Coffee production is economically and environmentally important for Mexico with 748,284 planted ha generating 1,336,882 t and more than 600 million US$ trading per year (SAGARPA et al. 2012). In Mexico, and particularly in the state of Veracruz, coffee is commonly cultivated under shade of a wide variety of trees, which provide an additional source of income for many producers (Licona-Vargas et al. 2006; Hernández-Martínez et al. 2009). This kind of shade coffee agro-ecosystems, with both structural and species diversity, also affords a refuge for many animal species and contributes to groundwater recharge and to carbon capture, thus providing important environmental services (Beer et al. 2003; Gordon et al. 2007; López-Gómez et al. 2008; Manson et al. 2008; Jha et al. 2011). However many of these environment friendly coffee agro-ecosystems are threatened in Veracruz by a syndrome called coffee corky-root disease (locally named “corchosis”) linked to the presence of Meloidogyne spp. according to reports from the 1960’s (Téliz-Ortíz et al. 1993). Coffee corky-root disease has been expanding and is now considered a severe threat to coffee production in Mexico, since up to 35 % of the annual Coffea arabica plant losses in Veracruz and Chiapas, the main coffee producing States of Mexico, are attributed to this disease (INIFAP et al. 2005).

In Veracruz this disease in coffee plantations has been attributed to M. incognita, the only species reported to date on coffee in Mexico (Téliz-Ortíz et al. 1993; García et al. 1997). These previous identifications of root-knot nematode (RKN) populations found in coffee in Mexico were based on perineal pattern observations. However, recent studies have shown that identification of Meloidogyne based on such morphological characters is unreliable for some species, such as in the case of M. incognita and M. paranaensis which both exhibit a similar perineal pattern (Carneiro and Cofcewicz 2008). Actually, M. paranaensis is associated with similar symptoms of corky-root disease in coffee plantations in Brazil and Guatemala (Carneiro et al. 1996, 2004; Campos and Villain 2005; Villain et al. 2013). Accurate identification of the causative species of coffee corky-root disease is essential for designing efficient control strategies, especially when using plant resistances and biological control agents with some grade of specificity. Currently more reliable identification methods of Meloidogyne species have been developed based on isoenzyme phenotypes or molecular markers (Carneiro and Cofcewicz 2008). Species-specific SCAR (sequence-characterized amplified region) markers have been developed for the identification of main Meloidogyne species detected in coffee in Latin America: M. exigua, M. incognita, M. paranaensis, M. enterolobii, M. arabicida and M. izalcoensis (Randig et al. 2002; Tigano et al. 2010; Correa et al. 2013). This study sought to use SCAR marker identification of Meloidogyne populations associated with symptoms of corky-roots through the coffee production region of Veracruz, Mexico. We used primers of the three major and most distributed species on coffee in Latin America: M. exigua, M. incognita and M. paranaensis, since the three other species, M. enterolobii, M. arabicida and M. izalcoensis are marginal or present a very limited distribution area (Carneiro and Cofcewicz 2008; Villain et al. 2013). Populations of Meloidogyne from intercropped banana plants were also studied since this agronomic practice is very common in this region.

Sample collection was carried out on eight farms distributed in central Veracruz, which is the major coffee producing area of the state (more than 95 % of production) and the second most important in Mexico (Fig. 1). These farms on the southern slopes of the Sierra Madre Oriental and the eastern slopes of the Mexican Transvolcanic Belt, were identified from previous studies reporting the presence of corky-root disease or reports of some unidentified root problems by coffee growers and field technicians.

