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

Soil with nematodes collected from an infested carrot field from Santa Maria Actipan, Acatzingo, Puebla, Mexico (18˚ 58′ 486″ N; 97˚ 50′ 295″ W; 2246 masl) was used to establish a laboratory culture. The nematode culture was maintained in the greenhouse at 20 to 25°C. Two plastic plots were filled with 15 kg of soil, then 50 seeds of carrot cv. Christian were sown. After 54 days, one plot was used for the extraction of second stage juveniles (J2) and males by the centrifugal flotation technique (Jenkins, 1964). After 65 days, the other plot was used to obtain females, the roots were separated, gently washed, and put in a Petri dish for its examination under Motic stereoscopic microscope. Females were separated using a brush and kept at 5°C until use. Females were used for morphology and morphometry. The cysts were extracted by the Fenwick method (Fenwick, 1940).

Males and second stage juveniles (J2) were killed in a water bath at 65°C for 5 min, fixed with a mixture of ethanol, acetic acid, and formalin (20:6:1) and processed into glycerin and the specimens were mounted in permanent slides with anhydrous glycerin and paraffin seal for examination (S’Jacob and Van Bezooijen, 1984; Southey, 1986). The measures were taken for length, width at mid body, stylet length, labial region height and width, dorsal oesophageal gland to spear knobs base, anterior end to median bulb, anterior end to excretory pore, oesophageal length, width at anus and hyaline region, tail length, hyaline part of tail length, and spicules length (males) (S’Jacob and Van Bezooijen, 1984; Southey, 1986). For the observation of the vulval cone, cysts were soaked in lactic acid 45% for 15 min, transferred to water and then, the posterior region was dissected and mounted in glycerin with paraffin seal for its observation and analysis (S’Jacob and Van Bezooijen, 1984; De la Jara-Alcocer et al., 1994). The measurements taken were vulval slit length, fenestral length and width. All measurements were made on Olympus CX31 microscope with Infinity Analyze software v6.5.2.

J2, females, males and cysts were processed. The nematode phases were fixed in 4% glutaraldehyde, post fixed in 1% osmium tetraoxide, and dehydrated in different concentrations of ethanol (30–100%). Samples were critical point dried and coated with gold palladium for its analysis in a scanning electron microscope (Shepherd and Clark, 1986). The anterior region of the different nematode stages was observed and for the J2 and males the number of incisures in the lateral field were counted.

Nine plant species from five families, which are known as hosts from H. carotae and H. cruciferae were used in the test: Apiaceae, carrot (Daucus carota L.); Asteraceae, lettuce (Lactuca sativa L.); Brassicaceae, broccoli (Brassica oleracea var. italica L.), cabbage (B. oleracea var. capitata L.), cauliflower (B. oleracea var. botrytis L.), radish (Raphanus sativus L.) and Brussels sprouts (B. oleracea var. gemmifera L.); Chenopodiaceae, beetroot (Beta vulgaris L.), and Poaceae, wheat (Triticum aestivum L.) (Goodey et al., 1959; Mugniery and Bossis, 1988; Evans and Russell, 1993). Plants growing in pots with 3 kg of sterilized soil were inoculated with 12,000 eggs and J2. Each plant species was tested in three replicates. After 70 days, the plants were removed and carefully washed with tap water, the roots were examined under a stereoscopic microscope for the search of white females and cysts. Then roots were stained by the acid fuchsin lactoglycerol technique to observe nematode stages inside of roots (Bybd et al., 1983). If white females or cysts were found on roots, the plant was considered as a host.

