The lychee is native to Southern China and Southeast Asia (^{Coates et al., 1994}), and is commercialized as a fresh, dried or canned fruit. China, India, Southeast Asia and South Africa are the main regions of production on a global scale (^{Menzel and Simpson, 1994}). Approximately 700,000 tons of fresh lychee are consumed annually in Asia and India, along with an important amount also being processed in the form of canned fruit or juice. The growing demand for the fruit has incentivized several countries to grow it, including Mexico, which grows this crop in at least 4,250 ha, with an annual harvest of 28,000 t and an average growth of 7.7% between 2012 and 2018 (^{SIAP, 2018}). Veracruz is the state with the highest production, with 9,223.47 t, accounting for 50% of the country’s total, followed by Puebla, San Luis Potosí and Oaxaca, with 3,524.25 t, 1,957.65 t and 1,983.48 t, respectively (^{Torres-Becerril et al., 2019}).

The lychee crop faces numerous problems in Mexico during production, mostly regarding postharvest handling and commercialization (^{Torres-Becerril et al., 2019}). However, on the field, there are a series of phytosanitary problems caused by anthracnose (^{Campbell and Campbell 2001}; ^{Martínez-Bolaños et al., 2015}) and some insect-related pests (^{Xu et al., 2005}). On the other hand, another important pest are nematodes, and their importance in tropical fruits is related to the induction of symptoms such as chlorosis, foliar deformations, shortening of internodes, reduction of growth and of the production (^{Ravichandra, 2019}). Several authors report such nematodes in lychee as Helicotylenchus dihystera, Helicotylenchus indicus, Hemicriconemoides litchi, Hoplolaimus indicus, Meloidogyne incognita, Rotylenchulus reniformis, Tylenchorhynchus leviterminalis, Xiphinema brevicolle,Xiphinemasp., (^{Nath et al., 2008}), Hemicriconemoides mangiferae, Trichodorus pakistanensis, Xiphinema inequale (^{Nisha et al., 2000}) and Helicotylenchus dihystera(Nisha et al., 2000; Nath et al., 2008). Out of these species, only H. mangiferae and X. brevicolle are considered pathogens, since they cause symptoms of defoliation, chlorosis, apical necrosis of leaves, reduction of flowering and the falling of fruits (Nisha et al., 2000; Nath et al., 2008).

In Oaxaca, Mexico, lychee orchards cover a surface of 1,500 ha, with an average yield of 4.6 t ha^-1; the Brewster and Mauritius varieties are grown commercially (^{Maldonado-Peralta et al., 2012}). During the 2010-2012 production cycles in trees of both varieties, symptoms of chlorosis and low yields were observed, possibly due to the damage caused by nematodes. Due to this, it is important to carry out studies that help identify the nematode, as well as the symptoms it causes in the lychee crop. The aim of the present investigation was to identify the main genera of nematodes related to the lychee crop and determine its relation with the foliar chlorosis symptoms.

Four commercial lychee plots were sampled (two with the Brewster variety and two with Mauritius), after having selected one plot per location or municipal area: San José Chiltepec (Brewster), Santa María Jacatepec (Brewster), San Juan Cotzocón (Mauritius) and Santiago Yaveo (Mauritius), in the state of Oaxaca, Mexico. In each plot, a surface of one hectare was considered, and from it, five trees (8 to 10 years in age) with symptoms of chlorosis and five asymptomatic plants were chosen (in a directed manner) and labelled.

In the dripping zone of each labelled tree (15-30 cm depth; the first 15 cm were eliminated) five 200g soil subsamples were taken (soil and roots). These subsamples were then placed on plastic and mixed. The roots and soil were separated with a sieve and 200 g of soil were taken from the sieved sample (representative composite sample for each tree). In each sample collection, ten composite samples were taken for each plot, five from symptomatic trees and five from asymptomatic trees (^{Barker, 1978}). Each sample was labelled and kept in an ice cooler for its analysis in the laboratory. Samples were taken in the months of March, May and August of 2013, corresponding to the phenological stages of flowering, harvesting and vegetative development of lychee.

Every soil sample was processed with the sieving-centrifuging technique (^{Ayoub, 1977}; ^{Hooper, 1986}). For this, 200 cm³ were placed in a 1 L graduated cylinder, and diluted with distilled water for a final volume of 1000 mL. The soil solution was poured in a plastic container, mixed, left to rest for 15 seconds and then sieved using 60, 100, 200, 325 and 400-mesh sieves. The material collected in the 100, 200, 325 and 400-mesh sieves for each sample was placed in 50 mL centrifuge tubes. Each sample was added 1 g of kaolin and then centrifuges at 2,500 rpm for 5 min. The sedimentary fraction was suspended in a 46% saccharose solution (460 g sugar x L of water^-1) and then centrifuged at 2,500 rpm for one minute. The collected supernatant was sieved using a 400-mesh sieve and water was added to avoid the plasmolysis of the nematodes. From each soil sample, 20 mL of suspension were collected and kept in glass jars to later analyze.

Glass jars with 10 mL of the extracted nematode suspension were placed in a double bath at 80 °C; each jar was then added a FTW solution (Formaldehyde 40%, Triethanolamine and distilled water) (^{Shurtleff and Averre III, 2000}) and left to rest for 24 h to then count the nematode population using a stereoscopic microscope.

