Eur J Plant Pathol https://doi.org/10.1007/s10658-017-1364-9 Description and molecular phylogeny of one new and one known needle nematode of the genus Paralongidorus (Nematoda: Longidoridae) from grapevine in Portugal using integrative approach Carlos Gutiérrez-Gutiérrez & Manuel Mota & Pablo Castillo & Margarida Teixeira Santos & Juan E. Palomares-Rius Accepted: 11 October 2017 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2017 Abstract A new and a known longidorid nematode, scanning electron microscopy studies, a very long and Paralongidorus lusitanicus n. sp. and Paralongidorus flexible odontostyle (180.0–223.0 μm), guiding ring lo- plesioepimikis, are described and illustrated from popula- cated at 28.0–41.5 μm from anterior end, vulva anterior to tions extracted from soil associated with grapevine (Vitis the mid-body (34–41%), a dorsally convex-conoid tail vinifera L.) from Escaroupim and Pó (central-Western with rounded terminus (29–42 μm long), bearing two or Portugal), respectively. The new needle nematode three pairs of caudal pores and males common (ratio 1:1.6 P. lusitanicus n. sp. is characterised by a very large body females) with spicules ca 80 μm long. Morphological and size (8072–12,022 μm), an expanded and rounded lip morphometric traits for P. plesioepimikis fit well with the region, ca 30 μm wide, with a clear constriction followed original description, and is reported for the first time in by a depression posterior to the amphidial aperture, Portugal. Integrative diagnosis of both species was com- amphidial fovea very large (11.0–19.0 μm), stirrup- pleted with molecular data obtained using D2-D3 expan- shaped, with conspicuous slit-like aperture as shown in sion segments of 28S rDNA, ITS1-rDNA and partial 18S–rDNA. The phylogenetic relationships of these spe- cies with other Paralongidorus spp. using these three C. Gutiérrez-Gutiérrez (*) : M. Mota molecular markers indicated that P. lusitanicus n. sp. NemaLab/ICAAM, Instituto de Ciências Agrárias e Ambientais clustered together with other Paralongidorus spp. Mediterrânicas & Departamento de Biologia, Universidade de forming a sister clade with P. plesioepimikis, both of them Évora, Núcleo da Mitra, Ap. 94, 7002-554 Évora, Portugal sharing a large body, long odontostyle, an anteriorly lo- e-mail: [email protected] cated vulva and an expanded and rounded lip region with M. Mota a clear constriction followed by a depression posterior to Departamento Ciências da Vida, Universidade Lusófona de the amphidial aperture. Humanidades e Tecnologias, EPCV, C. Grande 376, 1749-024 Lisbon, Portugal . : Keywords Bayesian inference Longidorids P. Castillo J. E. Palomares-Rius Paralongidorus . Phylogeny. rDNA . Taxonomy Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Campus de Excelencia Internacional Agroalimentario, ceiA3, Avenida Menéndez Pidal s/n, 14004 Córdoba, Spain Introduction M. T. Santos Instituto Nacional de Investigação Agrária e Veterinária (INIAV), Longidoridae is one of the largest families of soil nem- Quinta do Marquês, 2780-159 Oeiras, Portugal atodes in term of species diversity given that they Eur J Plant Pathol comprise a total of seven genera and more than 750 nematodes: partial 28S rRNA gene (He et al. 2005, species (Decraemer & Robbins 2007). The taxonomic Palomares-Rius et al. 2008, 2013,Pedrametal.2012, and systematic position of the genus Paralongidorus Kornobis et al. 2015, Barsi & De Luca 2017), 18S Siddiqi, Hooper & Klan, 1963 within the family rRNA gene (Palomares-Rius et al. 2008, 2013, Pedram Longidoridae (Thorne, 1935) Meyl, 1961 appears to et al. 2012, Kornobis et al. 2015) and internal tran- be accepted by the scientific community, however the scribed spacer (ITS) region of the rRNA genes species composition are continuously being subject of (Palomares-Rius et al. 2008, 2013,Pedrametal.2012, debate (Hunt 1993, Siddiqi et al. 1993,Coomans1996, Kornobis et al. 2015, Barsi & De Luca 2017). Kornobis Escuer & Arias 1997, Decraemer & Robbins 2007). et al. (2015)designedaPCR–RFLP of the D2D3 region This genus, member of the commonly known needle of 28S rRNA gene with five restriction enzymes to nematodes, is quite diverse with about 90 valid species establish diagnostic profiles from P. rex Andrássy, of migratory ectoparasites that parasitise a wide range of 1986. Recently, Barsi & De Luca (2017)designeda agronomic crops, ornamentals, and forest trees (Taylor PCR–RFLP of ITS region based on six restriction en- &Brown1997, Decraemer & Robbins 2007, zymes for genotyping of species-specific variations for Palomares-Rius et al. 2013,Kornobisetal.2015,Barsi P. francolambertii Barsi & De Luca, 2017. D2-D3 ex- &DeLuca2017). This group of phytopathogenic spe- pansion segments of 28S rRNA and ITS rRNA have