First Report of Carrot Cyst Nematode Heterodera Carotae in Mexico: Morphological, Molecular Characterization and Host Range Study

JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2018-021 Issue 2 | Vol. 50

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. Subbotin,3,4 and Alejandro Tovar-Soto1* Abstract 1Laboratorio de Nematología Agrícola, During 2008 to 2016 in several nematological surveys in the Tepeaca Departamento de Parasitología, Valley, Puebla, Mexico, carrot cyst nematode, Heterodera carotae Escuela Nacional de Ciencias was found parasitizing carrots, Daucus carota. The nematode was Biológicas, Instituto Politécnico present in 61% of the sampled fields with high population densities, Nacional, México, CP 11340. causing severe carrot yield losses in the Tepeaca Valley. The aim of this work was to study morphology, morphometrics, host range, and 2 Central de Instrumentación de molecular characterization of the nematode. The morphological and Microscopía, Escuela Nacional morphometric characterization was made using light and scanning de Ciencias Biológicas, Instituto electron microscopy of the second stage juveniles, females, males Politécnico Nacional, México, CP and cysts, and the host range study, was performed with nine different 11340. plants from five families. The molecular identification was made by 3Plant Pests Diagnostic Center, sequencing and analysing the ITS rRNA and partial COI genes. It was California Department of Food and shown that using presently available molecular tools it is not possible Agriculture. 3294 Meadowview Road, to make an accurate differentiation of H. carotae from H. cruciferae. Sacramento, CA 95832. The host range test allowed to distinguish these species from each other. Our study showed that male stylet length is longer for H. carotae 4Center of Parasitology of A.N. compared with that for H. cruciferae. This is an example of importance Severtsov Institute of Ecology and of combination of several methods for the correct identification of cyst Evolution of the Russian Academy of nematodes. To our knowledge, this is the first report of H. carotae in Sciences, 117071, Moscow, Russia. Mexico. *E-mail: [email protected]. This paper was edited by Zafar Key words Ahmad Handoo. Carrot, COI gene, Daucus carota, Morphometrics, Phylogeny, ITS rRNA gene. Received for publication December 8, 2017.

The carrot cyst nematode Heterodera carotae Jones, first producer of carrots at national level with an annual 1950a is a highly specialized parasite infecting only production of 75,950 t (SIAP, 2017). The Tepeaca Valley wild and cultivated carrots (Daucus carota L.) and an in this state is known as an important vegetable proucing herbaceous plant known as the hedge parsley (Torilis area and it is constituted by 13 municipalities. From 2008 leptophylla L.) (Jones, 1950b; Mugniery and Bossis, to 2016 during several nematological surveys in six mu- 1988). This nematode might cause serious problem nicipalities of the Tepeaca Valley, white females of a cyst in the carrot growing areas in Italy leading to yield loss nematode of the genus Heterodera parasitizing carrot around 20 to 90% in carrots (Greco et al., 1994). Dis- roots were found in 61% of sampled fields (Tovar-Soto tribution of this nematode is mostly restricted to Eu- et al., 2009, 2010; Escobar-Avila et al., 2017). In this rope and South Africa, however, in North America study, the cyst nematode was identified asH. carotae. it has been found in Ontario, Canada, and Michi- Although carrot yield losses caused by this nematode gan, United States (Graney, 1985; Berney and Bird, is not exactly estimated, however, the growers have 1992; Subbotin et al., 2010; Madani et al., 2018; Yu et been forced to stop sowing carrots where the nema- al., 2017). In 2016, the Puebla State of Mexico was the tode is present. The aim of this work was to conduct

