Biodiversity of phytoparasitic nematodes associated with Musaceae and fruit crops in Colombia
Wide distributions of economically important plant-parasitic nematodes belonging to the genera Meloidogyne, Pratylenchus and Radopholus have been recorded in plantain and fruit crops in Colombia. Despite of significant economic losses, taxonomic research on these taxa in Colombia is limited. This thesis focuses mainly on two composition components of biodiversity (species and genes) as a contribution to the analyses of biodiversity of economically important plant-parasitic nematodes in Colombia. Samples were taken from 16 different regions of the country. Identification, characterization, and description of known and new nematode species should be based on a combination of as many data as possible, using different approaches. Morphometrical and morphological studies were based on LM and SEM observations, combined with other taxonomical methods such as analyses of proteins and use of species-specific primers (only for Meloidogyne populations), molecular analyses of sequences and phylogenetic analyses. In the current study, morphometric and/or morphological features allowed to separate populations into small species groups as a first step towards identification. Hereby, features of perineal pattern of females or head shape of males in Meloidogyne; head annulation number in Pratylenchus and tail length of Radopholus were the most helpful features at species level and reduced the number of species to be taken into account. From the combination of these analyses it turned out that several species ocurred together in the field. Thus, purification of populations is recommended as a second step in the identification process. In this study, only three Meloidogyne “populations” could be successfully purified. However, to overcome the mixed species problem for all other species, Meloidogyne identification was based on a single young egg-laying female (protein analysis) and DNA (for species-specific PCR and sequence analyses) was always extracted from a single juvenile. The highest diversity was analyzed in the genus Meloidogyne since a large number of populations (24) were available for this study. Six Meloidogyne species: M. incognita, M. javanica, M. arenaria, M. hapla, M. paranaensis and M. konaensis were found associated with four fruit crops and plantain in Colombia. The last two species had never been recorded previously in Colombia but since they show morphological features of M. incognita, they were probably misidentified in the past. Only the use of combined methods allows accurate identification to species level. Analyses of esterase and malate dehydrogenase resulted in a profile that is typical of both species, P1 fitted both M. paranaensis and M. konaensis. Though, the morphological feature, number of projections protruding from the stylet shaft of males allowed their separation. Isozyme analysis also recovered phenotypes that could not be connected to known Meloidogyne species. E3N1, E3N2 phenotypes were found in populations P5a, P19a and P19b. These phenotypes represent a new species that is currently under investigation based on populations from Iran, Africa, Chile and Brazil by Karssen, Carneiro and collaborators (personal communication; December 10, 2007). Furthermore, the phenotype E1bN1b (population P14a) matched with M. hapla but the Mdh band was very slow and morphological features of this population agreed with M. javanica. Populations P3a, P10 and P11 yielded two esterase bands (E2N2): one slow band, and one fast band with a migration rate largely similar to the I1 band of the M. incognita phenotype. E1bN1b and phenotypes probably represent a new species. However additional specimens are needed for accurate morphological and morphometrical studies. Analyses of Colombian Meloidogyne populations (based on 65 nematode specimens) in combination with Meloidogyne populations from GenBank at different rDNA regions (D2D3, 18S and ITS1-5.8-ITS2) yielded phylogenetic trees with similar topologies. All of the generated trees contained three identical clades, strongly supported by high PP values. Meloidogyne populations containing mainly temperate species (as obtained from GenBank) were consistently placed as a separate clade (clade 0). Clade 1 contained M. hapla populations; clade 2 grouped six populations (P1, P2, P5a, P15, P22 and P23) not identified at species level and therefore with unknown reproductive strategies, and clade 3 contained M. incognita, M. javanica and M. arenaria. These topologies agreed with morphological and morphometric data, protein analyses and species-specific PCR. However, populations P1 and P15 formed exceptions. The tree topology and analyses of individual sequences showed that species-specific PCR results of populations P1 and P15 were