Bermudagrass plants (Cynodon dactylon cv. 419) from a soccer field in Santa Ana, San Jose Province, Costa Rica, were found with symptoms of decline with dried leaves and leaf yellowing in patches in February 2012. Roots of the affected plants presented small and smooth galls and protruding egg masses similar to those associated with root-knot nematodes (RKN), Meloidogyne spp. Morphometric and molecular analyses were conducted to identify the species present. Morphological measurements from 30 second-stage juveniles and perineal patterns from 10 adult females matched the description of Meloidogyne marylandi Jepson and Golden. Body length averaged 429.5 ± 18.5 (range: 392 to 459) μm, mean width averaged 16.0 ± 1.3 (range: 13.2 to 18.0) μm, stylet lengths averaged 10.7 ± 1.0 (range: 9.1 to 13.4) μm, dorsal gland orifice from stylet base averaged 2.8 ± 0.3 (range: 2.4 to 3.4) μm, tail lengths averaged 60.9 ± 4.9 (range: 45.5 to 74.7) μm, and the hyaline region of the tails averaged 12.4 ± 1.4 (range: 8.6 to 14.8) μm. Lower average tail length and hyaline tail terminal differentiate M. marylandi from the closely related species M. graminis, the grass root-knot nematode, that can also parasitize bermudagrass (1). Hemizonid position was posterior to the excretory pore. The overall shape of the perineal patterns was ovoid, and the dorsal arch was medium high with lateral lines and coarse striae. Males were rarely observed. DNA was extracted from 10 single mature females. Amplification and sequencing of the mitochondrial DNA region between COII and the lRNA gene was accomplished with primers C2F3 (5′-GGTCAATGTTCAGAAATTTGTGG-3′) and 1108 (5′-TACCTTTGACCAATCACGCT-3′) (2). A PCR product approximately 520 bp in length was obtained and the sequence (GenBank Accession Nos. KC473862 and KC473863) was compared with those in GenBank using BLAST and showed 99 to 100% identity with known sequences of M. marylandi (JN241916.1, JN241905.1, JN241917.1, JN241921.1, and JN241955.1) (3). Phylogenetic analysis with maximum likelihood (MEGA v.5.0) (4) of those sequences placed the Meloidogyne sp. from Costa Rica in a clade (100% bootstrap support) that included only M. marylandi sequences available from the GenBank database, thus confirming its identity. In addition, digestion of the PCR product with SspI restriction enzyme produced a four band pattern similar to what has been reported for M. marylandi from Israel and United States (Maryland) and this approach is considered useful for the separation of M. marylandi from M. graminis (3). To our knowledge, this is the first report of the root-knot nematode M. marylandi in Costa Rica.
References: (1) A. M. Golden. J. Nematol. 21:453, 1989. (2) T. O. Powers and T. S. Harris. J. Nematol. 25:1, 1993. (3) M. A. McClure et al. Plant Dis. 96:635, 2012. (4) K. Tamura et al. Mol Biol Evol. 28:2731, 2011.