DNA was extracted from single cysts of H. carotae fixed in ethanol. Protocols for DNA extraction, polymerase chain recaction (PCR), cloning and sequencing were as described in Tanha Maafi et al. (2003) and Subbotin (2015). The forward primer TW81 (5′-GTT TCC GTA GGT GAA CCT GC-3′) and the reverse primer AB28 (5′-ATA TGC TTA AGT TCA GCG GGT-3′) (Tanha Maafi et al., 2003) were used for amplification of the ITS1-5.8S-ITS2 of rRNA and the forward Het-coxiF (5′-TAG TTG ATC GTA ATT TTA ATG G 3′) and the reverse Het-coxiR (5′- CCT AAA ACA TAA TGA AAA TGW GC-3′) (Subbotin, 2015) was used for amplification of the partial COI mtDNA gene. For COI gene study the sequences of H. carotae from Italy, France, Switzerland and H. cruciferae from Russia, Moscow region and USA, California were also included. The new sequences were deposited in the GenBank under accession numbers: MG563227 to MG563237.

The newly obtained ITS rRNA and COI gene sequences were aligned with corresponding published sequences of species from the Goettingiana group (Subbotin et al., 2001; Tanha Maafi et al., 2003; Chizhov et al., 2009; Toumi et al., 2013; Madani et al., 2018; Vovlas et al., 2017 and others) using ClustalX 1.83 with default parameters. The sequence datasets were analysed with Bayesian inference (BI) using MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001) under the HKY+G model for COI and GTR+G+I model for ITS rRNA gene as they were obtained using jModeltest 0.1.1 (Posada, 2008). BI analysis was initiated with a random starting tree and was run with four chains for 1.0 × 10⁶ generations. The Markov chains were sampled at intervals of 100 generations. Two runs were performed for each analysis. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities are given on appropriate clades. The COI sequence alignment was also used to construct phylogenetic network estimation using statistical parsimony as implemented in POPART software (Bandelt et al., 1999).

Patches with poorly developed carrots were observed in the infected fields of the Tepeaca Valley, Puebla, Mexico (Fig. 1A,B). White females and cyst attached to secondary roots (Fig. 1C, D) sometimes giving the roots a “bearded” appearance. The foliage was stunted, chlorotic and in some plants reddish (Fig. 1E, F). These symptoms correspond to those reported previously for H. carotae (Jones, 1950a; Greco, 1986, 1987).

Fig. 1

Tests of nine plant species from five families for infection of H. carotae revealed that only carrot was host for the Mexican population (Table 2). In the roots of carrots all nematode stages from J2 to adults (male, female and cysts) were observed. No penetration of the nematode and any stages were observed in roots of the other plants tested. These results agree with those reported for H. carotae that has a narrow host range: carrots and Torilis leptophylla, in contrast with H. crucifeae that infects different species of Brassicaceae (Subbotin et al., 2010).

The ITS rRNA gene sequences obtained from the Mexican population of H. carotae were different in 4 bp (0.4%) from each other and differed from others H. carotae up to 1.2% and from H. cruciferae up to 1.0%. Phylogenetic relationships of the Mexican population of H. carotae with other populations and species of the Goettingiana group is given in Fig. 5. Sequences of H. carotae are not formed a single clade within the tree.

Fig. 5

The COI gene sequence of the Mexican population of H. carotae was identical to those of the Californian population of H. cruciferae and H. carotae from France and Canada. Total seven unique COI haplotypes were identified for H. carotae. The Mexican population of H. carotae differed in 1 to 5 bp (0.2–0.7%) from the majority of sequences of H. carotae from Canada, Italy, Switzerland and France. Phylogenetic relationships of the Mexican population of H. carotae with other populations and species of the Goettingiana group as inferred from Bayesian inference and Statistical parsimony are given in Fig. 6A and B, respectively.

Although, the Mexican population of H. carotae fits to the morphological and morphometrical characters of H. carotae published by other authors, nevertheless, using presently available molecular markers such as the ITS rRNA and partial COI gene sequence do not allow to make a reliable and accurate identification of H. carotae and H. cruciferae without knowing plant hosts and stylet length of males.

Fig. 6

Up to now, 15 species of cyst nematodes from four genera, Globodera, Punctodera, Cactodera, and Heterodera have been known in Mexico and with our new finding of H. carotae, total number became 16.

