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

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1 DOI /s A new haplotype of Candidatus Liberibacter solanacearum identified in the Mediterranean region Warrick R. Nelson & Venkatesan G. Sengoda & Ana O. Alfaro-Fernandez & Maria I. Font & James M. Crosslin & Joseph E. Munyaneza Accepted: 17 October 2012 # US Government 2012 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 W. R. Nelson The New Zealand Institute for Plant & Food Research Ltd., Private Bag 4704, Christchurch 8140, New Zealand V. G. Sengoda : J. E. Munyaneza (*) Yakima Agricultural Research Laboratory, United States Department of Agriculture-Agricultural Research Service, Wapato, WA 98951, USA Joseph.Munyaneza@ars.usda.gov A. O. Alfaro-Fernandez : M. I. Font Grupo Virologia Vegetal, Instituto Agroforestal Mediterraneo, Universidad Politecnica de Valencia, Cno. Vera s/n, Valencia, Spain J. M. Crosslin Vegetable and Forage Crops Research Unit, United States Department of Agriculture-Agricultural Research Service, Prosser, WA 99350, USA 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. Keywords Candidatus Liberibacter solanacearum. Haplotype. Psyllids. Bactericera cockerelli. Bactericera trigonica. Trioza apicalis. Zebra chip. Carrot disease Candidatus Liberibacter solanacearum (also known as Ca. L. psyllaurous), a phloem-limited and Gramnegative bacterium spread from infected to healthy plants by psyllid insect vectors, is an economically important pathogen of solanaceous crops, carrot and celery (Hansen et al. 2008; Liefting et al. 2009; Secor et al. 2009; Munyaneza 2010, 2012; Munyaneza et al.

2 Table 1 GenBank accession numbers showing geographic source and haplotype designation for each gene region. This table extends the number of sequences in haplotype C and adds haplotype D (Nelson et al. 2011). Accessions marked in bold are from this study GenBank accession Source Geographic source Haplotypes Eur J Plant Pathol 16S ISR-23S 50S EU Solanum tuberosum California A A EU Solanum lycopersicon New Zealand A FJ Solanum tuberosum Colorado B FJ Solanum tuberosum Colorado B FJ Solanum tuberosum Mexico B GU Daucus carota Finland C HM Daucus carota Finland C JX Daucus carota Finland C GU Daucus carota Finland C JX Daucus carota Finland C JN Daucus carota Norway C JX Daucus carota Norway C JN Daucus carota Norway C JX Daucus carota Norway C JN Trioza apicalis Sweden C JN Daucus carota Sweden C JX Daucus carota Sweden C JN Trioza apicalis Sweden C JN Daucus carota Sweden C JX Daucus carota Sweden C HQ Daucus carota Canary Islands D HQ Daucus carota Canary Islands D HQ Daucus carota Canary Islands D HQ Daucus carota Canary Islands D HQ Bactericera trigonica Canary Islands D HQ Daucus carota Canary Islands D HQ Daucus carota Canary Islands D HQ Daucus carota Canary Islands D HQ Daucus carota Canary Islands D HQ Bactericera trigonica Canary Islands D HQ Daucus carota Spain D HQ Daucus carota Spain D HQ Daucus carota Spain D JX Daucus carota Spain D HQ Daucus carota Spain D HQ Daucus carota Spain D HQ Daucus carota Spain D JX Daucus carota Spain D JX Bactericera cockerelli Oregon A JX Bactericera cockerelli Washington A JX Bactericera cockerelli Washington A JX Bactericera cockerelli Washington A