The nematodes were dehydrated according to the procedure described by ^{Seinhorst (1959}). In each jar, 50% of the volume of FTW was replaced with the same volume of solution A (20 parts of 96% ethanol/1 part glycerin/79 parts distilled water). Afterwards, the nematode suspension was transferred to a Petri dish and incubated for 24 h in a chamber with 96% ethanol at 40 °C. Solution B (93 parts of 96% ethanol/ 7 parts glycerin) was added to the liquid remaining from the nematode suspension until the Petri dish was filled, and incubated for 24 h at 26 °C. After two thirds of the volume of alcohol evaporated, the remaining volume was replaced with the C solution (80 parts of 96% Ethanol / 20 parts glycerin) and incubated at room temperature (26 °C) for 24 h. After the alcohol evaporated, the Petri dish was placed in an oven at 40 °C for 48 hours and then transferred to a dehydration chamber with calcium chloride to eliminate the remaining water.

Permanent (paraffin) and temporary (water -agar 3%) preparations were made from the nematode samples (fixed and dehydrated) collected. In order to characterize and identify the nematodes, observations were carried out on an Olympus CX3 compound microscopes with magnifications of 10X, 40X and 100X; taxonomically important characteristics were photographed using an Olympus E330 camera. The images of the specimens were measured and analyzed using the program Axion Vision LE^® for their identification with the use of taxonomic keys to determine the genus (^{Mai et al., 1996}; ^{Shurtleff y Averre III, 2000}) and species (^{Siddiqi, 1963}; ^{Boag and Jairajpuri, 1985}; ^{Siddiqui, 2000}; ^{Euon et al., 2002}; ^{Castillo and Volvias, 2007}).

Subsamples (3 mL) of the extracted nematodes were taken from each sample. They were placed in counting boxes and observed under a compound microscope, considering five repetitions per sample. The population count of the specimens per genus was carried out according to the specimen morphology, month in which samples were taken (phenological stage), variety and symptomatology. Three factors were evaluated, along with their relationship with nematode populations. The factors were three phenological stages (vegetative development, flowering and harvesting), two varieties (Brewster and Mauritius) and two symptoms (asymptomatic and chlorotic plants). The data were processes using an analysis of variance (ANOVA) and a Duncan average comparison test with a reliability of 0.05%.

According to the results obtained, 13 nematode genera were identified from lychee soil samples from commercial plots with Brewster and Mauritius varieties in Oaxaca, Mexico. The morphological and morphometric characteristics corresponded to the Aphelenchus, Ditylenchus, Helicotylenchus, Hemicriconemoides, Longidorus, Mesocriconema, Pratylenchus, Psilenchus, Rotylenchus, Trichodorus, Tylenchorhynchus, Tylenchus and Xiphinema genera (Figures 1 and 2). Some species within these genera have been reported to be related to the lychee crop (^{Nisha et al., 2000}). It is worth mentioning that some of these genera are, not only considered phytoparasitic, but they are nematodes with mycophagous feeding habits (Aphelenchus, Tylenchus, Ditylenchus and Psilenchus), and therefore their presence indicate that they are related, although this does not imply that they affect the crop.

The total nematode population during the flowering stage was higher in the Brewster variety. However, no significant differences (p=0.05) were observed between the evaluated varieties (Table 1). In the same populations evaluated, no direct relation was observed between the total population of nematodes and the symptom of chlorosis for the different phenological stages evaluated (Table 1).

The genus Mesocriconema displayed the highest number of individuals in the different phenological stages evaluated. Although this genus has been reported as a possible phytoparasite in guava (Psidium guajava), papaya (Carica papaya) (^{Castellano et al., 2012}) and passionfruit (Passiflora sp.) (^{Souza and Pala, 2016}), there are no reports on its possible role as a phytopathogen in lychee. The Hemicriconemoides and Tylenchus populations were significantly higher to those of the remaining genera, only in harvesting. For the Brewster variety, the main nematode populations corresponded to the genera Mesocriconema, Ditylenchus, Hemicriconemoides and Pratylenchus, whereas for the Mauritius variety, it was the genera Aphelenchus and Xiphinema (Table 2). Despite the genera Helicotylenchus, Hemicriconemoides (^{McSorley et al., 1980}) and Xiphinema having been reported to be related to symptoms of chlorosis in mango (Mangifera indica) and lychee plants, for the present study, the populations of both genera showed no significant differences between chlorotic and asymptomatic plants (Table 2). Only the genus Trichodorus displayed a significant difference between plant symptomatology, making the asymptomatic plant population the largest one (Table 2); this genus has been reported as a pathogen in sugarcane (^{Shahina and Firoza, 2007}).

In conclusion, thirteen nematode genera were identified as being related to two lychee varieties (Brewster and Mauritius) in the stages of flowering, harvesting and vegetative development in the state of Oaxaca, Mexico: Aphelenchus, Ditylenchus, Helicotylenchus, Hemicriconemoides, Longidorus, Mesocriconema, Pratylenchus, Psilenchus, Rotylenchus, Trichodorus, Tylenchorhynchus, Tylenchus and Xiphinema. The genera with ectoparasitic habits predominated. The symptom of chlorosis in lychee plants was not related with the population density of total nematodes or with any specific genus.

Figure 1. Distinctive morphological characteristics of genera of ectoparasitic nematodes related to the lychee crop (Litchi chinensis) in commercial plantations in the state of Oaxaca, Mexico. A) Aphelenchus, B) Longidorus, C) Mesocriconema, D) Trichodorus, E) Tylenchus and F) Xiphinema.  

Figure 2. Distinctive morphological characteristics of genera of nematodes related to the lychee crop (Litchi chinensis) in commercial plantations in the state of Oaxaca, Mexico. A) Helicotylenchus, B) Hemicriconemoides, C) Pratylenchus, D) Psilenchus, E) Rotylenchus and F) Tylenchorhynchus.