JOURNAL of NEMATOLOGY First Report of Carrot Cyst

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. The Markov chains were sampled at intervals of lemon shaped cysts, ambifenstrate, without bullae, 100 generations. Two runs were performed for each long vulval slit and J2 with 4 incisures in the lateral analysis. The topologies were used to generate a 50% field. It has been identified as belonging to the Goet- majority rule consensus tree. Posterior probabilities are tingiana group (Subbotin et al., 2010). After reviewing given on appropriate clades. The COI sequence align- the morphological characters and morphometrics, ment was also used to construct phylogenetic network it most resembles to H. carotae (Jones, 1950a; estimation using statistical parsimony as implemented Mathews, 1975) and H. cruciferae (Franklin, 1945; in POPART software (Bandelt et al., 1999). Stone and Rowe, 1976). Subbotin et al. (2010) stated that H. carotae differed from H. cruciferae by a Results and discussion longer average hyaline part of tail region (28–31 vs. 21–29 μ m) in J2 and a longer average vulval slit Symptoms (47 vs. 37–46 μ m) in cysts. However, the Mexican population has average hyaline part of tail region from Patches with poorly developed carrots were observed 21 to 31 μ m and vulval slit from 39 to 56 μ m which in the infected fields of the Tepeaca Valley, Puebla, does not make possible to differentiate between Mexico (Fig. 1A,B). White females and cyst attached to these two species using this characters. From all secondary roots (Fig. 1C, D) sometimes giving the roots the population measurements taken, only the male a “bearded” appearance. The foliage was stunted, stylet length showed clear differences in these two chlorotic and in some plants reddish (Fig. 1E, F). These species. The Mexican population male stylet length symptoms correspond to those reported previously for (29–36 μ m) (Table 1) fit with populations of Heterodera H. carotae (Jones, 1950a; Greco, 1986, 1987). carotae. According to Jones (1950a) and Mathews (1975) the male stylet of H. carotae is 28.8 and 31 Nematode description to 38 µm, respectively. In contrast with H. cruciferae that has a shorter male stylet, i.e., 22 to 28 µm Cysts (Chizhov et al., 2009), 20 to 24.3 µm (Jabbari and Niknam, 2008), and 25 to 28 µm (Bello et al., 1999) Light brown lemon shaped with small vulval cone. (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).

3 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 Heterodera cruciferae from different reported populations. All measurements are in micrometer.

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

J2 n = 30 – n = 100 n = 25 n = 28 – n = 30 Length 394 453.8 ± 422 431 ± 19 426 ± 31.8 351 ± 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 Figure 1: A, Carrot field infected with Heterodera carotae; B, Healthy carrot field; C and D, Carrot Labial region 9 (8–10) – 10 (9–10) – – – – secondary roots with white females and cysts (indicated by arrows); E and F, Symptoms of an diameter infected carrot. Chlorosis and reddish foliage. DGO 5 (4–6) – 5–6 5.5 ± 0.7 3–4 5–6 5.2 Anterior end 61 (52–73) – 66 (61–72) 68.3 ± 3.1 70 ± 4.1 – – Molecular characterization of H. carotae differed in 1 to 5 bp (0.2%–0.7%) from to valve of (62–81) the majority of sequences of H. carotae from Canada, median bulb The ITS rRNA gene sequences obtained from the Italy Switzerland and France. Phylogenetic relation- Anterior end 87 (78–98) – 99 101.6 ± 4.8 103 ± 6.2 95–110 – Mexican population of H. carotae were different in 4 bp ships of the Mexican population of H. carotae with to excretory (91–117) (0.4%) from each other and differed from others H. other populations and species of the Goettingiana pore carotae up to 1.2% and from H. cruciferae up to 1.0%. group as inferred from Bayesian inference and Sta- Oesophageal 131 – – – 95 ± 5.9 – – Phylogenetic relationships of the Mexican population tistical parsimony are given in Fig. 6A,B, respectively. length (104–148) (85–106) of H. carotae with other populations and species of the Although, the Mexican population of H. carotae fits Goettingiana group is given in Fig. 5. Sequences of H. Body diameter 21 (19–22) – 20 (19–21) 20.8 ± 1.5 21 ± 2.2 – – to the morphological and morphometrical characters at mid-body (12–27) carotae are not formed a single clade within the tree. of H. carotae published by other authors, neverthe- Body diameter 13 (11–14) – 12.4 13.6 ± 0.7 13 ± 0.8 – – The COI gene sequence of the Mexican population less, using morphology and even molecular markers at anus (11.1–13.4) (11–14) of H. carotae was identical to those of the Californian such as the ITS rRNA and partial COI gene sequence population of H. cruciferae and H. carotae from France do not allow to make a reliable and accurate identifica- Body diameter 8 (7–9) – 7.1 (5.9–8.3) – – – – and Canada. Total seven unique COI haplotypes tion of H. carotae and H. cruciferae without knowing at hyaline region were identified forH. carotae. The Mexicanz population plant hosts.