229 © The Society of Nematologists 2018. First Report of Carrot Cyst Nematode Heterodera carotae in Mexico: Morphological, Molecular Characterization morphological, morphometrics and molecular studies post fixed in 1% osmium tetraoxide, and dehydrat- of H. carotae from Mexico. ed in different concentrations of ethanol (30–100%). Samples were critical point dried and coated with Materials and methods gold palladium for its analysis in a scanning electron microscope (Shepherd and Clark, 1986). The anterior region of the different nematode stages was Nematode population observed and for the J2 and males the number of incisures in the lateral field were counted. Soil with nematodes collected from an infested carrot field from Santa Maria Actipan, Acatzingo, Puebla, Mexico (18˚ 58¢ 486² N; 97˚ 50¢ 295² W; 2246 masl) was used Host range study to establish a laboratory culture. The nematode cul- Nine plant species from five families, which are known ture was maintained in the greenhouse at 20 to 25°C. as hosts from H. carotae and H. cruciferae were Two plastic plots were filled with 15 kg of soil, then 50 used in the test: Apiaceae, carrot (Daucus carota L.); seeds of carrot cv. Christian were sown. After 54 days, Asteraceae, lettuce (Lactuca sativa L.); Brassicaceae, one plot was used for the extraction of second stage broccoli (Brassica oleracea var. italica L.), cabbage juveniles (J2) and males by the centrifugal flotation tech- (B. oleracea var. capitata L.), cauliflower (B. oleracea nique (Jenkins, 1964). After 65 days, the other plot was var. botrytis L.), radish (Raphanus sativus L.) and used to obtain females, the roots were separated, gently Brussels sprouts (B. oleracea var. gemmifera L.); washed, and put in a Petri dish for its examination under Chenopodiaceae, beetroot (Beta vulgaris L.), and Po- Motic stereoscopic microscope. Females were sepa- aceae, wheat (Triticum aestivum L.) (Goodey et al., rated using a brush and kept at 5°C until use. Females 1959; Mugniery and Bossis, 1988; Evans and Russell, were used for morphology and morphometry. The cysts 1993). Plants growing in pots with 3 kg of sterilized were extracted by the Fenwick method (Fenwick, 1940). soil were inoculated with 12,000 eggs and J2. Each plant species was tested in three replicates. After 70 Light microscopy days, the plants were removed and carefully washed Males and second stage juveniles (J2) were killed with tap water, the roots were examined under a in a water bath at 65°C for 5 min, fixed with a mix- stereoscopic microscope for the search of white ture of ethanol, acetic acid, and formalin (20:6:1) and females and cysts. Then roots were stained by the processed into glycerin and the specimens were acid fuchsin lactoglycerol technique to observe nema- mounted in permanent slides with anhydrous glyc- tode stages inside of roots (Bybd et al., 1983). If white erin and paraffin seal for examination (S’Jacob and females or cysts were found on roots, the plant was Van Bezooijen, 1984; Southey, 1986). The meas- considered as a host. ures were taken for length, width at mid body, stylet length, labial region height and width, dorsal Molecular characterization 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 as described in Tanha Maafi et al. (2003) and Southey, 1986). For the observation of the vulval cone, Subbotin (2015). The forward primer TW81 (5¢-GTT cysts were soaked in lactic acid 45% for 15 min, trans- TCC GTA GGT GAA CCT GC-3¢) and the reverse prim- ferred to water and then, the posterior region was dis- er AB28 (5¢-ATA TGC TTA AGT TCA GCG GGT-3¢) sected and mounted in glycerin with paraffin seal for its (Tanha Maafi et al., 2003) were used for amplification of observation and analysis (S’Jacob and Van Bezooijen, the ITS1-5.8S-ITS2 of rRNA and the forward Het-coxiF 1984; De la Jara-Alcocer et al., 1994). The measure- (5¢-TAG TTG ATC GTA ATT TTA ATG G 3¢) and the re- ments taken were vulval slit length, fenestral length verse Het-coxiR (5¢- CCT AAA ACA TAA TGA AAA and width. All measurements were made on Olympus TGW GC-3¢) (Subbotin, 2015) was used for amplifi- CX31 microscope with Infinity Analyze software v6.5.2. cation of the partial COI mtDNA gene. For COI gene study the sequences of H. carotae from Italy, France, Scanning electron microscopy Switzerland and H. cruciferae from Russia, Moscow region and USA, California were also included. The J2, females, males and cysts were processed. The new sequences were deposited in the GenBank under nematode phases were fixed in 4% glutaraldehyde, accession numbers: MG563227 to MG563237.

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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., 2018; 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 tree and was run with four chains for 1.0 × 106 genera- The species of Heterodera found in the Tepeaca tions. The Markov chains were sampled at intervals of Valley, Puebla, Mexico and parasitizing carrots has 100 generations. Two runs were performed for each lemon shaped cysts, ambifenstrate, without bullae, analysis. The topologies were used to generate a 50% long vulval slit and J2 with 4 incisures in the lateral field. majority rule consensus tree. Posterior probabilities are It has been identified as belonging to theGoettingiana given on appropriate clades. The COI sequence align- group (Subbotin et al., 2010). After reviewing the mor- ment was also used to construct phylogenetic network phological characters and morphometrics, it most re- estimation using statistical parsimony as implemented sembles to H. carotae (Jones, 1950a; Mathews, 1975) in POPART software (Bandelt et al., 1999). and H. cruciferae (Franklin, 1945; Stone and Rowe, 1976). Subbotin et al. (2010) stated that H. carotae differed from H. cruciferae by a longer average hya- Results and discussion line part of tail region (28–31 vs. 21–29 μ m) in J2 and a longer average vulval slit (47 vs. 37–46 μ m) in cysts. Symptoms However, the Mexican population has average hyaline part of tail region from 21 to 31 μ m and vulval slit from Patches with poorly developed carrots were observed 39 to 56 μ m which does not make possible to differen- in the infected fields of the Tepeaca Valley, Puebla, tiate between these two species using these charac- Mexico (Fig. 1A,B). White females and cyst attached to ters. From all the population measurements taken, only secondary roots (Fig. 1C,D) sometimes giving the roots the male stylet length showed clear differences in these a “bearded” appearance. The foliage was stunted, two species. The Mexican population male stylet length chlorotic and in some plants reddish (Fig. 1E,F). These (29–36 μ m) (Table 1) fits with those of other populations symptoms correspond to those reported previously for of Heterodera carotae. According to Jones (1950a) H. carotae (Jones, 1950a; Greco, 1986, 1987). and Mathews (1975) the male stylet of H. carotae is 28.8 and 31 to 38 µm, respectively. Madani et al. Nematode description (2018) reported stylet length of 31–35 μm for Italian populations. In contrast with H. cruciferae that has a Cysts shorter male stylet, i.e., 22 to 28 µm (Chizhov et al., 2009), 20 to 24.3 µm (Jabbari and Niknam, 2008), and Light brown lemon shaped with small vulval cone. 25 to 28 µm (Bello et al., 1999) (Table 1). Ambifenestrate. Sub-crystalline layer usually not observed; bullae absent. Eggs in a cyst = 125 (50–322) Host range (Table 1; Fig. 2A,B). Tests of nine plant species from five families for infec- Females tion of H. carotae revealed that only carrot was host for the Mexican population (Table 2). In the roots of White lemon shaped with large egg sac almost of the carrots all nematode stages from J2 to adults (male, size of the female; vulval slit on cone terminus. Labial female and cysts) were observed. No penetration of disc fusioned (Table 1; Fig. 3A,B). the nematode and any stages were observed in roots of the other plants tested. These results agree with Males those reported for H. carotae that has a narrow host range: carrots and Torilis leptophylla, in contrast with Vermiform with a short rounded tail, cuticle annulated, H. crucifeae that infects different species of Brassi- stylet well developed, labial disc slightly oval with six caceae (Subbotin et al., 2010).

