Molecular and Morphological Characterisation of Species

Nematology, 2011, Vol. 13(3), 295-306

Molecular and morphological characterisation of species within the Xiphinema americanum-group (Dorylaimida: Longidoridae) from the central valley of Chile ∗ Pablo MEZA 1,2, ,ErwinABALLAY 1 and Patricio HINRICHSEN 2 1 Faculty of Agronomy, Universidad de Chile, Avenida Santa Rosa 11315, Santiago, Chile 2 Biotechnology Laboratory, INIA La Platina, Avenida Santa Rosa 11610, Santiago, Chile Received: 7 January 2010; revised: 21 June 2010 Accepted for publication: 21 June 2010

Summary – Species of the Xiphinema americanum-group are among the most damaging nematodes for a diverse range of crops. This group includes 51 nominal species throughout the world. They are very difficult to identify by traditional taxonomic methods. Despite its importance in agriculture, the species composition of this group in many countries, including Chile, remains unknown. In order to identify the species in the central valley of Chile, we studied the morphological, morphometric and molecular diversity of 13 populations. Through classical taxonomic methods two species, X. inaequale and X. peruvianum, were identified with clear differences in the shape of the lip region. The DNA sequences of the ITS of ribosomal genes revealed divergences in the nucleotide sequences of the two species from 7.3% in ITS1 to 14.7% in ITS2. These results confirmed the presence of two distinct species, namely X. peruvianum and X. inaequale, in the northern and southern parts of the central valley of Chile, respectively. PCR-RFLP was developed for rapid species identification of these two species. Keywords – molecular, morphology, morphometrics, taxonomy, Xiphinema californicum, Xiphinema inaequale, Xiphinema peruvianum.

The Xiphinema americanum-group comprises 51 nom- nologies has opened a new spectrum of possibilities in ne- inal species found all over the world (Lamberti et al., matode taxonomy. The growing accumulation of DNA se- 2002, 2004). Species identification is very challenging quences in GenBank has made genetic testing a comple- and often unreliable by traditional methods (Luc et al., mentary classification methodology (Blaxter, 2004; Grif- 1998; Chen et al., 2005). Although several keys have fiths et al., 2006). One of the most used molecular mark- been published (Lamberti & Carone, 1991; Lamberti et ers is the ITS region. The versatility, specificity and han- al., 2000, 2004), ambiguities still persist due to poorly dling ease of the experimental analysis of this genomic defined species boundaries and overlapping morphomet- region have been established since the late 1990s (Cherry rics (Luc & Baujard, 2001). Even the latest polytomous et al., 1997; Powers et al., 1997) and have allowed for key, published by Lamberti et al. (2004), demonstrated the construction of phylogenetic trees, estimation of popu- limited success in the classification of species found in lation diversity and determination of taxonomic relation- Florida (USA) (Gozel et al., 2006) and Serbia (Barsi & ships (Vrain et al., 1992; Cherry et al., 1997; Subbotin De Luca, 2008). Despite these difficulties, there has been et al., 2000; Gozel et al., 2006). Currently, 12 species a strong interest in the study and classification of species of the X. americanum-group have nucleotide sequences in this group. Research performed in Brazil (Oliveira et of ITS fragments in GenBank: X. brevicollum Lordello al., 2004), Taiwan (Chen et al., 2005), the USA (Gozel et & Da Costa, 1961, X. incognitum Southey, 1973, X. dif- al., 2006) and Serbia (Barsi & De Luca, 2008) has demon- fusum Lamberti & Bleve-Zacheo, 1979, X. oxycaudatum strated significant progress in this field. In these studies, Lamberti & Bleve-Zacheo, 1979, X. thornei Lamberti & molecular technologies have been used in combination Golden, 1986, X. pachtaicum Tulaganov, 1938, X. rivesi with traditional methods. The incorporation of these tech- Dalmasso, 1969, X. citricolum Lamberti & Bleve-Zacheo,

∗ Corresponding author, e-mail: [email protected]