4 JOURNAL OF NEMATOLOGY

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

5 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 Stylet length 27 (23–32) – 27 (25–31) 26.5 ± 1.5 – 20.1 ± 4 – (43.5–58.6) (38–54) (34.1–58.9) (15.6–25) Hyaline region 26 (21–31) – 28.3 25.2 ± 2.7 24 ± 3.3 21.3 ± 1.9 27.3 Anterior end 94 (80–116) – 81–119 93 ± 20 – 112.5–143.7 –0.5 length (20.3–31.8) (17–30) (16–23.4) (20.7–33.9)0.5 to excretory Population Mexico Isle of Ireland, UK (Stone Moscow Tabriz, Iran Spain (Bello et pore Characters (original) Ely, UK England and Rowe, region, (Jabbari and al., 1999) Population Mexico Isle of Ireland, UK (Stone Moscow Tabriz, Iran Spain (Bello et (Jones, and Italy 1976) Russia Niknam, 2008) Characters (original) Ely, UK England and Rowe, region, (Jabbari and al., 1999) 1950a) (Mathews, (Chizhov et (Jones, and Italy 1976) Russia Niknam, 2008) 1975) al., 2009) 1950a) (Mathews, (Chizhov et Males n = 30 – n = 40 n = 25 n = 24 – n = 11 1975) al., 2009) Length 1180 1119 ± 1154 1170 ± 79 1156 ± 132.1 961 ± 83(828.6– 1149 Cysts n = 30 – n = 150 n = 25 n = 25 – n = 30 (1114–1219) 14 (1090–1220) (718–1343) 1028.6) (1081–1283) Length 423 210–680 408 429 ± 67 557 ± 71.6 505.4 ± 58.5 555 (427–624) a 37 – – 40.8 ± 5.8 35.3 ± 5.8 38.2 ± 2.5 44.5 (34.3 (excluding (286–575) (218–625) (355–690) (400–609.5) (33.9–42.8) (18.9–47.2) (34.1–41.2) –43.8) neck) b 6.7 (4.7–7.7) – – – 8.2 ± 0.9 8.3 ± 0.9 – Width 331 150–530 309 333 ± 56 398 ± 72.3 398 ± 63.2(305– 399 (326–464) (5.2–9.5) (8.1–10) (217–472) (165–500) (300–460) 571.4) L/W ratio 1.28 – – – 1.4 ± 0.3 1.3 ± 0.1 – b¢ 5.8 (4.6–6.5) – – – 6.1 ± 0.4 5.3 ± 0.6 (5–6) – (5.2–6.8) (1.22–1.31) (0.6–1.8) (1.1–1.5) Stylet length 32 (29–36) 28.8 ± 35 (31–38) 24.9 ± 1.1 25 ± 1.7 23 ± 1.6 27.1 (25–28) Neck length 80 (63–120) – – 76 ± 18 66 ± 17.4 66.5 ± 15 – 0.36 (22–28) (20–24.3) (40–90) (50–84.2) Labial region 5 (4–6) – 7 – 6–8 – – Fenestral lenth 44 (32–58) – 31 (27–36) 34.5 ± 5.9 41 ± 5.5 – 38.9 (22–58.8) height (27–53) Labial region 11 (9–13) – 11 – 10–12 – – Fenestral width 50 (38–67) – 39 (35–42) – – 36.6 ± 5 38.4 (30–50) diameter (31.2–44) DGO 5 (4–6) – 5–7 7.2 ± 0.8 4–6 6–7 – 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) Anterior end 103 – 90 (85–105) 97.8 ± 4.9 96 ± 8.7 – – to valve of (101–106) (68–107) median bulb Anterior end 157 – 153 154.4 ± 12.3 159 ± 10.8 – – to excretory (146–169) (148–161) (140–182) pore Body diameter 32 (26–36) 31.4 ± 0.6 30 (27–33) 29.1 ± 1.38 33 ± 5.4 – – at mid-body (23–43) Oesophageal 175 – – – 144 ± 14.3 – – length (144–238) (119–168) Anterior 203 – – – 192 ± 14.8 – – end to (172–267) (163–208) oesophageal 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% – 59% 50% – 48 ± 14.1 –0.5 (39–57%) (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) – – – –