wrong. For the latter populations the absence of autapomorphic characters typical of M. hapla species allowed to recognize the error. Only one Pratylenchus population was obtained, but morphological analyses were completed with comprehensive molecular divergence and phylogenetic analyses (based on eight nematode specimens from Colombia and additional Genbank material). Their outcome resulted in the description of a new species, indicated here as Pratylenchus sp. n. The new species is closely related to three Pratylenchus species but clearly differs from the other species within genus. It shares with P. coffeae, P. loosi and P. jaehni morphology features: amphimictic lesion nematodes with two lip annuli, and lip sectors fused together and also fused with the oral disc to a smooth face. Molecular distinctions between Pratylenchus n. sp., P. coffeae, P. jaehni and P. loosi are more clear-cut than are morphological differences. The sequence comparison of the D2D3 rDNA expansion region clearly separates the new species from P. jaehni, P. loosi and P. coffeae. Pratylenchus n. sp. had a sister relation with P. jaehni. P. loosi and P. coffeae were more distinctly related. Clear autapomorphic characters of the new species were present in all specimens studied, differentiating its D2D3 expansion region of 28S rDNA from the observations of its sister species P. jaehni. Our results fulfill the requirements of the evolutionary species concept and phylogenetic species concept sensu Adams (1998). Additionally, our new species is a typological morphospecies compared with most species included in the genus; morphometric characters overlap only with P. jaehni and P. loosi. In general, Pratylenchus species are polyphagous, but differences in host preference occur among the species. Type host for Pratylenchus n. sp. is Musa ABB (plantain, hartón cultivar), a host also reported for P. coffeae. So far, P. jaehni and P. loosi have not yet been recorded on Musa plants. Morphological data of two obtained Radopholus populations were supplemented with sequence divergence and phylogenetic analyses (D2D3, 18S and ITS1-5.8-ITS2) based on 23 specimens of nematodes of Colombia and additional Genbank material. All specimens were identified as R. similis. With respect to morphological characters and morphometric data, Colombian populations of R. similis females only slightly extend the inferior limits of the other R. similis populations described from different localities around the world, particularly with respect to body length, diameter at mid body and anus, length of the neck region, hyaline part of tail and length of the ovaries. The topology of ITS1-5.8-ITS tree positioned Colombian populations of R. similis together with populations from different countries and hosts in the same clade. Percentage of divergence yielded similar results except for the Costa Rican population which showed 11.7% divergence with respect to Colombian populations from the same clade. The populations of R. similis did not cluster together according to geographical distribution, nor did they for host. For example, accessions from the same host (banana plants) and from the same country (Sudan) were positioned in different clades and showed the largest differences in percentage of divergence. The populations analyzed by Elbadri (2000) from Sudan, Uganda, Cuba and Germany and sharing clade with Colombian populations in phylogenetic tree, were found to be the most pathogenic populations for banana among all the tested populations by this author. The phylogenetic position of Radopholus species in the ITS1-5.8-ITS tree, more particularly of R. arabocoffee, P. duriophilus, R. similis from Sudan and Belgium suggests that “R. similis” could represent a species complex; more research is needed to unravel these relationships. In conclusion, all retrieved Meloidogyne species were widely distributed and present in most of the fruit crops. R. similis and Pratylenchus n. sp. were restricted to Musa plants, but an incomplete distribution picture related to flawed sampling/transported processing can not be excluded for the Pratylenchidae. Correct species identification is crucial for reliable crop management because of differences in host range and virulence between species. Our results suggest that if more hosts and localities would be included in future studies, the number of new species, not only of Meloidogyne but also of other plant parasitic nematodes, will increase in correspondence with the high biodiversity of fauna and flora in Colombia. Additionally, studies on biodiversity represent a long-term multidisciplinary task, including descriptions of the new species, data on distribution, host associations, potential damage and relationships with other ecosystem components.
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