1979, X. georgianum Lamberti & Bleve-Zacheo, 1979, X. Materials and methods laevistriatum Lamberti & Bleve-Zacheo, 1979, X. floridae Lamberti & Bleve-Zacheo, 1979 and X. tarjanense Lam- Thirteen nematode populations were collected between berti & Bleve-Zacheo, 1979. 2006 and 2008 from the rhizosphere of various fruit trees In Chile, the earliest records of the X. americanum- located in the central valley of Chile, between 26◦ and group were presented by Gonzalez and Valenzuela (1968) 36◦S (regions III to VII; Table 1). Some fixed specimens and Allen et al. (1971), who sampled these nematodes and DNA samples of X. californicum (Lamberti & Bleve- from vineyards and orchards of the central valley, where Zacheo, 1979) from UC-Davis (Davis, CA, USA) were most of the agriculture is developed. At the end of the included for comparison. 1980s, Lamberti et al. (1988) reported six species: X. cal- Nematodes were extracted by means of Cobb’s wet ifornicum Lamberti & Bleve-Zacheo, 1979, X. floridae, X. sieving technique. DNA was extracted from single fe- inaequale Khan & Ahmad, 1975, X. peruvianum Lamberti males of each population. Based on Oliveira et al. (2004), & Bleve-Zacheo, 1979, X. pachtaicum and X. utahense the following protocol was developed: adult females were Lamberti & Bleve-Zacheo, 1979. However, these identi- transferred in a solution of 2 M NaCl, placed in individual microtubes (0.6 ml) containing 20 μl of 0.25 M NaOH fications have not been confirmed by subsequent studies, ◦ leading to a lack of consensus among taxonomists about and incubated overnight at 25 C. Afterwards, they were incubated at 99◦C for 3 min, followed by the addition of which species are present in the country (Doucet et al., 20 μl of extraction buffer (5 μlof0.5MTris-HCl,10μl 1998; Lamberti et al., 2000). Nonetheless, species identi- of 0.25 M HCl and 5 μl of 2% Triton X-100) and an in- fication within the X. americanum-group in Chile is very cubation for 3 min at the same temperature. Then, 30 μl important due to their considerable potential to damage − of 10% Chelexª and 5 μl of proteinase K (5 mg ml 1) various crops. The objective of this study was to iden- were added, followed by incubation at 55◦Cfor2h.Af- tify the species within the X. americanum-group in the ter that, the enzyme was inactivated at 95◦C for 10 min, central valley of Chile based on morphological, morpho- 2 μlofRNase(10mgml−1) was added and the sam- metric and molecular data from 13 populations collected ples were incubated at 37◦C for 30 min. Finally, the tubes from fruit trees between 2006 and 2008. These identi- were centrifuged at the maximum speed in an Eppendorf fications may provide the basis for adequate phytosan- microfuge for 15 min. The DNA obtained was stored at itary regulation and would be the starting point for the −20◦C until use. study of the biology, pathogenicity and spread of viral dis- Two primers that amplify the ITS region (ITS1, 5.8S eases. and ITS2), as well as short sections of the 18S and

Table 1. Origin of the populations of Xiphinema americanum s.l. used in this study. Population Region Locality Host P1 III Rodeo, Chile Table grape (Vitis vinifera) P2 III Tierras Amarillas, Chile Table grape (V. vinifera) P3 IV Huatulame, Chile Citrus (Citrus macrophylla) P4 IV Punitaqui, Chile Wine grape (V. vinifera) P5 IV Ovalle, Chile Citrus (C. macrophylla) P6 IV El Palqui, Chile Citrus (C. macrophylla) P7 V Los Andes, Chile Table grape (V. vinifera) P8 V Casablanca, Chile Wine grape (V. vinifera) P9 R.M. Codigua, Chile Wine grape (V. vinifera) P10 R.M. Alhué, Chile Wine grape (V. vinifera) P11 VI Totihue, Chile Plum (Prunus cerasifera) P12 VI Nancagua, Chile Table grape (V. vinifera) P13 VII Longovilo, Chile Apple (Malus domestica B.) XC USA Davis, CA, USA Table grape (V. vinifera)

R.M. = Metropolitan Region.

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