6 JOURNAL OF NEMATOLOGY

Stylet length 27 (23–32) – 27 (25–31) 26.5 ± 1.5 – 20.1 ± 4 – (15.6–25) Anterior end 94 (80–116) – 81–119 93 ± 20 – 112.5–143.7 –0.5 to excretory pore Population Mexico Isle of Ireland, UK (Stone Moscow Tabriz, Iran Spain (Bello et Characters (original) Ely, UK England and Rowe, region, (Jabbari and al., 1999) (Jones, and Italy 1976) Russia Niknam, 2008) 1950a) (Mathews, (Chizhov et 1975) al., 2009) 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) Width 331 150–530 309 333 ± 56 398 ± 72.3 398 ± 63.2(305– 399 (326–464) (217–472) (165–500) (300–460) 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 width 50 (38–67) – 39 (35–42) – – 36.6 ± 5 38.4 (30–50) (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 Heterodera have been known in Mexico and with our new finding of H. carotae, total number became 16. Up to now, 15 species of cyst nematodes from four Four species of the genus Globodera are reported genera, Globodera, Punctodera, Cactodera, and in this country. The golden cyst nematode Globodera

7 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. rostochiensis has been reported in Coahuila, Guanajuato, was also found in potato Mexican fields (Quiñonez Hidalgo, Mexico City, Mexico State, Nuevo Leon, Puebla, and Sosa-Moss, 1977; Sosa-Moss, 1986, 1987). Tlaxcala, and Veracruz (Brodie, 1998). This nema- Globodera bravoae infected creeping false holly tode is considered one of the most important patho- (Jaltomata procumbens (Cav.) J.L. Gentry) in Mexico City gens in Mexico because causes potato yield losses (Franco-Navarro et al., 2000). Mexican cyst nematode, of 40% to 70% (Santamaria-Cesar and Teliz-Ortiz, Globodera mexicana (Campos-Vela, 1967; Subbotin et 1984; Tovar-Soto et al., 2006). Globodera tabacum al., 2010) was described from Buffalo burr, Solanum

8 JOURNAL OF NEMATOLOGY

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 μ m; D and E, 35 μ m. rostratum Dunal in Mexico in Central Highlands of The Mexican corn cyst nematode, Punctodera Mexico, Toluca Valley in Mexico State and Huamantla chalcoensis, is distributed in Jalisco, Mexico State, Valle, Tlaxcala State and later found in Municipality of Michoacan, Puebla, Queretaro, Tlaxcala and Veracruz. Amecameca in Mexico State (Subbotin et al., 2010). The yield losses by this nematode can be up to 90%.

9 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.