231 First Report of Carrot Cyst Nematode Heterodera carotae in Mexico: Morphological, Molecular Characterization

Table 1. Morphometrics of J2, males, females, and cysts from Heterodera carotae and H. cruciferae from different populations. All measurements are in micrometer.

Species Heterodera carotae Heterodera cruciferae Population Mexico Isle of Ireland, UK, Moscow Tabriz, Iran Spain Characters (original) Ely, UK England (Stone region, (Jabbari and (Bello (Jones, and Italy and Russia Niknam, et al., 1999) 1950b) (Mathews, Rowe, (Chizhov 2008) 1975) 1976) et al., 2009)

J2 n = 30 – n = 100 n = 25 n = 28 – n = 30 Length 394 453.8 ± 422 431 ± 19 426 ± 31.8 351 ± 15 438 (407–479) (360–443) 1.38 (375–452) (377–504) (333–381) a 18.9 – 21.4 20.7 ± 1.1 20.5 ± 2.7 20 ± 1.5 22.4 (20–25.2) (18.8–20.14) (18.2–22.7) (14.5–31.8) (17.5–24) b 3 – – – 4.5 ± 0.2 4 ± 0.4 3.4 (2.7–7.4) (2.99–3.46) (4.1–5.1) b¢ 2.52 – – – 2.4 ± 0.2 2.4 ± 0.4 – (2.5–2.81) (2.1–3.1) (2.06–2.7) c 8.2 – – – 9.1 ± 0.5 8.4 ± 0.7 9 (7.9–12.5) (8.18–8.52) (8.2–10) (7.5–10) c¢ 3.7 (3.7–4) – – – 3.7 ± 0.3 4 ± 0.3 (3.5–4.4) – (3.2–4.4) Stylet length 25 (24–26) – 24 (22–25) 24.1 ± 1.6 23 ± 0.9 21 ± 0.8 23.5 (21–25) (20–22.4) (21.9–25.4) Labial region 5 (4–6) – 5 (4–6) 4.1 ± 0.7 – 3–4 – height Labial region 9 (8–10) – 10 (9–10) – – – – diameter Figure 1: A, Carrot field infected with Heterodera carotae; B, Healthy carrot field; C and D, Carrot secondary roots with white females and cysts (indicated by arrows); E and F, Symptoms of an DGO 5 (4–6) – 5–6 5.5 ± 0.7 3–4 5–6 5.2 infected carrot. Chlorosis and reddish foliage. Anterior end 61 (52–73) – 66 (61–72) 68.3 ± 3.1 70 ± 4.1 – – to valve of (62–81) Molecular characterization H. carotae differed in 1 to 5 bp (0.2–0.7%) from the ma- median bulb jority of sequences of H. carotae from Canada, Italy, Anterior end 87 (78–98) – 99 101.6 ± 4.8 103 ± 6.2 95–110 – The ITS rRNA gene sequences obtained from the Switzerland and France. Phylogenetic relationships to excretory (91–117) Mexican population of H. carotae were different in 4 bp of the Mexican population of H. carotae with other pore (0.4%) from each other and differed from others H. populations and species of the Goettingiana group as Oesophageal 131 – – – 95 ± 5.9 – – carotae up to 1.2% and from H. cruciferae up to 1.0%. inferred from Bayesian inference and Statistical parsi- length (104–148) (85–106) Phylogenetic relationships of the Mexican population mony are given in Fig. 6A and B, respectively. Body diameter 21 (19–22) – 20 (19–21) 20.8 ± 1.5 21 ± 2.2 – – of H. carotae with other populations and species of the Although, the Mexican population of H. carotae at mid-body (12–27) Goettingiana group is given in Fig. 5. Sequences of H. fits to the morphological and morphometrical charac- Body diameter 13 (11–14) – 12.4 13.6 ± 0.7 13 ± 0.8 – – carotae are not formed a single clade within the tree. ters of H. carotae published by other authors, never- at anus (11.1–13.4) (11–14) The COI gene sequence of the Mexican population theless, using presently available molecular markers Body diameter 8 (7–9) – 7.1 (5.9–8.3) – – – – of H. carotae was identical to those of the Californian such as the ITS rRNA and partial COI gene sequence at hyaline do not allow to make a reliable and accurate identifica- population of H. cruciferae and H. carotae from France region and Canada. Total seven unique COI haplotypes were tion of H. carotae and H. cruciferae without knowing identified forH. carotae. The Mexican population of plant hosts and stylet length of males.