3 Table 1 (continued) GenBank accession Source Geographic source Haplotypes 16S ISR-23S 50S JX Solanum tuberosum Idaho A JX Solanum tuberosum Idaho A JX Solanum tuberosum Idaho A JX Solanum tuberosum Oregon A JX Solanum tuberosum Oregon A JX Solanum tuberosum Oregon A JX Solanum tuberosum Washington A JX Solanum tuberosum Washington A JX Solanum tuberosum Washington A 2010b; Buchman et al. 2011; EPPO 2012). Ca. L. solanacearum (referred to as Lso hereafter) was first identified in 2008 (Hansen et al. 2008; Liefting et al. 2008) and shown to be associated with zebra chip disease of potato, which was linked to the tomato/potato psyllid Bactericera cockerelli (Šulc) (Hemiptera: Triozidae) for the first time in 2007 by Munyaneza et al. (2007a, b). First reported in Mexico in 1994, zebra chip was documented as causing serious economic damage in parts of southern Texas in 2000 (Secor et al. 2009). The disease is widespread in the central and western United States, Mexico, Central America, and New Zealand (Munyaneza et al. 2007a, b;liefting et al. 2009; Munyaneza et al. 2009a; Secor et al. 2009; Crosslin et al. 2010; Munyaneza 2010; Rehman et al. 2010; Crosslin et al. 2012a, b; Munyaneza 2012). This liberibacter species also severely affects tomato, pepper, eggplant, and tamarillo crops (Liefting et al. 2009; Munyaneza et al. 2009b, c; Brown et al. 2010). Recently, Munyaneza et al. (2010a, b) detected Lso in carrots affected by the carrot psyllid Trioza apicalis Förster (Hemiptera: Triozidae) in Finland, which constitutes the first report of liberibacter in Europe and Lso in a non-solanaceous species. Following these findings, well defined haplotypes of Lso were established (Nelson et al. 2011). These Lso haplotypes were designated A, B and C. LsoA and LsoB are described from North and Central America and New Zealand, and are present in solanaceous crops and associated with B. cockerelli. LsoC is present in Finland and described from carrot crops in association with T. apicalis. These haplotypes are described from single nucleotide polymorphisms (SNPs) and synchronous across three gene regions, namely the available partial sequences of 16S, 16S/23S intergenic spacer region (ISR) and 50S rrna genes. Haplotypes represent discrete and readily identified genetic changes that can be used to assist in epidemiological studies and offer a means of separating and defining slight differences in the bacteria which are not readily defined, for example tracking movement over time or slight differences in their impact on fitness of the host organisms. Subsequently, carrot crops in Norway and Sweden have also been reported exhibiting similar symptoms to those in Finland and Lso was found associated with both the carrots and T. apicalis (Munyaneza et al. 2012a, b). More recently, symptomatic carrot crops associated with infestations of the carrot psyllid Bactericera trigonica Hodkinson (Hemiptera: Triozidae) in the Canary Islands and mainland Spain have also proven positive for Lso (Alfaro-Fernández et al. 2012a, b). Also, Lso was recently reported on celery crops in Spain (EPPO 2012). The 16S rdna sequence from this Spanish material suggested the existence of a fourth Lso haplotype. Therefore, the objective of the present study was to determine the SNPs across the same genes as used in the earlier study (Nelson et al. 2011) to describe this putative fourth Lso haplotype. Similarly to the study by Nelson et al. (2011), the partial 16S and 50S rdna sequences from the present study were downloaded from GenBank (NCBI). The sequences, plus representative sequences of the three previously described Lso haplotypes, were aligned within gene regions using ClustalX and the SNPs identified visually and described as previously by Nelson et al. (2011). The 16S and 50S sequences from