Several Cactodera spp. were reported. Cactodera 1997) and Cactodera evansi in carnation (Dianthus cacti is a parasite of cacti and was found in several caryophyllus L.) in Villa Guerrero, Mexico State (Cid del states of Mexico with warm temperatures and lower Prado-Vera and Rowe, 2000). Catodera galinsogae elevations (Baldwin and Mundo-Ocampo, 1991), Cac- (Tovar-Soto et al., 2003) and Cactodera rosae (Cid todera amaranthi, in spinach (Spinacia oleracea L.) del Prado-Vera and Miranda, 2008) are parasites and some other Amarantheaceae and Chenopo- of barley in the High Valleys of Hidalgo State. In diaceae in the Mesa Central (Sossa-Moss, 1986, Texcoco, Cactodera torreyanae was found in romeritos 1987), Cactodera salina in Sonora infecting dwarf (Suaeda torreyana S. Watson) which is a plant that is saltwort (Salicornia bigelovii Torr) (Baldwin et al., prepared in a traditional Mexican dish (Cid del Prado-

10 JOURNAL OF NEMATOLOGY

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

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

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. (2017).

State (Sosa-Moss, 1987). A report of Heterodera Table 2. Host range test results of salixophila from Mexico (Tovar-Soto et al., 2006) Heterodera carotae from the Tepeaca should be confirmed (Subbotin et al., 2010). Sev- Valley, Puebla, Mexico. eral unidentifiedHeterodera species were reported in Michoacan State and associated with corn (Zea mays L.), bean (Phaseolus vulgaris L.), broad beans (Vicia faba L.), oat (Avena sativa L.), potato (Solamun Commun Latin name Nematodes tuberosum L.) and wheat (Triticum aestivum L.) name (Santacruz-Ulibarri and Pedroza, 1983). Heterodera sp. in sugar beet in Mexico City (Alcocér-Gómez Apiaceae and Gotwald, 1963), and Heterodera from the Goettingiana group in the High Valleys of Hidalgo Daucus carota L. Carrot + (Tovar-Soto et al., 2006). Asteraceae In this study using morphology, morphometry, Lactuca sativa L. Lettuce – analysis of the ITS rRNA, and partial COI gene Brassicaceae sequence and host range test we report the presence of the carrot cyst nematode, Heterodera carotae, in Brassica oleracea Cauliflower – Mexico for the first time. Our study also showed dif- var botrytis L. ference between the male stylet length, being longer Brassica oleracea Cabbage – for H. carotae and that accurate differentiation be- var capitata L. tween H. carotae and H. cruciferae could be only Brassica oleracea Brussels – done based on a multidisciplinary approach using the var gemmifera L. sprouts knowledge of plant-host. Brassica oleracea Broccoli – var italica L. Acknowledgement Raphanus sativus L. Radish – Chenopodiaceae The first author acknowledges CONACyT for the Beta vulgaris L. Beetroot – financial support provided for her PhD studies. Poaceae Triticum aestivum L. Wheat – References +All developmental stages were observed (J2 to adults); – Non developmental stages were observed. Alcocér-Gómez, L., and Gotwald, G.C. 1963. Determinación de nematodos fitoparásitos en México. Fitófilo 39: 3–26. Vera and Subbotin, 2014). Subbotin et al. (2011) Baldwin, J.G., and Mundo-Ocampo, M. 1991. Het- reported two identifiedCactodera from Mexico. eroderinae, cyst and noncyst forming nematodes, in Currently, there are no reliable reports on the Nickle, W.R. (Ed.), Manual of Agricultural Nematology, economic losses caused by representatives of the genus Marcel Dekker Inc., New York, NY, pp. 275–362. Cactodera. Baldwin, J.G., Mundo-Ocampo, M., and McClure, Four valid and several unidentified species of the M.A. 1997. Cactodera salina n. sp. from the estuary genus Heterodera were found in Mexico. Heterodera plant, Salicornia bigelovii, in Sonora, Mexico. Journal of schachtii is present in Chalco, Mexico State in sugar Nematology 29: 465–73. beet (Sosa-Moss, 1986), Heterodera cyperi in Cype- Bandelt, H., Forster, P., and Röhl, A. 1999. Median- rus spp. in Tlaxcala and Nayarit (Sosa-Moss, 1987), joining networks for inferring intraspecific phylogenies. Heterodera humuli in the Toluca Valley, Mexico Molecular Biology and Evolution 16: 37–48.