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Table 1. Morphometrics of J2, males, females, and cysts from Heterodera carotae and H. cruciferae from different populations. All measurements are in micrometer.

233 First Report of Carrot Cyst Nematode Heterodera carotae in Mexico: Morphological, Molecular Characterization

Tail length 48 (44–52) – 51.8 50 ± 2.7 47 ± 4 41 ± 5 (26.2–47) 49.2 Width 331 150–530 309 333 ± 56 398 ± 72.3 398 ± 63.2 399 (326–464) (43.5–58.6) (38–54) (34.1–58.9) (217–472) (165–500) (300–460) (305–571.4) Hyaline region 26 (21–31) – 28.3 25.2 ± 2.7 24 ± 3.3 21.3 ± 1.9 27.3 L/W ratio 1.28 – – – 1.4 ± 0.3 1.3 ± 0.1 – length (20.3–31.8) (17–30) (16–23.4) (20.7–33.9) (1.22–1.31) (0.6–1.8) (1.1–1.5) Males n = 30 – n = 40 n = 25 n = 24 – n = 11 Neck length 80 (63–120) – – 76 ± 18 66 ± 17.4 66.5 ± 15 – Length 1180 1119 ± 1154 1170 ± 79 1156 ± 132.1 961 ± 83 1149 (40–90) (50–84.2) (1114–1219) 14 (1090–1220) (718–1343) (828.6–1028.6) (1081–1283) Fenestral lenth 44 (32–58) – 31 (27–36) 34.5 ± 5.9 41 ± 5.5 – 38.9 (22–58.8) a 37 – – 40.8 ± 5.8 35.3 ± 5.8 38.2 ± 2.5 44.5 (34.3 (27–53) (33.9–42.8) (18.9–47.2) (34.1–41.2) –43.8) Fenestral 50 (38–67) – 39 (35–42) – – 36.6 ± 5 38.4 (30–50) b 6.7 – – – 8.2 ± 0.9 8.3 ± 0.9 – width (31.2–44) (4.7–7.7) (5.2–9.5) (8.1–10) Vulval slit 48 (39–56) – 47 (43–51) 45.5 ± 5.5 46 ± 6.3 42.3 ± 5 36.9 length (32–55) (30.2–50) (28.8–47.7) b¢ 5.8 – – – 6.1 ± 0.4 5.3 ± 0.6 (5–6) – (4.6–6.5) (5.2–6.8) Stylet length 32 (29–36) 28.8 ± 35 (31–38) 24.9 ± 1.1 25 ± 1.7 23 ± 1.6 27.1 (25–28) 0.36 (22–28) (20–24.3) Labial region 5 (4–6) – 7 – 6–8 – – height Labial region 11 (9–13) – 11 – 10–12 – – diameter DGO 5 (4–6) – 5–7 7.2 ± 0.8 4–6 6–7 – Anterior end 103 – 90 (85–105) 97.8 ± 4.9 96 ± 8.7 – – to valve of (101–106) (68–107) median bulb Anterior end to 157 – 153 154.4 ± 12.3 159 ± 10.8 – – excretory pore (146–169) (148–161) (140–182) Body diameter 32 (26–36) 31.4 ± 30 (27–33) 29.1 ± 1.38 33 ± 5.4 – – at mid-body 0.6 (23–43) Oesophageal 175 – – – 144 ± 14.3 – – length (144–238) (119–168) Anterior end to 203 – – – 192 ± 14.8 – – oesophageal (172–267) (163–208) gland base Spicules 36 (32–39) – 34 (31–36) 34.5 ± 2.4 34 ± 2.4 21 ± 4.4 32.2 (29–38) (16–26.2) (25.4–32.3) T(%) 48 (39–57) – 59 50 – 48 ± 14.1 – (13.5–68) Females n = 30 – n = 10 n = 20 – – – Length 474 – 451 – – 365.6 ± 28.15 – (388–686) (375–513) Width 295 – 203 – – 172.9 ± 18 – (229–430) (168–238) a 1.6 (1.6–1.7) – 2.3 (1.9–2.8) – – – – Stylet length 27 (23–32) – 27 (25–31) 26.5 ± 1.5 – 20.1 ± 4 – (15.6–25) Anterior end to 94 (80–116) – 81–119 93 ± 20 – 112.5–143.7 – excretory pore Cysts n = 30 – n = 150 n = 25 n = 25 – n = 30 Length 423 210–680 408 429 ± 67 557 ± 71.6 505.4 ± 58.5 555 (427–624) (excluding (286–575) (218–625) (355–690) (400–609.5) neck)

