09 Valadas_169 28-12-2011 12:29 Pagina 169 Nematol. medit. (2011), 39: 169-178 169 THREE SPECIES OF ENTOMOPATHOGENIC NEMATODES OF THE FAMILY STEINERNEMATIDAE (NEMATODA: RHABDITIDA) NEW TO CONTINENTAL PORTUGAL V. Valadas11*, Z. Mrácˇek2, S. Oliveira3 and M. Mota1 1 NemaLab-ICAAM & Departamento de Biologia, Universidade de Évora, Apartado 94, 7002-554 Évora, Portugal 2 Laboratory of Insect Pathology, Institute of Entomology, Biology Centre, Czech Academy of Sciences, Branisˇovská 31, 370 05, Cˇeské Budeˇjovice, The Czech Republic 3 Laboratorio de Microbiologia do Solo-ICAAM & Departamento de Biologia, Universidade de Évora, 7002-554 Évora, Portugal Summary. A survey was conducted to determine the species of entomopathogenic nematodes occurring in continental Portugal. Nematodes were recovered from soil samples collected from Alentejo (South) and central Portugal from 2006 to 2009. Nematode isolates were identified based on morphology and sequence analysis. Phylogenetic analysis was based on sequences of partial 28S (D2D3 domain), internal transcribed spacer (ITS) and cytochrome C oxidase subunit I gene (COI). Nematode isolates 59F, 15G, 20F and 2B were characterized in detail. In the collected samples, nematodes from the genera Steinernema and Heterorhabditis were identified, namely Heterorhabditis bacteriophora (1.9%), Steinernema feltiae (11.5%), S. kraussei (0.32%), S. intermedium (0.32%) and Steinernema sp. (0.63%) glaseri-group. Isolate 2B shows morphological characteristics identical to S. intermedium, which is a member of the affine/intermedium-group and is characterized by the presence of strongly curved and robust spicules with a distinct rostrum in the male, and dorsal tail depression in third-stage infective junveniles (IJ). However, the phylogenies based on the three molecular markers revealed that isolate 2B is more closely related to S. affine than to S. intermedium. Based on morphological observations, isolate 20F was identified as S. kraussei, which is a member of the kraussei/feltiae-group, character- ized by IJs with a straight body of medium length (mean = 700-950 µm), lateral field mostly with eight ridges, rather broad, flatly rounded and continuous cephalic region, excretory pore at mid-pharynx level; males with mucronate tail, yellowish spicules ca. 50 µm long and wide manubria; females with short conoid tail with pointed non-mucronate tip. Based on morphology and sequence analysis, isolates 59F and 15G were considered conspecific and identified as a species belonging to the glaseri-group. More de- tailed studies are necessary to decide whether these isolates represent a new species. Key words: Steinernema intermedium, S. kraussei, Steinernema sp. glaseri-group, cytochrome oxidase c, ribosomal sequences, phy- logeny. Entomopathogenic nematodes (EPNs) of the families infective juveniles (IJ) of these families are non-feeding, Steinernematidae Chitwood and Chitwood, 1937 and free living stages in the soil where they can survive for Heterorhabditidae Poinar, 1976 are lethal parasites of in- extended periods. They are safe to non-target organisms sects, being widely distributed in soils worldwide (Ho- and to the environment, can be mass produced, formu- minick et al., 1996). These nematodes have been known lated and easily applied as bio-pesticides (Georgis and since the XVIIth century and include more than 30 fami- Kaya, 1998), being compatible with some insecticides lies (Kaya and Stock, 1997). Hunt (2007) reported 55 (Chen et al., 2003), and have a broad host range of in- valid species of the genus Steinernema Travassos, 1927 sect pests in a variety of habitats (Kaya and Gaugler, and eleven species of the genus Heterorhabditis (Hunt, 1993; Gaugler, 2002). 2007). However, after 2007 many more entomopatho- Insects play an important role in agricultural produc- genic nematode species have been described, mainly tion and have acquired resistance to insecticides during from Africa and Asia (Khatri-Chhetri et al., 2011). recent decades (Ahmad et al., 2003) following the in- Nematodes of the families Steinernematidae and crease usage of pesticides. Entomopathogenic nema- Heterorhabditidae are potential candidates to be used todes represent an alternative to insecticides, the identi- as bio-control agents due to their capacity for control- fication of new species in different countries and ecosys- ling insect pests worldwide (Kaya and Stock, 1997; Bed- tems being of major importance in pursuing this objec- ding, 1998; Shapiro-Ilan et al., 2002). Classified as para- tive. These nematodes have a complex life cycle inside sitoids and pathogens, they have many attributes that the insect host. When infective juveniles locate a poten- give them the ability to be used as bio-pesticides: they tial host in the