12 JOURNAL OF NEMATOLOGY

Bello, A., Escuer, M., and Sanz, R. 1999. El género sp. (Tylenchida: Heteroderidae) from Mexico. Interna- Heterodera en hortalizas en España. Boletín de Sanidad tional. Journal of Nematology 10: 169–76. Vegetal. Plagas 25: 321–34. Franklin, M.T. 1945. On Heterodera cruciferae n. sp. Berney, M.F., and Bird, G.W. 1992. Distribution of of brassicas, and on a Heterodera strain infecting clo- Heterodera carotae and Meloidogyne hapla in Michi- ver and dock. Journal of Helminthology 21: 71–84. gan carrot production. Journal of Nematology 24(4S): Goodey, J.B., Franklin, M.T., and Hooper, D.J. 1959. pp. 776–8. Supplement to: The nematode parasites of plants cat- Brodie, B.B. 1998. Potato cyst nematodes (Globod- alogued under their hosts 1955–1958. Farnham, Royal: era species) in Central and North America, in Marks, Commonwealth Agricultural Bureaux, 66 pp. J.R., and Brodie, B.B. (Eds), Potato Cyst Nematodes Graney, L.S.O. 1985. Observation on the morphol- Biology, Distribution and Control, CAB International. ogy of Heterodera carotae and Heterodera avenae in Wallingford, Oxon, pp. 317–31. Michigan. Journal of Nematology 17: 519. Bybd, D.W. Jr, Kirkpatrick, T., and Barker, K.R. Greco, N. 1986. The carrot cyst nematode, in 1983. An improved technique for clearing and staining Lamberti, F., and Taylor, C.E. (Eds), Cyst nematodes. plant tissues for detection of nematodes. Journal of Plenum Press, London, UK, pp. 333–46. Nematology 15: 142–3. Greco, N. 1987. Heterodera carotae: A destruc- Campos-Vela, A. 1967. Taxonomy, life cycle and tive nematode of carrot. Nematology circular No. 140. host range of Heterodera mexicana. n. sp. (Nematode: Gainesville, FL: Florida Department of Agriculture and Heteroderidae). Ph.D. Thesis, University of Wisconsin. Consumer Services. Division of Plant Industries. 4 pp. Chizhov, V.N., Pridannikov, M.V., Nasonova, L.N., and Greco, N., D´Addabbo, T., Brandonisio, A., and Elia, Subbotin, S.A. 2009. Heterodera cruciferae Franklin, F. 1994. Damage to Italian crops caused by cyst-forming 1945, parasite of Brassica oleraceae L. from floodland nematodes. Journal of Nematology 25(4S): 836–42. fields in the Moscow region, Russia.Russian Journal of Huelsenbeck, J.P., and Ronquist, F. 2001. Nematology 17: 107–13. MrBAYES: Bayesian inference of phylogenetic trees. Cid del Prado-Vera, I., and Miranda, B.L. 2008. Bioinformatics 17: 754–5. A second cyst-forming nematode parasite of barley Jabbari, H., and Niknam, G. 2008. SEM observa- (Hordeum vulgare