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 aboveground. 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 (Sekimoto et al. 2017; Subbotin et al. 2010). The primer sets TW81/AB28 (Subbotin et al. 2001), D2A/D3B (De Ley et al. 1999), and 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 rRNA (28S), and the partial mitochondrial cox1 gene (cox1), respectively. The resulting sequences were submitted to GenBank (ITS, MT020783; 28S, MT010295; and cox1, MT007852). A BLASTn search of the ITS, 28S, and 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 the United States (MK093174), respectively. A greenhouse essay was established to confirm the reproduction of H. trifolii on each host. Using 10 replicates (individual pots) per treatment (hosts), pots were inoculated with 1,000 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 was 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.
Read full abstract