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Width 331 150–530 309 333 ± 56 398 ± 72.3 398 ± 63.2 399 (326–464) (217–472) (165–500) (300–460) (305–571.4) L/W ratio 1.28 – – – 1.4 ± 0.3 1.3 ± 0.1 – (1.22–1.31) (0.6–1.8) (1.1–1.5) Neck length 80 (63–120) – – 76 ± 18 66 ± 17.4 66.5 ± 15 – (40–90) (50–84.2) Fenestral lenth 44 (32–58) – 31 (27–36) 34.5 ± 5.9 41 ± 5.5 – 38.9 (22–58.8) (27–53) Fenestral 50 (38–67) – 39 (35–42) – – 36.6 ± 5 38.4 (30–50) width (31.2–44) Vulval slit 48 (39–56) – 47 (43–51) 45.5 ± 5.5 46 ± 6.3 42.3 ± 5 36.9 length (32–55) (30.2–50) (28.8–47.7)

Figure 2: Light microscopic photos. A, Cysts; B, Vulval cone. Scale bar: A, 0.5 mm; B, 20 μ m.

Cyst nematodes in Mexico (Jaltomata procumbens (Cav.) J.L. Gentry) in Mexico City (Franco-Navarro et al., 2000). Mexican cyst nematode, Up to now, 15 species of cyst nematodes from four Globodera mexicana (Campos-Vela, 1967; Subbotin et genera, Globodera, Punctodera, Cactodera, and al., 2010) was described from Buffalo burr, Solanum Heterodera have been known in Mexico and with our rostratum Dunal in Mexico in Central Highlands of new finding of H. carotae, total number became 16. Mexico, Toluca Valley in Mexico State and Huamantla Four species of the genus Globodera are reported Valley, Tlaxcala State and later found in Municipality of in this country. The golden cyst nematode Globode- Amecameca in Mexico State (Subbotin et al., 2010). ra rostochiensis has been reported in Coahuila, Gua- The Mexican corn cyst nematode, Punctodera najuato, Hidalgo, Mexico City, Mexico State, Nuevo chalcoensis, is distributed in Jalisco, Mexico State, Leon, Puebla, Tlaxcala, and Veracruz (Brodie, 1998). Michoacan, Puebla, Queretaro, Tlaxcala and Veracruz. This nematode is considered one of the most impor- The yield losses by this nematode can be up to 90%. tant pathogens in Mexico because causes potato yield Several Cactodera spp. were reported. Cactodera losses of 40 to 70% (Santamaria-Cesar and Teliz-Ortiz, cacti is a parasite of cacti and was found in several 1984; Tovar-Soto et al., 2006). Globodera tabacum states of Mexico with warm temperatures and lower was also found in potato Mexican fields (Quiñonez elevations (Baldwin and Mundo-Ocampo, 1991), Cac- and Sosa-Moss, 1977; Sosa-Moss, 1986, 1987). todera amaranthi, in spinach (Spinacia oleracea L.) Globodera bravoae infected creeping false holly and some other Amarantheaceae and Chenopo-

235 First Report of Carrot Cyst Nematode Heterodera carotae in Mexico: Morphological, Molecular Characterization

Figure 3: Scanning electron microscopic photos. A, Entire body of female; B, Vulval cone; C, En face view of male; D, Posterior region of male with bidentate spicules; E, Midbody of J2 with lateral field; F, Tail of J2. diaceae in the Mesa Central (Sossa-Moss, 1986, del Prado-Vera and Miranda, 2008) are parasites 1987), Cactodera salina in Sonora infecting dwarf of barley in the High Valleys of Hidalgo State. In saltwort (Salicornia bigelovii Torr) (Baldwin et al., Texcoco, Cactodera torreyanae was found in romeritos 1997) and Cactodera evansi in carnation (Dianthus (Suaeda torreyana S. Watson) which is a plant that is caryophyllus L.) in Villa Guerrero, Mexico State (Cid del prepared in a traditional Mexican dish (Cid del Prado- Prado-Vera and Rowe, 2000). Catodera galinsogae Vera and Subbotin, 2014). Subbotin et al. (2011) re- (Tovar-Soto et al., 2003) and Cactodera rosae (Cid ported two unidentified Cactodera from Mexico.

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Figure 4: Light microscopic photos. A, Entire body of second stage juvenile (J2); B, Head of J2; C, Tail of J2; D, Head of male; E, Tail with spiclules of male. Scale bar: A, 45 μ m; B and C, 25 μm; D and E, 35 μ m.

Currently, there are no reliable reports on the eco- schachtii is present in Chalco, Mexico State in sugar nomic losses caused by representatives of the genus beet (Sosa-Moss, 1986), Heterodera cyperi in Cype- Cactodera. rus spp. in Tlaxcala and Nayarit (Sosa-Moss, 1987), Four valid and several unidentified species of the Heterodera humuli in the Toluca Valley, Mexico genus Heterodera were found in Mexico. Heterodera State (Sosa-Moss, 1987). A report of Heterodera