L. var. Esmeralda) from Mexico. tions and morphometrics of the cabbage cyst nema- Nematropica 38(2): 105–14. tode, Heterodera cruciferae Franklin, 1945, collected Cid del Prado-Vera, I., and Rowe, J.A. 2000. Cac- where Brassica spp. are grown in Tabriz, Iran. Turkish todera evansi sp. n. and Meloidodera astonei sp. n. Journal of Zoology 32: 253–62. (Tylenchida: Heteroderidae) from Mexico. International Jenkins, D.W. 1964. A rapid centrifugal-flotation Journal of Nematology 10: 159–68. technique for separating nematodes from soil. Plant Cid del Prado-Vera, I., and Subbotin, S.A. 2014. A new Disease Reporter 48: 692. cyst nematode, Cactodera torreyanae sp. n. (Tylenchida: Jones, F.G.W. 1950a. A new species of root eelworm Heteroderidae), parasitising romerito, Suaeda torreyana, attacking carrots. Nature 165: 81. in Texcoco, Mexico. Nematology 16: 163–74. Jones, F.G.W. 1950b. Observations on the beet De la Jara-Alcocer, F., Zeron-Bravo, F., Torres-Coronel, eelworm and other cyst-forming species of Heterodera. R., and Tovar-Soto, A. 1994. Manual de Prácticas de Annals of Applied Biology 37: 407–40. Nematología Agrícola, 2ª Edición. Escuela Nacional de Madani, M., Palomares-Rius, J.E., Vovlas, N., Castillo, Ciencias Biológicas-IPN. México, DF. P., and Tenuta, M. 2017. Integrative diagnosis of carrot Escobar-Avila, I.M., Medina-Canales, M.G., and cyst nematode (Heterodera carotae) using morphology Tovar-Soto, A. 2017. Distribution, life cycle and histo- and several molecular markers for an accurate iden- logical changes by Heterodera sp. in carrot in Puebla. tification. European Journal of Plant Pathology 150: Mexican. Journal of Phytopathology 35: 304–13. 1023–39. Evans, K., and Russell, M.D. 1993. The population Mathews, H.J.P. 1975. Heterodera carotae. CIH dynamics in microplots of brassica and beet cyst nem- Descriptions of plant parasitic nematodes, Set 5. atode in rotations which include oilseed rape. Nemato- No. 61. St. Albans: Commonwealth Institute of logica 39(3): 411– 4. Helminthology. Fenwick, D.W. 1940. Methods for the recovery and Mugniery, D., and Bossis, M. 1988. Heterodera carotae counting of cysts of Heterodera schachtii from soil. Jones, 1950. 1. Gamme d’hetes, vitesse de développe- Journal of Helminthology 18: 155–72. ment, cycle. Revue de Nématologie 11: 307–13. Franco-Navarro, F., Cid del Prado-Vera, I., and Posada, D. 2008. jModelTest: Phylogenetic model Lamothe-Argumedo, R. 2000. Globodera bravoae n. averaging. Molecular Biology and Evolution 25: 1253–6.

