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Coffee (Coffea arabica L. cv. Catuai) seedlings with abundant small root galls caused by an unknown root-knot nematode were found in southern Costa Rica. Morphology, esterase and malate dehydrogenase isozyme phenotypes and DNA markers differentiated this nematode from known Meloidogyne spp. A new species, M. lopezi n. sp., with common name Costa Rican root-knot nematode, is suggested. Meloidogyne lopezi n. sp. is distinguished from other coffee-associated Meloidogyne spp. by size of female lips and stylet, male body length and stylet and second-stage juvenile body and tail morphology. The region of the mitochondrial genome between COII and 16S rRNA showed a unique amplicon size of 1370 bp, and digestions with restriction enzymes HinfI, AluI, DraI and DraIII revealed characteristic PCR-RFLP patterns that differed from the tropical root-knot nematode species M. arabicida, M. incognita, M. izalcoensis, M. javanica and M. paranaensis. Characterisation of the protein-coding map-1 gene and phylogenetic analyses suggested that M. lopezi n. sp. might reproduce by mitotic parthenogenesis. Phylogenies estimated using Bayesian analyses based on the region between the COII and 16S rRNA mitochondrial genes, as well as the 18S and 28S ribosomal nuclear genes, indicated that M. lopezi n. sp. is closely related to other tropical Meloidogyne spp. that infect coffee, especially M. arabicida, M. izalcoensis and M. paranaensis from Central and South America. Isozyme analyses and PCR-RFLP of the COII-16S rRNA mitochondrial gene region enable a clear diagnostic differentiation between these species.

Meloidogyne chitwoodi is a quarantine pathogen and a severe problem of potato. Intraspecific variation and genetic structure are not well characterised for M. chitwoodi, but are critical to avoid misidentification and to optimise management strategies. The objective of this study was to analyse the morphological and molecular variation of four M. chitwoodi isolates, representing all races and pathotypes currently known in the USA. Despite statistically significant morphological variation among adult females, morphometrics were not able reliably to distinguish M. chitwoodi isolates. In contrast to morphology, molecular traits that are determined by nuclear ribosomal genes were stable across aU isolates. Malate dehydrogenase, esterase and Superoxide dismutase isozyme phenotypes were conserved in all isolates, which is an important finding for diagnostics. To gain insight into the genetic structure of M. chitwoodi, we analysed a mitochondrial DNA segment including a partial region of COII, tRNA-His and 16S rRNA genes. Genetic structure was weak and marked by high haplotype and low nucleotide diversity. We found a high level of mitochondrial heteroplasmy in M. chitwoodi. Taken together, our results indicate that there is significant intraspecific morphological and molecular variation in M. chitwoodi. Consequences for resistance breeding in potato and directions for phylogeographic studies to trace the origin of M. chitwoodi are discussed.

The yam nematode, Scutellonema bradys, which can cause dry rot disease of yam (Dioscorea spp.), was recorded for the first time from Costa Rica in four species of yam occurring in the Atlantic and north regions. Morphometric measurements from two populations from each region using ten female and 11 male characters corresponded with previous descriptions of this species. Canonical discriminant analysis of the female morphometric data separated the populations by region, whereas no separation by region was evident using the male data. Analysis of DNA sequences from the ITS region indicated that populations from Costa Rica were monophyletic with S. bradys from West Africa and clearly distinct from other Scutellonema species. No genetic separation by geographic region or Dioscorea species host was observed between Costa Rica populations. Species-specific primers were developed from the ITS region and supported the identity of 17 populations from 15 locations in Costa Rica as S. bradys: 14 populations from D. alata (greater or water yam) and one each from D. trifida (white yampee), D. cayenensis (yellow yam) and D. rotundata (white yam). Yam production in Costa Rica began in the Atlantic region, where the yam nematode was likely introduced from the Caribbean, progressively spreading to other locations through the use of infected vegetative planting material.

Radopholus similis is an economically important pest of both banana and citrus in tropical regions. Here we present draft genomes from two populations of R. similis from Costa Rica that were created and assembled using short read libraries from Illumina HiSeq technology.

The morphology of Meloidogyne chitwoodi is relatively poorly characterised. In a recent study, morphological variation in adult females of M chitwoodi was analysed. However, comparable data are lacking for adult males and second-stage juveniles (J2). Therefore, our studies of four M. chitwoodi isolates from the western tJSA, representing all currently known races and pathotypes, were designed to characterise intraspecific morphological variability in J2 and males and to compare it to variability found in adult females. Multivariate analysis of variance indicated statistically significant variation among isolates for both J2 and males. Moreover, for adult males, canonical discriminant analysis grouped races with their respective pathotypes. In addition, we analysed two morphological features that were previously uncharacterised for M. chitwoodi, i.e., stylet knob width and distance from secretory-excretory pore to anterior end. Both of these characters may be useful in distinguishing M. chitwoodi from closely related species. SEM clearly showed the presence of areolations along the bodies of J2, thereby separating M. chitwoodi from M. fallax and M. minor. In summary, this study indicates that M. chitwoodi J2 and adult males show substantially more intraspecific morphological variabihty than previously known. Consequences for diagnostics are discussed.

This chapter presents procedures on polymerase chain reaction (PCR) amplification, protocols for PCR, cloning and sequencing, and mitochondrial genome annotation and gene identification for the characterization of nematodes.