Fig. 1
figure 1

Map of the major coffee cropping region of the State of Veracruz with location of the eight sampling sites. Code numbers of sampling sites are reported in Table 1 with their respective characteristics

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On each farm a composite sample of roots with corky-root symptoms was taken from 6 to 8 coffee plants distributed in the field and chosen by their chlorosis and/or decline aerial symptoms. In sites where banana was intercropped with coffee, roots of 6–8 Musa spp. plants closer to sampled coffee plants were also collected. Data of the geographical location, altitude, and host plants of each of the sampling sites are summarized in Table 1. Meloidogyne spp. eggs and 2nd stage juveniles (J2) were extracted from coffee and banana infested roots according to the methodology of blending in a 2 % chlorine solution described by Carneiro et al. (2004). For each population, DNA was extracted from 200 to 300 μl of eggs and J2 following the DNA Tissue and Insect MiniPrep Isolation Kit protocols (Zymo Research). Multiplex polymerase chain reaction (PCR) was performed using DNA extracted from each collected nematode population and three SCAR (Sigma-Aldrich) primers specific for M. exigua, M. incognita, and M. paranaensis (Randig et al. 2002), the three major species on coffee in Latin America (Campos and Villain 2005; Carneiro and Cofcewicz 2008; Villain et al. 2013). A positive control with reference DNAs of these same three species provided by the Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) was included as well as a negative control without DNA. The cycling program was as follows: 5 min at 94 °C, 30 cycles of 30 s at 94 °C, 45 s at 64 °C, and 1 min at 70 °C; and a final extension of 8 min at 70 °C according to Randig et al. (2002). The amplification products were separated by electrophoresis on a 3 % agarose gel stained with ethidium bromide (0.02 %) and visualized under UV light.

Table 1 Geographical data of collecting sites in coffee plantations of Veracruz State and SCAR-PCR diagnosed species from Coffea and Musa plants
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In the study sites, coffee plants presented different levels of decay from nutritional deficiency symptoms to chlorotic, defoliated, and dead stages (Fig. 2a) in agreement with previous observations (Téliz-Ortíz et al. 1993). In all sampling sites, these affected plants presented the same corky-root symptoms i.e. swellings with a corky and cracking aspect on the main roots including the tap root as far as the root collar (Fig. 2b, c). Cuttings of these root corky swellings showed the presence of numerous RKN females (Fig. 2d). On the sampled intercropped banana plants, we observed common root swelling with even some cortex cracking (not illustrated).

Fig. 2
figure 2

Symptoms of the corky-root disease on coffee plants; a. Rachitic and chlorotic 3 years old coffee plants with intercropped Musa AAA plants both infested by M. paranaensis; b. Root symptoms with corky swelling; c. Detail of corky and cracking aspect of a coffee root infested by M. paranaensis; d. Longitudinal cutting of a root corky swelling showing numerous females

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The SCAR-Multiplex-PCR analysis showed that neither of M. incognita and M. exigua was present in these eight coffee root samples even at sites 2, 4 and 6 where M. incognita was detected on intercropped banana plants (Fig. 3; Table 1). On the contrary, M. paranaensis was present in all eight root samples collected from coffee plants presenting corky-root symptoms (Fig. 3, Table 1). Since the eight sampling sites were covering most of the coffee cropping area of Veracruz State (Fig. 1), these results suggest that corky-root symptoms on coffee seem to be linked exclusively with the RKN species, M. paranaensis, in this State.

Fig. 3
figure 3

Amplification test for the three RKN species: M. exigua; M. incognita and M. paranaensis from corky-root of coffee plants (C coded populations) and from roots of intercropped Musa plants (B coded populations). Numbers of population codes refer to geographical sampling sites resumed in Fig. 1. PC is the positive control [a = M. exigua (562 bp), b = M. incognita (399 bp), and c = M. paranaensis (208 bp)], whereas CN is the negative control for the PCR. Flanking lines are the PhyX-174 DNA/Hae III molecular weight marker (Promega Corp.)

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These results are in agreement with the identifications of RKN related to corky-root symptoms on coffee in the adjacent country of Guatemala and in various states of Brazil where this species was first described (Carneiro et al. 1996; Carneiro et al. 2004, 2005; Campos and Villain 2005; Ferraz 2008; Villain et al. 2008; Barros et al. 2011; Villain et al. 2013). As far as we know, this is the first report of M. paranaensis in Mexico. Previous studies reported M. incognita as the causative RKN species of corky-root symptoms on coffee in Veracruz state (Téliz-Ortíz et al. 1993; García et al. 1997; Marbán-Mendoza 2009). However these previous identifications were based on perineal patterns which do not allow a reliable species distinction between M. incognita and M. paranaensis as mentioned before. These results reinforce the fact that particular attention should be focused on detection of this very pathogenic RKN on coffee throughout Latin America because of likely misidentifications (Campos and Villain 2005; Carneiro and Cofcewicz 2008; Elling 2013; Villain et al. 2013). Locally, the coffee industry of Mexico should be concerned by the presence of this very destructive RKN particularly at a prophylactic level, taking action in order to avoid more dissemination of this nematode through the distribution of contaminated nursery seedlings and soils.

