RESUMO
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.
RESUMO
AIM: Development of a PCR-RFLP assay that could reliably distinguish strains of Pythium myriotylum that are pathogenic to cocoyam from nonpathogens, as well as in planta detection of the pathogen. METHODS AND RESULTS: Sequences of the internal transcribed spacer regions of nuclear ribosomal DNA (rDNA-ITS) containing ITS1 and ITS2 of P. myriotylum isolates from cocoyam and other hosts were aligned and a restriction map was generated. rDNA-ITS alignment report revealed a new single nucleotide polymorphism (SNP; thymine/cytosine) downstream to previously published SNP (guanine/adenine) between isolates of P. myriotylum that are pathogenic to cocoyam and nonpathogenic strains. This new SNP is within the restriction site of the endonuclease AarI. Based on this SNP, a PCR-RFLP assay was developed for specific detection of P. myriotylum. The PCR amplicons of all isolates of P. myriotylum that infect cocoyam were cleaved by AarI, resulting to two bands (600/400 bp); but those from other hosts showed a single band (1000 bp), confirming the presence and specificity of the AarI restriction site. Also, the assay was effective in in planta detection of the pathogen on infected cocoyam roots without prior isolation of a pure culture. CONCLUSION: A PCR-RFLP method was developed that differentiates isolates of P. myriotylum that are pathogenic to cocoyam from nonpathogens as well as from other fungi commonly found in the cocoyam rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: Early and rapid detection of the pathogen could be of great importance in certifying planting materials as disease-free, enhancing sustainable management practices and limiting economic losses.
