©2018 Institute of Parasitology, SAS, Košice DOI 10.1515/helm-2017-0053 HELMINTHOLOGIA, 55, 1: 88 – 94, 2018 Research Note First report of the lesion nematodes: Pratylenchus brachyurus and Pratylenchus delattrei on tomato (Solanum lycopersicum L.) plants in Cape Verde Ł. FLIS1*, R. DOBOSZ2, K. RYBARCZYK-MYDŁOWSKA1, B. WASILEWSKA-NASCIMENTO3, M. KUBICZ1, G. WINISZEWSKA1 1Museum and Institute of Zoology, Polish Academy of Sciences, Wilcza 64, 00-679 Warszawa, Poland, E-mail: *lfl [email protected], [email protected]; [email protected]; [email protected]; 2Institute of Plant Protection-National Research Institute, Węgorka 20, 60-318, Poznań, Poland, E-mail: [email protected]; 3University of Cape Verde, Palmarejo, CP 279-Praia, Republic of Cape Verde, E-mail: [email protected] Article info Summary Received July 3, 2017 Roots of Solanum lycopersicum L. were collected in growing season of year 2015, on the island of Accepted September 28, 2017 Santiago in Cape Verde. Morphological, morphometric and molecular (18S rDNA and 28S rDNA) studies revealed the presence of Pratylenchus brachyurus and P. delattrei in root systems and root zones of tomato plants. To our knowledge, this is the fi rst record of the occurrence of these nematode species in Cape Verde. Keywords: Cape Verde; new geographic record; Pratylenchus brachyurus; Pratylenchus delattrei; 18S rDNA; 28S rDNA Introduction migratory endoparasites move within host root tissues causing necrosis and creating wounds, thus providing openings for soil- Tomato (Solanum lycopersicum L.) is one of the vegetable crops borne plant pathogens to enter and cause disease. In tropical and most widely grown on irrigated land in the Republic of Cape Verde. subtropical regions Pratylenchus spp. infect roots of several crops. Little is known about the plant-parasitic nematodes responsible for P. brachyurus has been recorded parasitising pinapple roots in Ha- pest infestations of this economically important crop in this area. waii (Godfrey, 1929), Uganda (Bafokuzara, 1982) and Brazil (Mon- So far only Rotylenchulus reniformis Linford & Oliveira, 1940 teiro & Lordello, 1972); citrus and forest trees in North America (Germani, 1978), Heterodera schachtii Schmidt, 1871 (Sturhan, (Brooks & Perry, 1967; Ruehle, 1971), coffee in Brazil (Lordello et 1993) as well as some representatives of the genus Meloidogyne al., 1968), rubber in India (Mukherjee et al., 2000) and many other Göldi, 1892 (Netscher & Taylor, 1976) and Meloidogyne incognita species of economically important plants (Luc et al., 2005; Castillo (Kofoid & White, 1919) Chitwood, 1949 (Flis et al., 2018) have & Vovlas, 2007; De Araujo Filho et al., 2014). P. brachyurus has been identifi ed as nematodes parasitising tomato roots in Cape also been reported from a few countries in Europe, in Bulgaria on Verde. During the study of plant parasitic nematodes associated tobacco (Katalan-Gateva & Nedechlev, 1983), on several crops in with tomato cultivation in Cape Verde, representatives of lesion Russia (Ryss, 1988) and Australia (Riley & Kelly, 2002). P. delattrei nematodes Pratylenchus (Filipjev, 1936) were found. Morpho- primarily described from Madagascar (Luc, 1958) has also been metric analysis and molecular study allowed to identify the two found on several crops in Vietnam (Ryss, 1988), on sugarcane in investigated species as Pratylenchus brachyurus (Godfrey, 1929) Sudan (Saadabi, 1988), in ornamental plants, like rose and rhapis, Filipjev et Schuurmans Stekhoven, 1941 and P. delattrei Luc, grown in Korea (Kim & Minagawa, 1996), in date palm in Oman 1958. Pratylenchus spp. have signifi cant economic impacts. Crop (Mani et al., 2005) and in several hosts and localities in India (Jothi damage and yield losses by these nematodes have been pre- et al., 2004). In this article we provide morphological description viously observed (Egunjobi, 1974; Talwana et al., 2016). Those and morphometric data of P. brachyurus and P. dellatrei from Cape * – corresponding author 88 Table 1. Morphometrics of Pratylenchus spp. from tomato in Cape Verde. All measurements are in μm and in format: mean ± S.D. (range). Character/ratios Pratylenchus brachyurus Pratylenchus delattrei (Godfrey 1929) Luc 1958 n1420 L 517 ± 45 (414 – 573) 532 ± 33 (498 – 586) a 24.2 ± 1.9 (20.1 – 26.7) 26.6 ± 2.2 (22.1 – 31.3) b 6.3 ± 0.5 (5.1 – 6.9) 6.6 ± 0.5 (6.1 – 7.7) b’ 3.7 ± 0.4 (2.9 – 4.1) 4.5 ± 0.4 (4.0 – 5.3) c 20.1 ± 2.4 (15.3 – 25.5) 21.9 ± 2.1 (18.5 – 25.1) c’ 2.2 ± 0.2 (1.7 – 2.5) 2.2 ± 0.2 (1.9 – 2.8) V 86 ± 1 (84 – 88) 76 ± 1 (75 – 78) Stylet length 18.3 ± 0.4 (17.8 – 18.9) 16.4 ± 0.4 (15.4 – 16.9) Dorsal gland opening 2.2 ± 0.3 (1.6 – 2.6) 2.9 ± 0.3 (2.4 – 3.1) O 12.0 ± 1.9 (8.6 – 14.6) 17.4 ± 1.7 (14.3 – 