JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2018-021 Issue 0 | Vol. 0 First Report of Carrot Cyst Nematode Heterodera carotae in Mexico: Morphological, Molecular Characterization, and Host Range Study Ilia Mariana Escobar-Avila,1 Edgar Óliver López-Villegas,2 Sergei A. Abstract Subbotin3,4 and Alejandro Tovar-Soto1* During 2008 to 2016 in several nematological surveys in the Tepeaca Valley, Puebla, Mexico, populations of carrot cyst nematode, Heterodera 1Laboratorio de Nematología carotae was found parasitizing carrots, Daucus carota. The nematode Agrícola, Departamento de was present in 61% of the sampled fields with high population Parasitología, Escuela Nacional densities, causing severe carrot yield losses by this nematode in de Ciencias Biológicas, Instituto the Tepeaca Valley. The aim of this work was to study morphology, Politécnico Nacional, México, CP morphometrics, host range, and molecular characterization of the 11340. nematode. The morphological and morphometric characterization 2Central de Instrumentación de was made using light and scanning electron microscopy of the second Microscopía, Escuela Nacional de stage juveniles, female, male and cyst, and the host range study, was Ciencias Biológicas, Instituto performed using nine different plants from five families. The molecular Politécnico Nacional, México, identification was made by sequencing and analysing the ITS rRNA CP 11340. and partial COI genes. It was shown that using morphological and molecular tools it was not possible to make an accurate differentiation 3Plant Pests Diagnostic Center, of H. carotae from Heterodera cruciferae. The only test that allowed to California Department of Food and distinguish these species was the host range study. This is an example Agriculture. 3294 Meadowview Road, of importance of combination of several methods for the correct Sacramento, CA 95832. identification of cyst nematodes. To our knowledge, this is the first 4Center of Parasitology of A.N. report of H. carotae in Mexico. Severtsov Institute of Ecology and Evolution of the Russian Academy of Key words Sciences, 117071, Moscow, Russia. Carrot, COI gene, Daucus carota, Morphometrics, Phylogeny, ITS *E-mail: [email protected]. rRNA gene. The carrot cyst nematode Heterodera carotae is a carrots at national level with an annual production of highly specialized parasite infecting only wild and cul- 75,950 t (SIAP, 2017). The Tepeaca Valley in this state is tivated carrots (Daucus carota L.) and an herbaceous known as an important vegetable producing area and plant known as the hedge parsley (Torilis leptophylla L.) it is constituted by 13 municipalities. In 2008 to 2016 (Jones, 1950b; Mugniery and Bossis, 1988). This during several nematological surveys in six munici- nematode might cause serious problem in the carrot palities of the Tepeaca Valley, white females of a cyst growing areas in Italy leading to yield loss around nematode of the genus Heterodera parasitizing carrot 20% to90% in carrots (Greco et al., 1994). Distribu- roots were found in 61% of sampled fields (Tovar-Soto tion of this nematode is mostly restricted to Europe et al., 2009, 2010; Escobar-Avila et al., 2017). In this and South Africa, however, in North America it has study, the cyst nematode was identified asH. carotae. been found in Ontario, Canada, and Michigan, United Although carrot yield losses caused by this nematode States (Graney, 1985; Berney and Bird, 1992; Subbotin is not exactly estimated, however, the growers have et al., 2010; Madani et al., 2017; Yu et al., 2017). In 2016, been forced to stop sowing carrots where the nema- the Puebla State of Mexico was the first producer of tode is present. The aim of this work was to conduct 1 © The Society of Nematologists 2018. First Report of Carrot Cyst Nematode Heterodera carotae in Mexico: Morphological, Molecular Characterization morphological, morphometrics, host-range, and in different concentrations of ethanol (30%–100%). molecular studies of H. carotae from Mexico. Samples were critical point dried and coated with gold palladium for its analysis in a scanning electron Materials and methods microscope (Shepherd and Clark, 1986). The anterior region of the different nematode stages was observed and from the J2 and males the number of Nematode population incisures in the lateral field were counted. Soil with nematodes collected from an infested carrot field from Santa Maria Actipan, Acatzingo, Puebla, Mexico Host range study (18˚ 58¢ 486² N; 97˚ 50¢ 295² W; 2246 masl) was used Nine plant species from five families, which are known to establish a laboratory culture. The nematode cul- as hosts from H. carotae and H. cruciferae were ture was maintained in the greenhouse at 20 to 25°C. used in the test: Apiaceae, carrot (Daucus carota L.); Two plastic plots were filled with 15 kg of soil, then 50 Asteraceae, lettuce (Lactuca sativa L.); Brassicaceae, seeds of carrot cv. Christian