Molecular Characterization of Canadian Populations of Potato Cyst
Total Page：16
File Type：pdf, Size：1020Kb
Can. J. Plant Pathol. (2010), 32(2): 252–263 Soilborne pathogens/Agents pathogènes telluriques MolecularTCJP characterization of Canadian populations of potato cyst nematodes, Globodera rostochiensis and G. pallida using ribosomal nuclear RNA and cytochrome b genes M.Potato cyst nematodes MADANI1, S. A. SUBBOTIN2, L. J. WARD1, X. LI1 AND S. H. DE BOER1 1Canadian Food Inspection Agency, Charlottetown Laboratory, 93 Mount Edward Road, Charlottetown, PE C1A 5T1, Canada 2Plant Pest Diagnostics Center, California Department of Food and Agriculture, 3294 Meadowview Road, Sacramento, CA 95832-1448, USA (Accepted 10 January 2010) Abstract: The mitochondrial cytochrome b gene (cytb), the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rRNA gene and D2-D3 expansion segments of the 28S rRNA gene were amplified, sequenced and used to characterize several populations of potato cyst nematodes, Globodera pallida and G. rostochiensis, collected from different areas in Canada. Diagnostic PCR-ITS-RFLP profiles with three restriction enzymes are provided for identification of both species. Sequences of ITS rRNA and cytb genes were compared with those in Genbank of other potato cyst nematode populations originating from Europe, South America, USA, Australia and New Zealand. The ITS rRNA sequences of Canadian G. rostochiensis were similar to those of all previously sequenced populations of this species. Sequence divergence of ITS rRNA for G. rostochiensis varied from 0 to 1.6%, whereas for G. pallida sequence divergence among populations reached 1.95%. Sequence and phylogenetic analysis of cytb and ITS rRNA genes using Bayesian inference revealed that Canadian G. pallida is almost identical to European and USA populations and formed a large clade with all these populations on the phylogenetic trees. The present molecular result with cytb confirmed the hypothesis that there are possibly several sibling species within G. pallida. Our study also supports a previously proposed hypothesis regarding introduction of G. pallida from a restricted area in Peru, firstly into Europe with subsequent spread to other continents including North America. Our sequence analysis revealed that several newly obtained sequences cannot be translated into functional cytb protein, because point indels disrupt the reading frame. Poly(T) variation in mtDNA genes in G. pallida might be explained by post-transcriptional editing mechanisms in Globodera mitochondria as well as by errors during PCR amplification of mononucleotide repeats within these genes. Keywords: molecular diagnostics, mitochondrial DNA, potato cyst nematode Downloaded By: [Agriculture & Agri-Food Canada] At: 12:37 6 July 2010 Résumé: Le gène mitochondrial cytochrome b (cytb), la région de l’espaceur transcrit interne (ITS1-5.8S-ITS2) du gène rARN et les segments du gène rARN 28S ont été amplifiés et utilisés pour caractériser plusieurs populations de nématodes à kystes de la pomme de terre, Globodera pallida et G. rostochiensis, collectés dans différentes régions du Canada. Les profils de diagnostics obtenus par PCR, IT et RFLP, traités avec trois enzymes de restriction, servent à identifier les deux espèces. Les séquences d’ITS des gènes rARN et cytb ont été comparées à celles de la GenBank ainsi qu’avec d’autres populations de nématodes à kystes de la pomme de terre provenant d’Europe, d’Amérique du Sud, des États-Unis, d’Australie et de Nouvelle-Zélande. Les séquences d’ITS du gène rARN de la souche canadienne de G. rostochiensis étaient semblables à celles de toutes les populations préalablement séquencées de cette espèce. La divergence dans les séquences de l’ITS rARN pour G. rostochiensis a varié de 0 à 1,6 %, tandis que, pour G. pallida, la divergence dans les séquences a atteint 1,95 %. L’analyse phylogénétique et celle de la séquence des gènes cytb et ITS rARN, basée sur l’inférence bayésienne, ont révélé que la souche canadienne de G. pallida est presque identique à l’européenne et à l’américaine, et formait une large variante sur les arbres phylogénétiques. Ces résultats découlant de l’analyse moléculaire avec cytb confirment l’hypothèse qu’il existe probablement plusieurs espèces sœurs chez G. pallida. Notre étude soutient également une hypothèse précédente qui avait été proposée en ce qui à trait à l’introduction de G. pallida provenant d’une région restreinte du Pérou, d’abord en Europe, puis de là, sur les autres continents, y compris l’Amérique du Nord. Notre analyse des séquences a Correspondence to: M. Madani. E-mail: [email protected] ISSN 0706-0661 print/ISSN 1715-2992 online © Her Majesty the Queen in Right of Canada (Canadian Food Inspection Agency). All rights reserved. Use without permission is prohibited. DOI: 10.1080/07060661003740033 Potato cyst nematodes 253 révélé que plusieurs nouvelles séquences ne peuvent être traduites en protéine cytb fonctionnelle parce que les indels perturbent le cadre de lecture. La variation Poly(T) dans les gènes de l’ADN mitochondrial de G. pallida pourrait être expliquée par les