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

Quiñonez, F.J.A., and Sosa-Moss, C. 1977. Carac- Subbotin, S.A., Vierstraete, A., De Ley, P., Rowe, terización morfométrica de tres poblaciones mexicanas J., Waeyenberge, L., Moens, M., and Vanfleteren, J.R. del género Globodera. Nematropica 7: 13–14. 2001. Phylogenetic relationships within the cyst-forming S’Jacob, J.J., and Van Bezooijen, J. 1984. A manual nematodes (Nematoda, Heteroderidae) based on analysis for practical work in nematology. Wageningen: Wagen- of sequences from the ITS regions of ribosomal DNA. ingen Agricultural University, 76 pp. Molecular Phylogenetics and Evolution 21: 1–16. Santacruz-Ulibarri, H., and Pedroza, M.A.A. 1983. Tanha Maafi, Z., Subbotin, S.A., and Moens, M. Estudio preliminar de la nematofauna en el estado de 2003. Molecular identification of cyst-forming nem- Michoacán. Nematropica 13: 120–1. atodes (Heteroderidae) from Iran and a phylogeny Santamaria-Cesar, P., and Teliz-Ortiz, D. 1984. based on the ITS sequences of rDNA. Nematology 5: Control de Globodera rostochiensis (Woll., 1923) de la 99 –111. papa Solanum tuberosum en Tlaxcala. XI Congreso Toumi, F., Waeyenberge, L., Viaene, N., Dababat, Nacional de la Sociedad Mexicana de Fitopatología, A., Nicol, J.M., Ogbonnaya, F., and Moens, M. 2013. San Luis Potosi, San Luis Potosi, México, 131p. Development of two species-specific primer sets to Shepherd, A.M., and Clark, S.A. 1986. Preparation detect the cereal cyst nematodes Heterodera avenae of nematodes for electron microscopy, in Southey, J.F. and Heterodera filipjevi. European Journal of Plant (Ed.), Laboratory Methods for Work with Plant and Soil Pathology 136: 613–24. Nematodes, Ministry of Agriculture, Fisheries and Food Tovar-Soto, A., Cid del Prado-Vera, I., Nicol, J.M., Reference Book 402, Her Majesty’s Stationery Office, Evans, K., Sandoval-Islas, J.S., and Martinez-Garza, A. London, pp. 121–31. 2003. Cactodera galinsogae n. sp. (Tylenchida: Het- SIAP. 2017. Secretaria de Información Agroalimenta- eroderidae) on barley (Hordeum vulgare L.) of the high riay Pesquera. HYPERLINK “http://www.siap.gob.mx” valleys of Mexico. Nematropica 33(1): 41–54. www.siap.gob.mx. Tovar-Soto, A., Cid del Prado-Vera, I., Sandoval-Islas, Sosa-Moss, C. 1986. Cyst nematodes in Mexico, J.S., Martinez-Garza, A., Nicol, J.M., and Evans, K. 2006. Central and South America. Nematologia mediterranea Los Nematodos Formadores de Quistes en México. 15: 13–19. Mexican Journal of Phytopathology 24: 145–51. Sosa-Moss, C. 1987. Cyst nematodes in Mexico, Tovar-Soto, A., Hernández-López, J.M., and Central and South America, in Lamberti, F., and Taylor, Torres-Coronel, R. 2009. Nematodos Formadores de C.E. (Eds), Cyst nematodes, Plenum Press, London, Quistes en la Zona Hortícola del Estado de Puebla. XI pp. 397–8. Congreso Internacional/XXXVI Nacional de la Sociedad Southey, J.F. 1986. Laboratory methods for work Mexicana de Fitopatología, A.C. Sociedad Mexicana with plant and soil nematodes. London: Ministery of de Fitopatología. Acapulco, Gro., México. 4p. Agriculture, Fisheries and Food, 402 pp. Tovar-Soto, A., Medina-Canales, M.G., and Torres- Stone, A.R., and Rowe, J.A. 1976. Heterodera Coronel, R. 2010. Nematodos Fitoparásitos cruciferae. CIH descriptions of plant-parasitic nematodes. Asociados a Hortalizas en el Valle de Tepeaca, Puebla, Set 6, No. 90. St. Albans: Commonwealth Institute of México. XII Congreso Internacional/XXXVII Nacional Helminthology. de la Sociedad Mexicana de Fitopatología, A.C. Subbotin, S.A. 2015. Heterodera sturhani sp. n. Sociedad Mexicana de Fitopatología. Mérida, Yuc. from China, a new species of the Heterodera avenae México. 4p. species complex (Tylenchida: Heteroderidae). Russian Vovlas, A., Santoro, S., Radicci, V., Leonetti, P., Journal of Nematology 23: 145–52. Castillo, P., and Palomares-Rius, J.E. 2017. Host- Subbotin, S.A., Cid Del Prado-Vera, I., Mundo- suitability of black medick (Medicago lupulina L.) and Ocampo, M., and Baldwin, J.G. 2011. Identification, additional molecular markers for identification of the phylogeny and phylogeography of circumfenestrate pea cyst nematode Heterodera goettingiana. European cyst nematodes (Nematoda: Heteroderidae) as inferred Journal of Plant Pathology 149: 193–9. from analysis of ITS-rDNA. Nematology 13: 805–24. Yu, Q., Ponomareva, E., Van Dyk, D., McDonald, Subbotin, S.A., Mundo-Ocampo, M., and Baldwin, M.R., Sun, F., Madani, M., and Tenuta, M. 2017. First J.G. 2010. Systematics of cyst nematodes (Nematoda: report of the carrot cyst nematode (Heterodera carotae Heteroderinae). Nematology monographs and per- Jones) from carrot fields in Ontario, Canada. Plant spectives. Vol. B. The Netherlands: Brill, 351 pp. Disease 101: 1056–6.