The determination of M. paranaensis as the causative RKN of corky-root disease in the study region is also of primordial importance for establishing control strategies. Actually, sources of resistance to M. paranaensis have already been identified among coffee germplasm and used for breeding of cultivars resistant to M. paranaensis. Especially, some new selected F1 hybrids of C. arabica inherit resistance to M. paranaensis from parent wild Ethiopian accessions (Anzueto et al. 2001; Bertrand and Anthony 2008). One C. canephora rootstock cv. Nemaya has also been selected for its resistance to the major RKN found in Central America including M. paranaensis (Anzueto et al. 2001; Bertrand et al. 2002). Indeed, two of the collected populations (C2 and C5) were collected on non-selected C. canephora rootstocks which presented serious corky-root symptoms in agreement with previous studies showing the susceptibility of most C. canephora accessions to M. paranaensis (Anzueto et al. 2001; Bertrand et al. 2002) and some similar observations in the field in Guatemala and Brazil (Villain et al. 2013; Barros et al. 2014). These results prove again the need to use selected resistant rootstocks such as cv. Nemaya in order to control this very pathogenic RKN. As observed previously in Guatemala (Villain et al. 2013), M. paranaensis proved to be able to parasitize coffee on a wide range of altitudes in Veracruz state from 660 to 1360 m and including the major part of the altitudinal range of coffee production (Table 1).

M. paranaensis was also detected in the five samples collected on intercropped banana plants from the sampled coffee plantations (Table 1). These infested banana plants all belong to Musa acuminata, some are AA diploids of the Pisang Mas subgroup, locally called “dominico” (sample B2) and others are AAA triploids cv. “Roatan” - Gros Michel subgroup - (samples B4, B5 and B6) and cv. “morado” - Red subgroup - (sample B7). The presence of M. paranaensis in all these AA and AAA Musa root samples indicates there are good hosts for this RKN. Thus bananas represent potentially important ways for dissemination of M. paranaensis since they are frequently vegetatively propagated by growers using banana suckers for plantation renovations.

Another interesting result is that M. incognita was detected in intercropped Musa AAA (B4 and B6) and Musa AA (B2) root samples from three plots, but not in the corresponding coffee root samples from the same sampling sites despite the already mentioned high sensitivity of the SCAR Multiplex-PCR analysis used in this study and despite the age of the plantations (more than 3 years old). Though M. incognita is reported as a very pathogenic RKN on coffee, particularly in Brazil (Campos and Villain 2005), these populations of M. incognita could not be pathogenic for coffee plants but this hypothesis must be confirmed. A similar observation was made in Guatemala in a coffee plantation where M. incognita was detected on the associated Musa AAB plants but not in the closer coffee plants furthermore infested by M. paranaensis (Villain et al. 2013).

Phylogeny and crossed pathogenicity studies for Coffea spp. and Musa spp. with populations of M. paranaenis collected in different states of Mexico from both crops will be carried on in order to characterize the intraspecific diversity among these populations and in relation to populations from other countries. Current studies are also being carried out to identify the fungi associated with these symptoms of corky-roots disease and determine their role in the etiology of the disease. In the case of coffee corky-root symptoms observed in Costa Rica, it was demonstrated that the presence of both pathogens M. arabicida and a non-identified f.sp of Fusarium oxysporum was necessary to induce such symptoms (Bertrand et al. 2000). In the case of the coffee corky-root disease observed in Veracruz State, various fungi such as Fusarium spp., Cylindrocladium, and Trichoderma have been observed in coffee roots presenting symptoms of the disease (Téliz-Ortíz et al. 1993). The role of these and other species of fungi in the etiology of this disease has to be clarified.