mécanismes d’édition post-transcriptionnelle dans les mitochondries de Globodera ainsi que par des erreurs qui se produisent durant l’amplification PCR des microsatellites dans ces gènes. Mots clés: ADN mitochondrial, diagnostics moléculaires, nématode à kystes de la pomme de terre Introduction (A. Boucher, personal communication). In Canada, G. pallida has only been found in the Botwood area of Potato cyst nematodes (PCN) Globodera pallida (pale Newfoundland in 1977 (Stone et al., 1977) and appears potato nematode) and G. rostochiensis (yellow or golden to be restricted to this area, although a field plot at the potato nematode) are considered among the most Avondale research sub-station of Agriculture and Agri-Food economically important nematode pests of potatoes and Canada was artificially inoculated from the Botwood are the subject of strict quarantine regulations in many sample with this species in order to test resistance of potato countries. More than 12% of average worldwide potato accessions to PCN (S. Wood, personal communication). yields have been reported as lost due to PCN (Bates et al., An integrated approach to PCN management is usually 2002). Annual costs due to these nematodes were estimated based mainly on chemical control, crop rotation and resist- at £50 million in the UK (Jones & Perry, 2004) and €440 ant cultivars (Trudgill et al., 1987). However, the two million in the European Union (Ryan et al., 2000). PCN species can react differently to control measures. In It is generally accepted that PCN originated from the field experiments, Whitehead et al. (1984) found that Andean region of South America, where these nematodes oxamyl controlled G. pallida less effectively than G. ros- parasitize wild potatoes and other Solanum species tochiensis. Presently, there is very little potato germplasm (Evans & Stone, 1977). From South America, PCN has available that is fully resistant to G. pallida, whereas a spread as the result of human activity into many regions high level of germplasm resistance is available for G. ros- of the world. Although potatoes were introduced into tochiensis. Thus, rapid identification of the PCN species Europe in about 1570, it is thought that PCN probably is critical for planning control measures and implement- came with tubers from South America into Europe only ing rational regulatory decisions. around 1850 (Evans et al., 1975). In Central and North DNA-based techniques developed during the last dec- America, both species of PCN have been reported in ades provide an attractive solution to problems associated Panama, Costa Rica, the USA, Canada, and Mexico, but with identification of nematodes. Comparisons of PCR- in these countries, PCN still has a rather restricted distri- ITS-RFLP profiles and sequences of the rRNA genes bution with small infested areas compared to the wide facilitate reliable and rapid identification of PCN and their infestations found in European countries. In the USA, differentiation from each other and closely related cyst G. rostochiensis was first found in 1941 in a potato field nematode species (Ferris et al., 1995; Thiéry & Mugniéry, on Long Island, New York (Cannon, 1941), and later in 1996; Orui, 1997; Blok et al., 1998; Subbotin et al., 2000; Steuben county, western New York (Spears, 1968) and Downloaded By: [Agriculture & Agri-Food Canada] At: 12:37 6 July 2010 Sirca & Urek, 2004; Madani et al., 2005, 2008; Uehara Delaware (Spears, 1969), whereas G. pallida was discov- et al., 2005; Skantar et al., 2007). Due to a high copy ered only recently in Idaho (Hafez & Sundararaj, 2007). number in individual cells, lack of recombination and In Canada, G. rostochiensis was first found in 1962 in strict maternal inheritance, mitochondrial genes (mtDNA) Newfoundland, and while it does not occur in significant can also be used as excellent markers in addition to the agricultural areas, G. rostochiensis was subsequently ITS rRNA for diagnostics and the study of phylogenetic shown to be present in home gardens in eastern and relationships. Recently, mtDNA has been used to study southern regions of the province near St. John’s and genetic relationships among Peruvian populations of Conception Bay (Farstad, 1962; Olsen & Mulvey, 1962; G. pallida and to identify the origin of Western European Morris, 1971). In the western part of the country, an populations of this species (Picard et al., 2007; Plantard infestation of G. rostochiensis was first reported in the et al., 2008). Using mtDNA gene, cytochrome b (cytb) Saanich Peninsula, Vancouver Island, British Columbia sequences, Plantard et al. (2008) showed that G. pallida and identified as pathotype R1A (Stone et al., 1977). In presently distributed in Europe was derived from a single addition to the ongoing persistence of G. rostochiensis on restricted area in the extreme south of Peru, located Vancouver Island (Rott et al., 2008), this species was between the north shore of the Lake Titicaca and Cusco. also recently found in potato fields in the Saint Amable This could possibly be considered the first case where the area of Quebec (Sun et al., 2007) and in Alberta M.