4 Table 2 Haplotypes and SNP differences with dbsnp ss# references. The reference sequence for the 16S and 23S genes is EU , and for 50S genes is EU Nucleotide numbers count from the beginning of the reference sequence, haplotypes A, B and C as previously described (Nelson et al. 2011) Description SS# Gene region Haplotypes A B C D g.116c>t S C C C T g.212t>g T G T T g.581t>c T C T T g.1049a>g A A G G g.1073g>a G G G A g.1742a>g ISR-23S A A A G g.1748c>t C C C T g.1858_1859insg G G g.1859_1860inst T g.1860_1861delt T T T g.1920t>c T T C T g.1943g>a G A G G g.2055c>t C T C C g.2081g>a G G G A g.2218g>a G A G G g.2260c>t C T C C g.583g>c S G G C G g.622a>g A A A G g.640c>t C C T C g.669g>c G C G G g.689c>t C C C T g.691g>t G T T G g.700a>g A A A G g.712g>t G T G G g.749c>a C C C A g.780_781insa A A g.786g>a G A G G g.850t>c T T T C g.909t>c T C C C g.920t>c T C C C g.920_921instgt TGT g.955g>t G G T G g.987t>g T G G G g.993a>g A A G A g.1041g>a G A A G g.1049a>g A G A A g.1072c>t C C C T g.1107g>a G A G G g.1111_1112insc C g.1122g>a G A A A g.1143g>a G A G G Norway and Sweden exhibited the same SNPs described for LsoC, with a single exception, the insertion at g.1111_1112insc on the 50S sequence is not represented.

5 The lack of 16S/23S ISR sequences and the unexpected discrepancy at a single locus between the Scandinavian isolates resulted in the decision to resequence material from the original Finnish investigation (Munyaneza et al. 2010a, b), as well as sequencing the Spanish material for the 16S/23S ISR as previously described by Nelson et al. (2011). Liberibacter from potato psyllids collected in Washington, USA was also sequenced to determine the haplotype of Lso in this region, following the first zebra chip outbreak in the Pacific Northwest late in 2011 (Crosslin et al. 2012a, b; Munyaneza 2012). Table 1 details the GenBank accessions and associated metadata used in this study, including representative examples of LsoA and LsoB. Table 2 is duplicated from the earlier study by Nelson et al. (2011) with the addition of descriptions of SNPs for this fourth Lso haplotype, designated LsoD.LsoDis currently represented from Spain and the Canary Islands, described from carrot crops and associated with the carrot psyllid B. trigonica. The re-sequenced Finnish material did not repeat SNP on the 50S gene indicating an insertion event (Table 2). The non-repeat finding of this single SNP does not change the evidence for LsoC across Finland, Norway, and Sweden, but does raise doubt that this specific SNP is common in the region. Somewhat unexpectedly during this study, Lso from psyllids collected in Washington and Oregon turned out as LsoA, a haplotype more prevalent in southwestern U.S., Mexico, and New Zealand. Previously, psyllids reported from Washington carried LsoB commonly found in south and central U.S. (Nelson et al. 2011), but these insects had come from a laboratory colony maintained at the USDA-ARS facility in Wapato originally collected from Texas. More recently, potato crops in Washington, Oregon, and Idaho with ZC symptoms have been found to be positive for Lso (Crosslin et al. 2012a, b) and also belongs to the A haplotype (this study Table 1). Psyllids collected from Washington potato crops carried LsoA (this study) and are also reported to be a different haplotype of psyllid from those commonly found west and east of the Rocky Mountains (Liu and Trumble 2007; Swisher et al. 2012). A cladogram (Fig. 1) on the 16S rrna gene indicates a genetic closeness of LsoA with LsoC. This is somewhat surprising considering their large geographic separation and the differences in both plant and insect hosts. In spite of the genetic separation between LsoA and LsoB, no known biological difference has yet been reported although they are both associated with zebra chip of potatoes and vectored by the psyllid B. cockerelli in North and Central America. The presence of Lso in both North and Central America and Europe cannot be explained by an incursion event, largely because of the difference in both psyllid species and psyllid plant hosts on either side of the Atlantic, in contrast to the obvious recent incursion event in New Zealand, possibly from western United States (Thomas et al. 2011). There is no suggestion that any of the three psyllid species are not in their native ranges, and similarly for the plant hosts of these psyllids (at least closely related native species if not the specific crop plant). The genetic similarity across these haplotypes presents an obvious anomaly considering they are a subset of the same species yet are geographically distant and have such different plant and insect hosts. Fig. 1 Cladogram of Candidatus Liberibacter solanacearum haplotypes on the 16S gene segment (1022 bp) with Ca. Liberibacter asiaticus (Las) as the out group, using the UPGMA algorithm. GenBank sequence references are indicated

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