237 First Report of Carrot Cyst Nematode Heterodera carotae in Mexico: Morphological, Molecular Characterization

Figure 5: Phylogenetic relationships within populations and species of the Goettingiana group of the genus Heterodera as inferred from Bayesian analysis using the ITS rRNA gene sequence dataset. Posterior probability more than 70% is given for appropriate clades. Newly obtained sequences are indicated in bold. *Identified as Heterodera goettingiana by Ou et al. (unpublished); **Identified as H. goettingiana by Huang et al. (unpublished); ***Identified as Heterodera sp. by Peng et al. (unpublished) in the GenBank. salixophila from Mexico (Tovar-Soto et al., 2006) Gotwald, 1963), and Heterodera from the Goettingi- should be confirmed (Subbotin et al., 2010). Sev- ana group in the High Valleys of Hidalgo (Tovar-Soto eral unidentifiedHeterodera species were reported et al., 2006). in Michoacan State and associated with corn (Zea In this study using morphology, morphometry, mays L.), bean (Phaseolus vulgaris L.), broad beans analysis of the ITS rRNA, and partial COI gene (Vicia faba L.), oat (Avena sativa L.), potato (Solamun sequence and host range test we report the pres- tuberosum L.) and wheat (Triticum aestivum L.) (San- ence of the carrot cyst nematode, Heterodera tacruz-Ulibarri and Pedroza, 1983), Heterodera sp. carotae, in Mexico for the first time. Our study also in sugar beet in Mexico City (Alcocér-Gómez and showed difference between the male stylet length,

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Figure 6: A, Phylogenetic relationships within populations and species of the Goettingiana group of the genus Heterodera as inferred from Bayesian analysis using the COI gene sequence dataset. Posterior probability more than 70% is given for appropriate clades. B, Statistical parsimony network showing the phylogenetic relationships between haplotypes of species of the Goettingiana group. Small black cycles represent missing haplotypes. Pie chart sizes are proportional to the number of samples with a particular haplotype. Newly obtained sequences are indicated in bold. *Originally identified as Heterodera pratensis by Toumi et al. (2013) and later corrected to Heterodera carotae by Madani et al. (2018).

239 First Report of Carrot Cyst Nematode Heterodera carotae in Mexico: Morphological, Molecular Characterization

Table 2. Host range test results of Nickle, W.R. (Ed.), Manual of Agricultural Nematology, Heterodera carotae from the Tepeaca Marcel Dekker Inc., New York, NY, pp. 275–362. Valley, Puebla, Mexico. Baldwin, J.G., Mundo-Ocampo, M., and McClure, M.A. 1997. Cactodera salina n. sp. from the estuary plant, Salicornia bigelovii, in Sonora, Mexico. Journal of Nematology 29: 465–473. Commun Bandelt, H., Forster, P., and Röhl, A. 1999. Medi- Latin name Nematodes name an-joining networks for inferring intraspecific phyloge- nies. Molecular Biology and Evolution 16: 37–48. Bello, A., Escuer, M., and Sanz, R. 1999. El género Apiaceae Heterodera en hortalizas en España. Boletín de Sani- Daucus carota L. Carrot + dad Vegetal. Plagas 25: 321–334. Asteraceae Berney, M.F., and Bird, G.W. 1992. Distribution of Lactuca sativa L. Lettuce – Heterodera carotae and Meloidogyne hapla in Mich- igan carrot production. Journal of Nematology 24: Brassicaceae 776–778. Brassica oleracea Cauliflower – Brodie, B.B. 1998. “Potato cyst nematodes (Glo- var botrytis L. bodera species) in Central and North America”, in Brassica oleracea Cabbage – Marks, J.R., and Brodie, B.B. (Eds), Potato Cyst Nem- var capitata L. atodes Biology, Distribution and Control, CAB Interna- Brassica oleracea Brussels – tional, Wallingford, Oxon, pp. 317–331. var gemmifera L. sprouts Bybd, D.W. Jr, Kirkpatrick, T., and Barker, K.R. Brassica oleracea Broccoli – 1983. An improved technique for clearing and staining var italica L. plant tissues for detection of nematodes. Journal of Raphanus sativus L. Radish – Nematology 15: 142–143. Chenopodiaceae Campos-Vela, A. 1967. Taxonomy, life cycle and host range of Heterodera mexicana. n. sp. (Nematode: Beta vulgaris L. Beetroot – Heteroderidae). Ph.D. Thesis, University of Wisconsin. Poaceae Chizhov, V.N., Pridannikov, M.V., Nasonova, L.N., Triticum aestivum L. Wheat – and Subbotin, S.A. 2009. Heterodera cruciferae Frank- lin, 1945, parasite of Brassica oleraceae L. from flood- +All developmental stages were observed (J2 to adults). land fields in the Moscow region, Russia.Russian Jour- –Non developmental stages were observed. nal of Nematology 17: 107–113. Cid del Prado-Vera, I., and Miranda, B.L. 2008. A second cyst-forming nematode parasite of barley (Hor- being longer for H. carotae and that accurate differ- deum vulgare L. var. Esmeralda) from Mexico. Nemat- entiation between H. carotae and H. cruciferae could ropica 38: 105 –114. be done based on a multidisciplinary approach using Cid del Prado-Vera, I., and Rowe, J.A. 2000. Cac- the knowledge of plant-host. todera evansi sp. n. and Meloidodera astonei sp. n. (Tylenchida: Heteroderidae) from Mexico. International Acknowledgments Journal of Nematology 10: 159–168. Cid del Prado-Vera, I., and Subbotin, S.A. 2014. A new The first author acknowledges CONACyT for the cyst nematode, Cactodera torreyanae sp. n. (Tylenchida: financial support provided for her PhD studies. Heteroderidae), parasitising romerito, Suaeda torreyana, in Texcoco, Mexico. Nematology 16: 163–174. De la Jara-Alcocer, F., Zeron-Bravo, F., Torres-Cor- References onel, R., and Tovar-Soto, A. 1994. Manual de Prácticas de Nematología Agrícola, 2ª Edición., Escuela Nacional Alcocér-Gómez, L., and Gotwald, G.C. 1963. De- de Ciencias Biológicas-IPN, México, DF. terminación de nematodos fitoparásitos en México. Escobar-Avila, I.M., Medina-Canales, M.G., and Fitófilo 39: 3–26. Tovar-Soto, A. 2017. Distribution, life cycle and histo- Baldwin, J.G., and Mundo-Ocampo, M. 1991. “Het- logical changes by Heterodera sp. in carrot in Puebla. eroderinae, cyst and noncyst forming nematodes”, in Mexican. Journal of Phytopathology 35: 304–313.

