato strain), which has been the most prevalent genotype in Spain. The Spanish PepMV population was not structured spatially or temporally. Also, isolates highly similar to those from nontomato hosts from Peru (Peruvian strain) or to isolate US2 from the United States (US2 strain) were detected at lower frequency relative to the European strain. These two strains were detected in peninsular Spain only in 2004, but the Peruvian strain has been detected in the Canary Islands since 2000. These results suggest that PepMV was introduced into Spain more than once. Isolates from the Peruvian and US2 strains always were found in mixed infections with the European tomato strain, and interstrain recombinants were detected. The presence of different strains of the virus, and of recombinant isolates, should be considered for the development of control strategies based on genetic resistance.
      Simultaneously with the first outbreaks of PepMV, a new tomato disorder was appeared in Spain. In the affected tomato plants, a sudden wilt was observed after a sudden change in temperature and light intensity. When the disorder is severe, the total plant “Collapse” occurs. Statewide surveys of commercial tomato crops were conducted in Spain from 2000 to 2005 to obtain information on the incidence and the causative agents of “Collapse”. Laboratory assays of wilted plants revealed that wilting was caused by a dual infection of PepMV and Olpidium virulentus. PepMV was detected in 85% of the plants analysed and O. virulentus was found in 88% of the feeder roots examined. Both pathogens were found in 86% of completely examined plants and occurred together in all the fields surveyed. Other pathogens were detected very rarely in collapsed tomatoes. To verify the implications of both pathogens in the tomato “Collapse”, two greenhouse trials were performed. We reproduced the disorder by simultaneous plant inoculation with four PepMV and two O. virulentus isolates, but a sudden change in the temperature regime took place. The collapse incidence varied from approximately 6.25% in the summer growing season to 56.25% in the winter growing season, both under controlled conditions. No significant changes in the genomic sequences were found between the PepMV isolates showing typical PepMV symptoms and those isolated from wilted plants. Collapse PepMV isolates did not cluster together in a specific genotype group. The results indicate that Collapse is well-established in tomato production areas, and that PepMV and O. virulentus pose a very serious challenge to tomato producers.
      More recently, a severe disease of greenhouse-grown tomato in Spain was observed. The new disorder is characterised by expanding necrotic patches on stems, leaves and green fruits or by expanding necrotic spots on the leaves of chlorotic tomato plants. Thus in order to finish the present work, we try to identify and characterize the causal agent of this necrosis. Two strains of PepMV were identified and described as the causal agents of necrosis on the basis of host range and symptomatology. Phylogenetic analysis, based on two complete regions of the genomes; the TGB and the CP gene; and a portion of the RdRp gene, revealed that Spanish necrotic isolates share high nucleotide sequence identity with isolates of the EU and CH2 genotypes.