21.3) Pharynx length 82.1 ± 1.7 (79.2 – 85.2) 80.1 ± 3.2 (73.1 – 84.2) Pharyngeal overlap 47.7 ± 7.9 (37.0 – 62.3) 39.0 ± 7.2 (29.8 – 49.0) Maximal body diameter 21.5 ± 1.9 (17.7 – 26.0) 20.2 ± 2.2 (16.8 – 23.7) Anal body diameter 12.2 ± 1.1 (15.9 – 20.9) 11.2 ± 1.2 (8.9 – 13.2) Tail length 25.9 ± 1.9 (22.0 – 28.3) 24.8 ± 2.3 (21.0 – 27.1) Tail annuli 18 ± 2 (16 – 23) 19 ± 2 (16 – 24) Phasmid to terminus 14.3 ± 1.5 (11.6 – 16.9) 10.6 ± 2.7 (6.0 – 14.9) Abbreviations are defi ned in Siddiqi (2000) Verde. Genetic characteristics of LSU (28S rDNA) and SSU (18S implemented in a phylogenetic analysis concerning relationships rDNA) from analysed populations and results of comparative study within clades IV (‘P. penetrans group’) and VI (‘P. zeae group’) of of these sequences are also supported. the genus Pratylenchus as defi ned by Subbotin et al. (2008) and Palomares-Rius et al. (2014). Initial multiple sequence alignment Material and Methods was performed using the BioEdit program (v. 7.2.5; Hall, 1999) and included publically available (GenBank) representatives of ‘P. pen- Eight tomato plants with soil surrounded root systems were har- etrans and P. zeae groups’. All available unique P. brachyurus and vested in June 2015 in the village of Achada Colaço (located at P. delattrei sequences were used. In order to root the tree two out- 15°06’35.3”N, 23°31’31.9”W). Nematodes from roots were isolated group sequences (FN433867 and JN967754) were chosen based by the modifi ed Baermann method, whereas specimens from soil on the total nematode phylogeny by Van Megen et al. (2009). The samples were extracted using the decanting and sieving method. fi nal multiple-sequence alignment contained 663 positions. Substi- Females assigned for morphological analysis were fi xed in a trieth- tution models were tested using “FindModel”, an online implemen- anolamine formalin water solution (TAF) and mounted in anhy- tation of the MODELTEST program (Posada & Crandall, 1998). drous glycerine (Seinhorst, 1959) on slides using the paraffi n-ring The General Time Reversible plus Gamma substitution model method. Morphological observations and morphometric analyses was selected. The Bayesian phylogeny was constructed with the were performed using a Leica light microscope with the Nomarski program MrBayes (v. 3.1; Ronquist & Huelsenbeck, 2003). Four differential interference contrast. Females selected for the genetic independent runs were performed with four Markov chains per run. analysis (three specimens for each species) were fi xed in a DESS The program was run for 800,000 generations with a sample fre- solution (Yoder et al., 2006). After washing in sterilized milli-Q quency of 200 generations. The sampled trees from each run were water, single nematode individuals were used for DNA extraction combined in a single 50 % majority-rule tree. Stabilisation of the according to the nematode lysis procedure, as described by Holter- likelihood and parameters was checked with the program Tracer man et al. (2006). 18S rDNA gene fragments were amplifi ed in (v. 1.6; Rambaut et al., 2014). PCR reactions with the aid of 988F and 1912R, as well as 1813F and 2646R primer combinations (Holterman et al., 2006). Amplifi - Results and Discussion cation of the partial 28S rDNA sequence was obtained using prim- ers 61F (Holterman et al., 2008) and MCB1R (Dobosz et al., 2013). Morphological and morphometric analyses of the investigated The 18S and 28S rDNA regions were sequenced by the Sanger females confi rmed their affi liation to P. brachyurus and P. delat- method on ABI 3500L genetic analyzer (Applied Biosystems, Fos- trei. The morphology of P. brachyurus from Cape Verde (14 fe- ter City, CA, USA). The newly obtained 28S rDNA sequences were males) was characterised by: low and angular lip region with two 89 Fig. 1. Pratylenchus brachyurus (Godfrey 1929) female from Cape Verde. a. pharyngeal region; b. anterior end; c. lateral fi eld at mid-body; d. ovary with single row of oocytes; e. vulval region; f. tail; g. tail, phasmid. (All scale bars = 10 μm) lips annuli, which is separated from body contour; long spear with (Fig. 1, Table 1). The morphology of P. delattrei from Cape Verde rounded, laterally directed knobs; lateral fi eld with four lines; a (20 females) was characterised by continuous body contour, more single row of oocytes; empty and rounded spermatheca; poste- or less truncate lip region with three annuli; medium spear, with rior vulva; undifferentiated and well developed postvulval uterine rounded and slightly anteriorly directed knobs; lateral fi eld with sac; conoid tail with rounded (to truncate), smooth or broadly an- four lines; two rows of oocytes; empty and rounded spermathe- nulated terminus.