were sown. After 54 days, broccoli (Brassica oleracea var. italica L.), cabbage one plot was used for the extraction of second stage (B. oleracea var. capitata L.), cauliflower (B. oleracea juveniles (J2) and males by the centrifugal flotation tech- var. botrytis L.), radish (Raphanus sativus L.) and nique (Jenkins, 1964). After 65 days, the other plot was Brussels sprouts (B. oleracea var. gemmifera L.); used to obtain females, the roots were separated, gently Chenopodiaceae, beetroot (Beta vulgaris L.), and washed, and put in a Petri dish for its examination under Poaceae, wheat (Triticum aestivum L.) (Goodey et al., Motic stereoscopic microscope. Females were sepa- 1959; Mugniery and Bossis, 1988; Evans and Russell, rated using a brush and kept at 5°C until use. Females 1993). Plants growing in pots with 3 kg of steri- were used for morphology and morphometry. The cysts lized soil were inoculated with 12,000 eggs and J2. were extracted by the Fenwick method (Fenwick, 1940). Each plant species was tested in three replicates. After 70 days, the plants were removed and carefully Light microscopy washed with tap water, the roots were examined Males and J2 were killed in a bath at 65°C for 5 min, under a stereoscopic microscope for the search of fixed with a mixture of ethanol, acetic acid, and for- white females and cysts. Then roots were stained by malin (20:6:1) and processed into glycerin and the the acid fuchsin lactoglycerol technique to observe specimens were mounted in permanent slides with nematode stages inside of roots (Bybd et al., 1983). anhydrous glycerin and paraffin seal for examination If white females or cysts where found on roots, the (S’Jacob and Van Bezooijen, 1984; Southey, 1986). plant was considered as a host. The measures were taken for length, width at mid body, stylet length, labial region height and width, dor- Molecular characterization sal oesophageal gland to spear knobs base, anterior end to median bulb, anterior end to excretory pore, DNA was extracted from single cysts of H. carotae oesophageal length, width at anus and hyaline re- fixed in ethanol. Protocols for DNA extraction, poly- gion, tail length, hyaline part of tail length, and spicules merase chain recaction (PCR), cloning and sequenc- length (males) (S’Jacob and Van Bezooijen, 1984; ing were described as in Tanha Maafi et al. (2003) Southey, 1986). For the observation of the vulval cone, and Subbotin (2015). The forward primer TW81 cysts were soaked in lactic acid 45% for 15 min, trans- (5¢-GTT TCC GTA GGT GAA CCT GC-3¢) and the re- ferred to water and then, the posterior region was dis- verse primer AB28 (5¢-ATA TGC TTA AGT TCA GCG sected and mounted in glycerin with paraffin seal for its GGT-3¢) (Tanha Maafi et al., 2003) were used for ampli- observation and analysis, (S’Jacob and Van Bezooijen, fication of the ITS1-5.8S-ITS2 of rRNA and the forward 1984; De la Jara-Alcocer et al., 1994). The measure- Het-coxiF (5¢-TAG TTG ATC GTA ATT TTA ATG G 3¢) ments taken were vulval slit length, fenestral length, and the reverse Het-coxiR (5¢- CCT AAA ACA TAA and width. All measurements were made on Olympus TGA AAA TGW GC-3¢) (Subbotin, 2015) was used for CX31 microscope with Infinity Analyze software v6.5.2. amplification of the partialCOI mtDNA gene. For COI gene study the sequences of H. carotae from Italy, Scanning electron microscopy France, Switzerland and H. cruciferae from Russia, Moscow region and USA, California were also J2, females, males and cysts were processed. The included. The new sequences were deposited in the nematode phases were fixed in 4% glutaraldehyde, GenBank under accession numbers: MG563227 to post fixed in 1% osmium tetraoxide, and dehydrated M G 56 3237. 2 JOURNAL OF NEMATOLOGY The newly obtained ITS rRNA and COI gene se- indistinct postlabial annuli. Spicules bidentate (Table 1; quences were aligned with corresponding published Figs. 3C,D; 4D, E). sequences of species from the Goettingiana group (Subbotin et al., 2001; Tanha Maafi et al., 2003; Chizhov Second stage juveniles et al., 2009; Toumi et al., 2013; Madani et al., 2017; Vovlas et al., 2017 and others) using ClustalX 1.83 with Vermiform, anterior region heavily sclerotized, strong default parameters. The sequence datasets were ana- stylet well developed. Tail acutely conical with round- lysed with Bayesian inference (BI) using MrBayes 3.1.2 ed tip usually with the presence of 1-3 spherical re- (Huelsenbeck and Ronquist, 2001) under the HKY+G fractive body sometimes with associated swelling. model for COI and GTR+G+I model for ITS rRNA gene Lateral field with four incisures forming three bands as they were obtained using jModeltest 0.1.1 (Posada, (Table 1; Figs. 3E,F; 4A–C). 2008). BI analysis was initiated with a random starting The species of Heterodera found in the Tepeaca tree and was run with four chains for 1.0 × 106 genera- Valley, Puebla, Mexico and parasitizing carrots has tions.