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.
The presence of Meloidogyne enterolobii on two hosts and in several locations in Costa Rica is reported. Meloidogyne spp. females were extracted from root of acerola and of wild and introduced guava and their perineal patterns were examined. Females from acerola showed round or dorso-ventrally ovoid perineal patterns, in some cases with lateral lines or with a moderately high to high dorsal arch as is characteristic of M. incognita. In guava, perineal patterns were similar, but with a low to moderate dorsal arch. PCR amplification of the mitochondrial DNA region between COII and the 16S ribosomal RNA yielded a unique product of 705 bp for the five populations extracted from both crops. In addition, the amplified product spanning the 63 bp repeat region of the mitochondrial genome was a single fragment of 320 bp in contrast to other tropical species, which typically amplify multiple bands. Size of PCR products of both mitochondrial regions and the sequence of the 63 bp repeat region supported the identity of these isolates as M. enterolobii (= M. mayaguensis).
In 2018, during a survey sampling for cyst-forming nematodes, two populations of Heterodera sp. were found in the north region of Cartago (Llano Grande and Oreamuno), Costa Rica. White females and cysts were attached to the plant roots of two weeds, white clover (Trifolium repens L.) and Rumex obtusifolius L. Plants were asymptomatic above ground. Cysts were extracted from soil samples using the Fenwick method (Fenwick 1940) and cut to release the second-stage juveniles (J2s). Cysts were lemon-shaped with a prominent vulval cone, light to dark brown coloring, and ambifenestrate with developed underbridge and bifurcated at the end. Measurements combining both populations are given in micrometers and provided in the following format: mean ± SD (range). Cysts (n = 20): length excluding neck = 744.3 ± 61.6 (648.5 to 837.4); width = 495.9 ± 60.5 (421.9 to 621.1); fenestral length = 50.7 ± 6.4 (40.3 to 61.3); and semifenestral width = 36.2 ± 5.6 (24.1 to 45.7). J2s (n = 60): length = 541.1 ± 25.6 (492.5 to 604.9); width = 22.9 ± 1.7 (20.5 to 27.3); stylet = 27.9 ± 0.9 (26.2 to 29.6); labial region height = 4.7 ± 0.3 (3.8 to 5.5); labial region diameter = 9.4 ± 0.5 (8.5 to 10.9); DGO = 5.9 ± 0.5 (4.6 to 7.3); anterior end to excretory pore = 122.2 ± 8.3 (106.4 to 148.7) and to median bulb valve = 81.2 ± 5.3 (67.3 to 92.6); tail length = 66.6 ± 4.9 (55.9 to 77.3); and hyaline region of tail length = 37.1 ± 3.7 (27.7 to 44.5). No males were found. The morphological and morphometrical features of the two Heterodera populations overlapped with each other and within other H. trifolii populations reported worldwide (Subbotin et al. 2010; Sekimoto et al. 2017). The primer sets TW81/AB28 (Subbotin et al. 2001), D2A/D3B (De Ley et al. 1999) and the HTcox1F2 (5´-GCTTCTGATCTTTCCTTTCCACGTA-3´, this study)/JB4 (Derycke et al. 2005) were used for amplification of the internal transcribed spacer (ITS) rRNA, the D2-D3 expansion segments of the 28S (28S) rRNA and the partial mitochondrial cox1 gene (cox1), respectively. The resulting sequences were submitted to the GenBank (ITS: MT020783, 28S: MT010295, and cox1: MT007852). A BLASTn search of the ITS, 28S and the cox1 gene sequences of H. trifolii from Costa Rica revealed a 100% identity with sequences of H. trifolii from Japan (LC208684), South Korea (MN720070) and USA (MK093174), respectively. A greenhouse essay was established to confirm the reproduction of H. trifolii on each host. Using ten replicates (individual pots) per treatment (hosts), pots were inoculated with 1000 individuals (eggs + infective J2s). The experiment was conducted twice. The average greenhouse temperature was 22.1 ± 3.8 °C and plants were harvested 75 days after inoculation. Results showed that H. trifolii was able to reproduce successfully on both hosts. Final average population (roots + soil) in R. obtusifolius and T. repens were 21,516 and 2,626 nematodes (all stages and cysts), respectively. These two weed species can be a source of inoculum for economically important crops. Therefore, studies on life cycle and pathogenicity assays are being performed on agricultural crops. To our knowledge, this is the first report of H. trifolii on white clover and R. obtusifolius in Costa Rica.
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