240 JOURNAL OF NEMATOLOGY

Evans, K., and Russell, M.D. 1993. The population Mathews, H.J.P. 1975. Heterodera carotae. CIH De- dynamics in microplots of brassica and beet cyst nem- scriptions of plant parasitic nematodes, Set 5. No. 61. atode in rotations which include oilseed rape. Nemato- St. Albans: Commonwealth Institute of Helminthology. logica 39: 411–414. Mugniery, D., and Bossis, M. 1988. Heterodera caro- Fenwick, D.W. 1940. Methods for the recovery and tae Jones, 1950. 1. Gamme d’hôtes, vitesse de dével- counting of cysts of Heterodera schachtii from soil. oppement, cycle. Revue de Nématologie 11: 307– 313. Journal of Helminthology 18: 155–172. Posada, D. 2008. jModelTest: Phylogenetic mod- Franco-Navarro, F., Cid del Prado-Vera, I., and Lam- el averaging. Molecular Biology and Evolution 25: othe-Argumedo, R. 2000. Globodera bravoae n. sp. 1253–1256. (Tylenchida: Heteroderidae) from Mexico. International Quiñonez, F.J.A., and Sosa-Moss, C. 1977. Car- Journal of Nematology 10: 169 –176. acterización morfométrica de tres poblaciones mex- Franklin, M.T. 1945. On Heterodera cruciferae n. sp. icanas del género Globodera. Nematropica 7: 13–14. of brassicas, and on a Heterodera strain infecting clo- S’Jacob, J.J., and Van Bezooijen, J. 1984. A manu- ver and dock. Journal of Helminthology 21: 71–84. al for practical work in nematology. Wageningen: Wa- Goodey, J.B., Franklin, M.T., and Hooper, D.J. 1959. geningen Agricultural University. Supplement to: The nematode parasites of plants cat Santacruz-Ulibarri, H., and Pedroza, M.A.A. 1983. alogued under their hosts 1955–1958. Farnham, Royal: Estudio preliminar de la nematofauna en el estado de Commonwealth Agricultural Bureaux, 66 pp. Michoacán. Nematropica 13: 120–121. Graney, L.S.O. 1985. Observation on the morphol- Santamaria-Cesar, P., and Teliz-Ortiz, D. 1984. ogy of Heterodera carotae and Heterodera avenae in Control de Globodera rostochiensis (Woll., 1923) de Michigan. Journal of Nematology 17: 519. la papa Solanum tuberosum en Tlaxcala. XI Congre- Greco, N. 1986. “The carrot cyst nematode”, in so Nacional de la Sociedad Mexicana de Fitopatología, Lamberti, F., and Taylor, C.E. (Eds), Cyst nematodes, San Luis Potosí, San Luis Potosí, México, pp. 131. Plenum Press, London, UK, pp. 333–346. Shepherd, A.M., and Clark, S.A. 1986. “Preparation Greco, N. 1987. Heterodera carotae: A destruc- of nematodes for electron microscopy”, in Southey, J.F. tive nematode of carrot. Nematology circular No. 140. (Ed.), Laboratory Methods for Work with Plant and Soil Gainesville, FL: Florida Department of Agriculture and Nematodes, Ministry of Agriculture, Fisheries and Food Consumer Services. Division of Plant Industries. 4 pp. Reference Book 402, Her Majesty’s Stationery Office, Greco, N., D´Addabbo, T., Brandonisio, A., and Elia, London, pp. 121–131. F. 1994. Damage to Italian crops caused by cyst-forming SIAP. 2017. Secretaria de Información Agroalimen- nematodes. Journal of Nematology 25: 836–842. taria y Pesquera. HYPERLINK “http://www.siap.gob. Huelsenbeck, J.P., and Ronquist, F. 2001. mx” www.siap.gob.mx. MrBAYES: Bayesian inference of phylogenetic trees. Sosa-Moss, C. 1986. Cyst nematodes in Mexico, Bioinformatics 17: 754–755. Central and South America. Nematologia mediterranea Jabbari, H., and Niknam, G. 2008. SEM observa- 15: 13–19. tions and morphometrics of the cabbage cyst nema- Sosa-Moss, C. 1987. Cyst nematodes in Mexico, tode, Heterodera cruciferae Franklin, 1945, collected Central and South America, in Lamberti, F., and Taylor, where Brassica spp. are grown in Tabriz, Iran. Turkish C.E. (Eds), Cyst nematodes, Plenum Press, London, Journal of Zoology 32: 253–262. pp. 397–398. Jenkins, D.W. 1964. A rapid centrifugal-flotation Southey, J.F. 1986. Laboratory methods for work technique for separating nematodes from soil. Plant with plant and soil nematodes. London: Ministery of Disease Reporter 48: 692. Agriculture, Fisheries and Food. Jones, F.G.W. 1950a. A new species of root eelworm Stone, A.R., and Rowe, J.A. 1976 Heterodera cru- attacking carrots. Nature 165: 81. ciferae. CIH descriptions of plant-parasitic nematodes. Jones, F.G.W. 1950b. Observations on the beet eel- Set 6, No. 90. St. Albans: Commonwealth Institute of worm and other cyst-forming species of Heterodera. Helminthology. Annals of Applied Biology 37: 407–440. Subbotin, S.A. 2015. Heterodera sturhani sp. n. Madani, M., Palomares-Rius, J.E., Vovlas, N., Castillo, from China, a new species of the Heterodera avenae P., and Tenuta, M. 2018. Integrative diagnosis of carrot species complex (Tylenchida: Heteroderidae). Russian cyst nematode (Heterodera carotae) using morphology Journal of Nematology 23: 145–152. and several molecular markers for an accurate iden- Subbotin, S.A., Cid Del Prado-Vera, I., Mundo- tification. European Journal of Plant Pathology 150: Ocampo, M., and Baldwin, J.G. 2011. Identification, 1023–1039. phylogeny and phylogeography of circumfenestrate

