Identificación morfológica, morfométrica y molecular de especies de Helicotylenchus (Tylenchida: Hoplolaimidae) asociadas a seis cultivos en Costa Rica

Plant-parasitic nematodes are pathogens, but their interactions with other disease-causing agents make it difficult to measure their true impact on crop yields and their estimation on a large scale. In this sense, the correct identification of species becomes an important task to determine which crops they may be associated with and their potential damage. The objective of the present study was to identify species of the nematode Helicotylenchus associated with pineapple crops in San Carlos, Alajuela; citrus in Los Chiles, Alajuela; grape in Santa Ana, San José; banana in Talamanca, Limón; banana in Jicaral, Puntarenas and rice in Paso Canoas, Puntarenas. Morphological, morphometric and molecular studies were carried out. A total of 3000 morphometric measurements were carried out, which facilitated the descriptive statistics of each nematode population. A total of 2500 microphotographs of females and males were taken to discriminate between the Helicotylenchus species associated with the sampled cultures. Polymerase chain reaction (PCR) was used to amplify the D2-D3 domain of the 28S ribosomal DNA (rDNA) region with primers D2A (5'-ACAAGTACCGTGAGGGAAAGTTG-3') and D3B (5'-TCGGAAGGAAGGAACCAGCTACTA-3'). Also, the ITS region between the nuclear ribosomal genes was amplified with primers TW81 (5'-GTTTCCGTAGGTGAACCTGC-3') and AB28 (5'-ACGAGCCGAGTGATCCACCG-3'). PCR products were purified prior to sequencing and each sequence was compared to other sequences reported for Helicotylenchus species deposited in GenBank®. The percentages of similarity and divergence between them were calculated. For phylogenetic analyses, the sequences of the 28S and ITS regions of the rDNA of the different Helicotylenchus species reported in GenBank® were used. Phylogenetic relationships between the species identified from the three molecular markers were established using the Bayesian Inference (BI) method. Molecular information was compared with morphological and morphometric information to obtain a correct identification of the species found in each crop. The species Helicotylenchus dihystera was found associated with pineapple, citrus, banana and grape plantations. The species H. multicinctus was identified in association with the banana crop. In the case of rice cultivation, it was not possible to identify the associated species, so it will be necessary to use other conserved regions of DNA molecularly to provide more information about the species information. The sequences obtained for H. dihystera showed 99% similarity with accessions corresponding to this species reported in mulberry and yam crops in China and Camellia sp. in Burma. As for the sequences of the species H. multicinctus, these showed 99% similarity with accessions corresponding to this species, found in the cultivation of Musa paradisiaca in China and cocoa in South Africa. In the case of the Helicotylenchus population present in the rice crop, the analysis of the amplification of the D2-D3 domain of the 28S ribosomal DNA region (rDNA) showed percentage similarity results of 96% with accessions corresponding to the species H. caudatus, below those necessary to confirm which species it belonged to. The phylogenetic tree corresponding to the analysis of the ITS region sequences showed H. dihystera grouping with accessions found in Japan. In addition, the sequence of the species H. multicinctus grouped with an accession reported in China. The correct description of species represents an important method for the generation of scientific information that allows pest and host records, their location and impact on the performance of plantations. In this way, sufficient information is available to be used in the development of quarantine programs and control strategies.