Four species of the genus Globodera are reported in this country. The golden cyst nematode Globodera rostochiensis has been reported in Coahuila, Guanajuato, Hidalgo, Mexico City, Mexico State, Nuevo Leon, Puebla, Tlaxcala, and Veracruz (Brodie, 1998). This nematode is considered one of the most important pathogens in Mexico because causes potato yield losses of 40 to 70% (Santamaria-Cesar and Teliz-Ortiz, 1984; Tovar-Soto et al., 2006). Globodera tabacum was also found in potato Mexican fields (Quiñonez and Sosa-Moss, 1977; Sosa-Moss, 1986, 1987). Globodera bravoae infected creeping false holly (Jaltomata procumbens (Cav.) J.L. Gentry) in Mexico City (Franco-Navarro et al., 2000). Mexican cyst nematode, Globodera mexicana (Campos-Vela, 1967; Subbotin et al., 2010) was described from Buffalo burr, Solanum rostratum Dunal in Mexico in Central Highlands of Mexico, Toluca Valley in Mexico State and Huamantla Valley, Tlaxcala State and later found in Municipality of Amecameca in Mexico State (Subbotin et al., 2010).

The Mexican corn cyst nematode, Punctodera chalcoensis, is distributed in Jalisco, Mexico State, Michoacan, Puebla, Queretaro, Tlaxcala and Veracruz. The yield losses by this nematode can be up to 90%.

Several Cactodera spp. were reported. Cactodera cacti is a parasite of cacti and was found in several states of Mexico with warm temperatures and lower elevations (Baldwin and Mundo-Ocampo, 1991), Cactodera amaranthi, in spinach (Spinacia oleracea L.) and some other Amarantheaceae and Chenopodiaceae in the Mesa Central (Sossa-Moss, 1986, 1987), Cactodera salina in Sonora infecting dwarf saltwort (Salicornia bigelovii Torr) (Baldwin et al., 1997) and Cactodera evansi in carnation (Dianthus caryophyllus L.) in Villa Guerrero, Mexico State (Cid del Prado-Vera and Rowe, 2000). Catodera galinsogae (Tovar-Soto et al., 2003) and Cactodera rosae (Cid del Prado-Vera and Miranda, 2008) are parasites of barley in the High Valleys of Hidalgo State. In Texcoco, Cactodera torreyanae was found in romeritos (Suaeda torreyana S. Watson) which is a plant that is prepared in a traditional Mexican dish (Cid del Prado-Vera and Subbotin, 2014). Subbotin et al. (2011) reported two unidentified Cactodera from Mexico. Currently, there are no reliable reports on the economic losses caused by representatives of the genus Cactodera.

Four valid and several unidentified species of the genus Heterodera were found in Mexico. Heterodera schachtii is present in Chalco, Mexico State in sugar beet (Sosa-Moss, 1986), Heterodera cyperi in Cyperus spp. in Tlaxcala and Nayarit (Sosa-Moss, 1987), Heterodera humuli in the Toluca Valley, Mexico State (Sosa-Moss, 1987). A report of Heterodera salixophila from Mexico (Tovar-Soto et al., 2006) should be confirmed (Subbotin et al., 2010). Several unidentified Heterodera 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 tuberosum L.) and wheat (Triticum aestivum L.) (Santacruz-Ulibarri and Pedroza, 1983). Heterodera sp. in sugar beet in Mexico City (Alcocér-Gómez and Gotwald, 1963), and Heterodera from the Goettingiana group in the High Valleys of Hidalgo (Tovar-Soto et al., 2006).

In this study using morphology, morphometry, analysis of the ITS rRNA, and partial COI gene sequence and host range test we report the presence of the carrot cyst nematode, Heterodera carotae, in Mexico for the first time. Our study also showed difference between the male stylet length, being longer for H. carotae and that accurate differentiation between H. carotae and H. cruciferae could be done based on a multidisciplinary approach using the knowledge of plant-host.