cies are of global interest because they cause directly proved to be more effective in species identification damage on the roots of the host plant attributable to their when compared to partial 18S rRNA, as both these ectoparasitic feeding and one species is able to transmit markers display more molecular variability than partial damaging nepoviruses (Taylor & Brown 1997, 18S rRNA (He et al. 2005, Palomares-Rius et al. 2008, Decraemer & Robbins 2007). Paralongidorus maximus 2013, Pedram et al. 2012,Kornobisetal.2015, Barsi & (Bütschli 1874) Siddiqi 1964 has important phytopath- De Luca 2017). Additionally, the mitochondrial DNA ological implications because it is a vector of Raspberry (mtDNA) markers have been used for species identifi- ringspot virus (RpRSV) (Jones et al. 1994, Taylor & cation, phylogeny and phylogeography in a large num- Brown 1997). This species is considered as a major pest ber of species inside Longidoridae (Lazarova et al. in Europe and other many parts of the world (Taylor & 2006, 2016, Gutiérrez-Gutiérrez et al. 2011, 2013a,b, Brown 1997), and for this reason is included in the A2 Kumari 2014, Subbotin et al. 2014, 2015), however list of pest recommended regulation of the European and there is a lack of the mitochondrial molecular data on Mediterranean Plant Protection Organization (EPPO, Paralongidorus spp. Only a small fraction of www.eppo.int/QUARANTINE/). Paralongidorus species (nine identified species) has so An accurate and detailed description of needle nem- far sequences in the GenBank database. These se- atodes is essential to establish unequivocal diagnosis in quences should be obtained by using voucher specimens order to discern virus vectors and/or quarantine patho- from the described species in order to perform molecular gen species, select appropriate management strategies species comparison and to differentiate molecularly for preventing the spread of them and establish efficient cryptic species (Palomares-Rius et al. 2014). Based on control measures. Morphometric and morphological our current knowledge, eleven valid Paralongidorus identification within this genus at species level is mainly species have been reported in several cultivated and wild based on characteristics of adult females (Escuer & plants in Europe and in the Mediterranean Basin (Bravo Arias 1997).However, the high intraspecific variability &Lemos1997, Escuer & Arias 1997, Taylor & Brown of some diagnostic features and the great diversity in 1997,Lišková & Brown 2003, Barsi & De Luca 2017): phenotypic plasticity make species identification based i) P. ma xi mu s is the most common Paralongidorus spp. on gross morphology and internal anatomical features a in Europe and it is frequently found parasitizing a technically difficult task even for experts. Recently, the large number of forest trees, grapevine, chestnut tree sequencing of RNA-based markers is an increasingly (Castanea spp.), walnut trees (Juglans regia L.) and powerful approach for the molecular diagnostics and for a wide range of vegetable crops in Europe(Heyns, understanding their inter- and intra-genetic variability 1975;Lambertiet al., 1983;Rocaetal. 1988; (Palomares-Rius et al. 2013,Kornobisetal.2015,Barsi Macara 1994;Lišková, 1995;Bravo&Lemos1997; & De Luca 2017). Several ribosomal RNA (rRNA) Taylor & Brown 1997;Lišková & Brown, 1998, 1999, genes are used for molecular characterizations of these 2003; Gutiérrez-Gutiérrez et al. 2016), ii) P. iberis Eur J Plant Pathol Escuer & Arias, 1997 and P. monegrensis Escuer & followed by a usually slight depression posterior to the Arias, 1997 in the rhizosphere of plants in the amphidial aperture (Palomares-Rius et al. 2013). These Rhamno-Cocciferelum ecosystem in Spain (Escuer & traits prompted us to undertake a detailed morphological Arias 1997), iii) P. litoralis Palomares-Rius, Subbotin, and molecular sequence-based comparative study with Landa, Vovlas & Castillo 2008, in the rhizosphere of previously described Paralongidorus species. These mastic tree (Pistacia lentiscus L.) in Spain (Palomares- observations indicated that one of the detected species Rius et al. 2008), iv) P. paramaximus Heyns, 1965 on appeared to be morphologically and morphometrically soil around plants in citrus orchards (Citrus aurantium unrelated to nominal Paralongidorus species and did L.) in Spain (Palomares-Rius et al. 2008), v) P. epimikis not fit with their descriptions. Detailed studies using Dalmasso, 1969 in grapevine in Algeria (Dalmasso light and scanning electron microscopy (SEM) and mo- 1969), vi) P. plesioepimikis Palomares-Rius, lecular analyses indicated that this population should be Cantalapiedra-Navarrete, Gutiérrez-Gutiérrez, Liébanas assigned to a new species and is described herein