In just a few short years, pepino mosaic disease has quickly become endemic in greenhouse tomatoes around the world. Although three genotypes of Pepino mosaic virus (PepMV) were identified in the United States, genetic composition of PepMV in greenhouse tomato crops in North America has not been determined. In this study, genetic variability and population structure of PepMV were evaluated through nucleotide sequence comparison and phylogenetic analysis of two genomic regions (helicase domain and TGB2-3) derived from 91 cDNA clones that were derived from 31 field-collected samples. These samples were collected from several major greenhouse tomato facilities in five states in the United States and two provinces in Canada. All four major genotypes of PepMV (EU, US1, US2, and CH2) were found in North America. Three distinct genotypes (EU, US1, and US2) were found in mixed infection in samples collected from Arizona and Colorado, two genotypes (EU and CH2) in Texas, and a single genotype (EU) in Alabama and California and the provinces of British Columbia and Ontario in Canada. The complexity of population genetics of PepMV in the United States poses an additional challenge to the greenhouse tomato industry because a tomato cultivar with durable resistance to multiple genotypes of PepMV may be harder to develop.
BackgroundPepino mosaic, once an emerging disease a decade ago, has become endemic on greenhouse tomatoes worldwide in recent years. Three distinct genotypes of Pepino mosaic virus (PepMV), including EU, US1 and CH2 have been recognized. Our earlier study conducted in 2006–2007 demonstrated a predominant EU genotype in Canada and United States. The objective of the present study was to monitor the dynamic of PepMV genetic composition and its current status in North America.ResultsThrough yearly monitoring efforts in 2009–2012, we detected a dramatic shift in the prevalent genotype of PepMV from the genotype EU to CH2 in North America since early 2010, with another shift from CH2 to US1 occurring in Mexico only two years later. Through genetic diversity analysis using the coat protein gene, such genotype shifting of PepMV in North America was linked to the positive identification of similar sequence variants in two different commercial tomato seed sources used for scion and rootstock, respectively. To allow for a quick identification, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) system was developed and demonstrated to achieve a rapid identification for each of the three genotypes of PepMV, EU, US1 and CH2.ConclusionThrough systemic yearly monitoring and genetic diversity analysis, we identified a linkage between the field epidemic isolates and those from commercial tomato seed lots as the likely sources of initial PepMV inoculum that resulted in genetic shifting as observed on greenhouse tomatoes in North America. Application of the genotype-specific RT-LAMP system would allow growers to efficiently determine the genetic diversity on their crops.
In the summer of 2008, tomato (Solanum lycopersicum) plants in a large greenhouse tomato facility located in Delta, British Columbia, Canada exhibited general stunting, chlorosis, and purple-leaf symptoms that were distinct from those of Pepino mosaic virus (PepMV) (1). Diseased plants were localized mainly in two rows in a section of the greenhouse and produced no fruits or only fruits with reduced size. Leaf samples were collected from four individuals among numerous diseased plants in this greenhouse. Screening samples by ELISA, PCR, or reverse transcription (RT)-PCR for PepMV, Tomato spotted wilt virus, Tomato yellow leaf curl virus, Tomato torrado virus, Tomato apex necrosis virus, and Begomovirus, Tobamovirus, and Pospiviroid species showed that all four plants had a mixed infection of both PepMV and a pospiviroid. RT-PCR with the pospiviroid-specific primers Pospil-RE and Pospil-FW (3) amplified the expected 196-bp products from these four samples. Each amplicon was cloned into the pCR4-TOPO vector (Invitrogen, Carlsbad, CA) and one individual cDNA clone from each isolate was sequenced. BLASTN analyses of nucleotide sequences of these clones showed 97 to 99% identity to Mexican papita viroid (MPVd) isolates currently in the NCBI Genbank. These four newly identified MPVd isolates were not identical; seven nucleotide substitutions or indels were identified in this region. The full viroid genome was obtained by RT-PCR in isolate VF2 with a new reverse primer MPVd-RE (5′ GATCCCTGAAGCGCTCCT 3′) in combination with the forward primer Pospil-FW (3). Using the same approach as stated above, this amplicon was cloned and sequenced. The nucleotide sequence of the 196-nt amplicon previously amplified and cloned from the isolate VF2 genome was identical to this region in the genomic clone. BLASTN analysis showed that the VF2 genome (GenBank Accession No. FJ824844) had >98% sequence identity to each of nine MPVd isolates (GenBank Accession Nos. L78454 and L78456–L78463), 94% identity to Tomato planta macho viroid (TPMVd) (GenBank Accession No. K00817) and ~80% identity to Tomato chlorotic dwarf viroid (GenBank Accession Nos. EF582392–EF582393). Prior to this find, MPVd had been identified only in papita (Solanum cardiophyllum) in Mexico and is considered a possible ancestor of TPMVd, Potato spindle tuber viroid (PSTVd), and possibly of other PSTVd-group viroids now infecting crop plants (2). The origin of MPVd in this greenhouse facility in Delta, British Columbia is unknown. The infected plants were destroyed by the grower. The pathogenicity of MPVd isolates characterized in this study was not evaluated on tomato because of quarantine regulations governing this viroid in the United States. The identification of MPVd infecting an important agricultural crop (tomato) outside its center of origin in Mexico indicates a potentially important major shift in the epidemiology of MPVd. To our knowledge this is the first report of MPVd from tomato in Canada. References: (1). K.-S. Ling et al. Plant Dis. 92:1683, 2008. (2) J. P. Martinez-Soriano et al. Proc. Natl. Acad. Sci. U.S.A. 93:9397, 1996. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.
Injury to Citrus Fruit Methods of Sampling 11 GENERAL INTRODUCTION CHAPTER I ' BIOLOGICAL STUDIES ON THE CITRUS TREE SNAIL .... water and not on nocturanl conditions. The author wishes to acknowledge the possibility that the snail may be dependent upon free water which develops on the leaf surface. Further studies examining this environmental factor are needed. Section 3. Effect of Snail Movement on Fruit Microbiota Using Scanning Electron Microscopy
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