María Isabel Font

A new haplotype of "Candidatus Liberibacter solanacearum" identified in the Mediterranean region

Abstract“Candidatus Liberibacter solanacearum”, a phloem-limited and Gram-negative bacterium that is spread from infected to healthy plants by psyllid insect vectors, is an economically important pathogen of solanaceous and carrot crops in the Americas, New Zealand and Europe. Three haplotypes of “Ca. L. solanacearum” have previously been described, two (LsoA and LsoB) in relation to solanaceous crops in the Americas and New Zealand and the third (LsoC) to carrots in Finland. Herein, we describe a fourth haplotype of this ‘Candidatus Liberibacter’ species (LsoD), also associated with carrots, but from Spain and the Canary Islands and vectored by the psyllid Bactericera trigonica. In addition, LsoC was confirmed in carrot and psyllid samples recently collected from Sweden and Norway. Phylogenetic analysis of the 16S rRNA gene suggests that two of the haplotypes, one in the Americas and the other in northern Europe are closer to each other in spite of a large geographic separation and host differences. Furthermore, during this study, potatoes with symptoms of zebra chip disease recently observed in potato crops in Idaho, Oregon and Washington states were analyzed for haplotype and were found to be positive for LsoA. This liberibacter haplotype was found in psyllids associated with the diseased potato crops as well. This finding contrasts with an earlier report of LsoB from psyllids in Washington which came from a laboratory colony originally collected in Texas.

FIRST REPORT OF SOUTHERN TOMATO VIRUS IN TOMATO IN THE CANARY ISLANDS, SPAIN

In October 2006, tomato plants with torrado disease were sampled in Spain. In a sample of cv. Mariana, originating from Gran Canaria, Tomato torrado virus (ToTV, genus Torradovirus) was detected (isolate GCN06; Alfaro-Fernandez et al., 2010). In 2013, the sample was further analysed using next-generation sequencing (NGS). Total RNA, extracted from ca. 150 mg of leaf tissues, was used for an RNA library suitable for Illumina HiSeq paired end sequencing. A de novo assembly (using CLC Genomic Workbench v.6.5) resulted in 1380 sequence contigs. BLAST searches revealed the presence of ToTV and Tomato chlorosis virus (ToCV, genus Crinivirus). One contig (3427 nts, 8901 reads), showed 99.85% identity with Southern tomato virus (STV, genus Amalgavirus), isolate Mexico-1 (GenBank accession No. EF442780), a dsRNA seed-transmitted virus (Sabanadzovic et al., 2009). Reference assembly with the Mexico-1 sequence resulted in a partial sequence of 3420 nts (GenBank KJ174690). The presence of STV in sample GCN06 was verified in RT-PCR using the specific primers STV-fw and STV-rev (Candresse et al., 2013) and sequencing of the obtained amplicon. A new sampling in May 2014 in Gran Canaria revealed the presence of STV in six out of seven tomato samples of cv Mariana. All these samples were co-infected with Pepino mosaic virus (PepMV, genus Potexvirus), Tomato spotted wilt virus (TSWV, genus Tospovirus), and Tomato yellow leaf curl virus (TYLCV, genus Begomovirus). Because STV was generally found in samples co-infected with other viruses, its symptomatology in tomato remains unclear. To our knowledge, this is the first report of STV in the Canary Islands and Spain.

Haplotypes of ‘Candidatus Liberibacter solanacearum’ identified in Umbeliferous crops in Spain

Abstract‘Candidatus Liberibacter solanacearum’ is a phloem-limited Gram-negative bacterium that causes serious damage to different crops of the botanical families Solanaceae and Apiaceae. Five haplotypes have been described: LsoA and LsoB are present in solanaceous crops in America and vectored by the tomato/potato psyllid Bactericera cockerelli; LsoC affects carrots from Northern and Central Europe, and is transmitted by the carrot psyllid Trioza apicalis; haplotypes LsoD and LsoE are present in Southern Europe and Morocco in carrot and celery, and are associated with the psyllid Bactericera trigonica. Thirty-four ‘Ca. L. solanacearum’ isolates were collected in six different regions of Spain from distinct Apiaceae hosts (carrot, celery, parsley and parsnip) in eight consecutive years and were analysed. Their haplotypes were determined by a sequence analysis of 16S ribosomal RNA, the 16S–26S ribosomal RNA intergenic spacer, and the 23S ribosomal RNA and rplJ and rplL genes. Both haplotypes LsoD and LsoE were found across Spain, and no host specificity appeared between these two haplotypes. This is the first report of ‘Ca. L. solanacearum’ associated with parsley and parsnip.

EVALUATION OF PCR AND NON-RADIOACTIVE MOLECULAR HYBRIDIZATION TECHNIQUES FOR THE ROUTINE DIAGNOSIS OF TOMATO LEAF CURL NEW DELHI VIRUS, TOMATO YELLOW LEAF CURL VIRUS AND TOMATO YELLOW LEAF CURL SARDINIA VIRUS

The begomovirus Tomato leaf curl New Delhi virus (ToLCNDV) has been reported as a causal agent of leaf curl disease in tomato and other solanaceous crops and, more recently, affecting different cucurbitaceous crops. ToLCNDV was first detected in Asia and recently in Europe, in 2013. In the present analysis, we have evaluated the PCR and the non-radioactive nucleic acids spot hybridization (NASH) techniques together with two nucleic acids extraction protocols, for the routine diagnosis of ToLCNDV and its discrimination from the closely related Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV). A protocol, designed to extract only the DNA, gave the best results with the PCR technique meanwhile the use of silica, which favors total nucleic acids extraction, was the best extraction protocol for the NASH. All allowed the specific detection of ToLCNDV but only some of the general begomovirus primers allowed the detection of all three viruses. The two ToLCNDV riboprobes analyzed by NASH, targeting the replicase and the coat protein genes, respectively, detected the virus with no cross-reaction with the TYLCV and TYLCSV infected extracts, obtaining a better detection limit with the replicase riboprobe. Direct comparison between the PCR and NASH techniques by the analysis of 42 field samples, revealed a good correlation between the two techniques. In addition, some samples were detected only by NASH due the presence of PCR inhibitors. The use of the PCR and NASH for the routine diagnosis of ToLCNDV is discussed.