soil, they move towards it and penetrate can be found under diverse ecological conditions in- the insect body, via natural openings or areas of thin cu- cluding cultivated fields, forests, grasslands, deserts and ticle. Once inside the body cavity, they release their ocean beaches (Hominick et al., 1996). The third-stage symbiotic bacteria (belonging to the genera Xenorhab- dus and Photorhabdus for steinernematids and het- erorhabditids, respectively), which multiply rapidly, causing insect death within 48 hours by septicemia * Corresponding author: [email protected] (Kaya and Koppenhofer, 1999). IJs feed on liquefying 09 Valadas_169 28-12-2011 12:29 Pagina 170 170 host tissues, and then mature into adults. During their habitats, such as forest, cultivated fields, grasslands, adult stages they complete their life cycle, producing woodlands, vineyards, and irrigated land. We tried to eggs. The eggs release juveniles and when food re- obtain samples from all the different habitats common sources drecrease, IJs emerge from the insect cadaver, to the different regions, but also typical habitats from searching for a new host. each region. From each sampling site, three or four sub- In Portugal, until 2006, the only region where sur- samples were taken from an area of c. 200 m2, totalling veys and studies of EPN had been conducted was the approximately 2 dm3 of soil. Soil samples were collected Azores archipelago (Simões et al., 1994; Rosa and from a depth of 0-20 cm and placed in a plastic bag Simões, 2004). Therefore, an extensive survey was initi- identified by GPS location, vegetation and date. To ex- ated in 2006 and concluded in 2009 to obtain more in- tract EPN from the soil, the G. mellonella L. (Lepi- formation on the distribution of EPN in continental doptera: Pyralidae) trapping method (Bedding and Portugal. Soil samples were randomly collected from se- Akhurst, 1975) was used. Therefore, ten last instar lar- lected ecosystems, especially in areas considered suit- vae of G. mellonella were placed inside perforated metal able habitats for the presence of steinernematids and tea bags, partly filled with soil, which were then embed- heterorhabditids, such as conifer forests, sandy soils, ded in the soil sample in each plastic bag. Soil samples cultivated fields, natural grasslands, roadsides with tree were stored at 25 ºC, and every four days dead G. mel- verges, cultivated fields, and typical habitats from each lonella were removed and replaced by new ones. This location in Portugal. The main goal of the survey was to procedure was carried out during 12 days. The dead G. evaluate the biodiversity in continental Portugal of en- mellonella larvae were transferred to White traps tomopathogenic nematodes, which hopefully could then (White, 1929) and IJs collected during the following be used as bio-control agents. Because the number of days and stored at 10 ºC in distilled water. To identify species of EPNs is growing fast, it has become difficult the nematodes, morphological, morphometrical and to identify them just by morphological characters and, molecular characterizations were made. therefore, it is necessary to use both morphological and molecular approaches, using different markers accord- Morphological and morphometrical characterization ing to the purpose. For scanning electron microscopy (SEM), adults and Previously, molecular and phylogenetic studies dauer juveniles were fixed in 4% formalin buffered with showed that the genus Steinernema contains some 0.1 M sodium cacodylate at pH 7.2 for 24 hours at 4-6 species which are difficult to discriminate using DNA ºC. They were then post-fixed with 2% osmium tetrox- sequence analysis (Nguyen et al., 2007). Therefore, in ide solution for 12 hours at 25 ºC, dehydrated in a grad- the present study, the four new EPN isolates found in ed ethanol series, critical point dried with liquid CO2, samples collected in Alentejo and Algarve, as well as in mounted on SEM stubs, and coated with gold (Nguyen central and northern continental Portugal, were firstly and Smart, 1995). The mounts were examined in a JE- identified morphologically. Then, phylogenetic analysis OL scanning electron microscope. based on three molecular markers, ITS (Internal Tran- For light microscopy (LM), the nematodes were scribed Spacer), the partial sequence of the D2D3 from heat-killed on glass slides in a drop of water, and 28S and cytochrome C oxidase subunit I gene (COI), mounted in aqueous media under a cover glass. Another were also used to confirm nematode identification. batch of nematodes was fixed in hot TAF (Southey, 1970), transferred to glycerin by the slow evaporation method and mounted permanently in anhydrous glyc- MATERIAL AND METHODS erin mounts (Seinhorst, 1959). Measurements and