241 First Report of Carrot Cyst Nematode Heterodera carotae in Mexico: Morphological, Molecular Characterization cyst nematodes (Nematoda: Heteroderidae) as inferred Tovar-Soto, A., Cid del Prado-Vera, I., Sandoval-Islas, from analysis of ITS-rDNA. Nematology 13: 805–824. J.S., Martinez-Garza, A., Nicol, J.M., and Evans, K. 2006. Subbotin, S.A., Mundo-Ocampo, M., and Baldwin, Los Nematodos Formadores de Quistes en México. J.G. 2010. Systematics of cyst nematodes (Nematoda: Mexican Journal of Phytopathology 24: 145–151. Heteroderinae). Nematology monographs and per- Tovar-Soto, A., Hernández-López, J.M., and spectives. Vol. B. The Netherlands: Brill. Torres-Coronel, R. 2009. Nematodos Formadores de Subbotin, S.A., Vierstraete, A., De Ley, P., Rowe, Quistes en la Zona Hortícola del Estado de Puebla. XI J., Waeyenberge, L., Moens, M., and Vanfleteren, J.R. Congreso Internacional/XXXVI Nacional de la Sociedad 2001. Phylogenetic relationships within the cyst-forming Mexicana de Fitopatología, A.C. Sociedad Mexicana nematodes (Nematoda, Heteroderidae) based on anal- de Fitopatología. Acapulco, Gro., México, pp. 4. ysis of sequences from the ITS regions of ribosomal Tovar-Soto, A., Medina-Canales, M.G., and DNA. Molecular Phylogenetics and Evolution 21: 1–16. Torres-Coronel, R. 2010. Nematodos Fitoparásitos Tanha Maafi, Z., Subbotin, S.A., and Moens, M. Asociados a Hortalizas en el Valle de Tepeaca, Puebla, 2003. Molecular identification of cyst-forming nem- México. XII Congreso Internacional/XXXVII Nacional atodes (Heteroderidae) from Iran and a phylogeny de la Sociedad Mexicana de Fitopatología, A.C. based on the ITS sequences of rDNA. Nematology 5: Sociedad Mexicana de Fitopatología. Mérida, Yuc. 99 –111. México. pp. 4. Toumi, F., Waeyenberge, L., Viaene, N., Dababat, A., Vovlas, A., Santoro, S., Radicci, V., Leonetti, P., Nicol, J.M., Ogbonnaya, F., and Moens, M. 2013. De- Castillo, P., and Palomares-Rius, J.E. 2017. Host-suit- velopment of two species-specific primer sets to detect ability of black medick (Medicago lupulina L.) and ad- the cereal cyst nematodes Heterodera avenae and Het- ditional molecular markers for identification of the pea erodera filipjevi. European Journal of Plant Pathology cyst nematode Heterodera goettingiana. European 136: 613–624. Journal of Plant Pathology 149: 193–199. Tovar-Soto, A., Cid del Prado-Vera, I., Nicol, J.M., Yu, Q., Ponomareva, E., Van Dyk, D., McDonald, Evans, K., Sandoval-Islas, J.S., and Martinez-Garza, A. M.R., Sun, F., Madani, M., and Tenuta, M. 2017. First 2003. Cactodera galinsogae n. sp. (Tylenchida: Het- report of the carrot cyst nematode (Heterodera carotae eroderidae) on barley (Hordeum vulgare L.) of the high Jones) from carrot fields in Ontario, Canada. Plant Dis- valleys of Mexico. Nematropica 33: 41–54. ease 101: 1056.

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