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JOURNAL of NEMATOLOGY Molecular Identification Of JOURNAL OF NEMATOLOGY Article | DOI: 10.2130/jofnem-2020-117 e2020-117 | Vol. 52 Molecular identification of Bursaphelenchus cocophilus associated to oil palm (Elaeis guineensis) crops in Tibu (North Santander, Colombia) Greicy Andrea Sarria1,*, Donald Riascos-Ortiz2, Hector Camilo Medina1, Abstract 1 3 Yuri Mestizo , Gerardo Lizarazo The red ring nematode (Bursaphelenchus cocophilus (Cobb) Baujard 1 and Francia Varón De Agudelo 1989) has been registered in oil palm crops in the North, Central 1Pests and Diseases Program, and Eastern zones of Colombia. In Tibu (North Santander), there Cenipalma, Experimental Field are doubts regarding the diagnostic and identity of the disease. Oil Palmar de La Vizcaína, Km 132 palm crops in Tibu with the external and internal symptoms were Vía Puerto Araujo-La Lizama, inspected, and tissue samples were taken from different parts of Barrancabermeja, Santander, the palm. The refrigerated samples were carried to the laboratory of 111611, Colombia. Oleoflores in Tibu for processing. The light microscopy was used for the quantification and morphometric identification of the nematodes. 2 Facultad de Agronomía de Specimens of the nematode were used for DNA extraction, to amplify la Universidad del Pacífico, the segment D2-D3 of the large subunit of ribosomal RNA (28S) Buenaventura, Valle del Cauca, and perform BLAST and a phylogeny study. The most frequently Campus Universitario, Km 13 vía symptoms were chlorosis of the young leaves, thin leaflets, collapsed, al Aeropuerto, Barrio el Triunfo, and dry lower leaves, beginning of roughening, accumulation of Colombia. arrows and short leaves. Bursaphelenchus, was recovered in most 3Extension Unit, Cenipalma, Tibu of the tissues from the samples analyzed: stem, petiole bases, Norte de Santander, 111611, inflorescences, peduncle of bunches, and base of arrows in variable Colombia. populations. The morphometric data and sequences obtained for the segment D2-D3 confirms to B. cocophilus as the causal agent of *E-mail: [email protected] red ring disease on oil palms in the study area. For our knowledge, This paper was edited by this study reports the first partial sequences of segment D2-D3 of Zafar Ahmad Handoo. B. cocophilus in oil palm in Colombia. Received for publication April 17, 2020. Keywords Bursaphelenchus cocophilus, Diagnostic, Molecular biology, Phylogenetic analysis. The oil palm industry is one of the most important in are considered as the main threat and have been the Colombian agricultural sector. Currently, Colombia responsible for crop losses, including the so-called is the fourth producer of palm oil and the first in Latin lethal ones such as sudden wilt (Phytomonas sp.), America, with more than 535,000 hectares planted lethal wilt (unknown etiology), and the red ring in 112 towns of 20 states, placing it as one of the disease associated with the migratory endoparasite main agricultural lines of the country (Fedepalma, nematode Bursaphelenchus cocophilus (Cobb, 1919; 2018; Sistema de información estadística del sector Goodey, 1960; Baujard, 1989) (=Rhadinaphelenchus palmero-SISPA, 2019). One of the most important cocophilus [Cobb]) (Martínez, 2010). aspects of Colombian palm production is related The red ring disease has been reported in Central to phytopathological problems. These diseases America (Guatemala, Nicaragua, Belize, Costa Rica, © 2020 Authors. This is an Open Access article licensed under the Creative 1 Commons CC BY 4.0 license, https://creativecommons.org/licenses/by/4.0/ Bursaphelenchus cocophilus associated to oil palm: Sarria et al. El Salvador, Honduras, Mexico, Panama), Latin act as intracellular parasites (Griffith, 1987; Griffith and America (Brazil, Ecuador, Guyana, French Guyana, Koshy, 1990), causing the interruption of the transport Peru, Venezuela, and Colombia), and in the southern of nutrients, water, and sap, which induces drying of Caribbean (Granada, San Vicente, Tobago, Suriname, the leaves. In palms with short leaves, the nematodes Trinidad, Dominican Republic) (Kraaijenga and den are found mainly in the leaf buds, which makes the Ouden, 1966; Kastelein, 1987; Giblin-Davis, 2001; palm generate shorter, deformed, yellow, and dry Sánchez and Cerda, 2002; Brammer and Crow, 2002a). leaves (Brammer and Crow, 2001; Oehlschlager et al., In Colombia, there is a history of the red ring since 2002). It have been reported that in raining season 1960s. In 1967, more than 10,000 ha of coconut palm the nematode populations increase and they move disappeared on the Pacific coast. In 1986, red ring toward arrows, central leaves, tissue in the leaf buds, disease was detected in oil palm in Palmeras de la Costa and the spear leaf (Volcy, 1998) and B. cocophilus plantation (El Copey, state of Cesar) and currently, in the achieves the central arrow may access to any petiole Eastern Plains and the state of North Santander, causing (Menjivar et al., 1988; Cuthbert, 1993; Ferris, 1999). in addition to the red ring, the short leaf syndrome Morphologically, males of B. cocophilus are (Varón de Agudelo and Granada, 1986; Chinchilla, 1992; characterized by having sclerotized cephalic region, Calvache et al., 1995; Aldana et al., 2015). paired spicules, absent gubernaculum, a conoid to The red ring disease has caused losses that sharply pointed tail, and strongly ventrally curved, reached 8 million dollars between 1990 and 2002 which contains a terminal bursa, presents two pairs in the North and Eastern production areas (Aldana of ventrosubmediales papillae very close at the et al., 2015). It has been registered mainly in crops from base of the bursa and a pair preanal, anterior to the North, Central, and Eastern zone of Colombia, where cloacal aperture (Opperman, 1995). The females are management strategies are mainly preventive through characterized by having a thin cuticle, marked with frequent monitoring and trapping of the Rynchophorus transverse striations, lateral fields with four incisures, palmarum nematode vector (Coleoptera: Curculionidae) the labial region is high and smooth, covered vulva (Blair and Darling, 1968; Griffith, 1969; Oehlschlager by a protuding anterior vulvar lip, presence of a long et al., 2002). Other management practices include early post-uterine sac, a narrow and elongated tail with a detection and timely eradication of plants, followed by clindroid and rounded terminus (Opperman, 1995). proper waste disposal (Van Hoof and Seinhorst, 1962; Currently, DNA sequences of segment D2- Griffith, 1987; Chinchilla, 1992; Chinchilla and Escobar, D3, Internal Transcribed Spacer-(ITS) of ribosomal 2007; Saenz, 2016). RNA and Cytochrome oxidase subunit I-COI of The red ring symptoms in oil palm are varied in mitochondrial DNA have been obtained for B. Colombia, in the case of the Eastern Plains the main cocophilus in coconut palm and deposited in public symptom is the shortening of leaves. In the North Zone, molecular databases, which are used as a reference there is shortening and copper coloration of the leaflets. for the identification of the nematode (Ye et al., 2007; In some cases, there is accumulation of spear leaf and Silva et al., 2016). However, to date in Colombia no when they open are very short and chlorotic (Chinchilla, molecular characterization or phylogenetic analysis of 1992; Calvache et al., 1995). However, external B. cocophilus from oil palm has been performed. symptoms are not considered a diagnostic method of In Tibu, oil palm plants with symptoms similar the disease because there are other pathogens that to those induced by the disease known as the red induce similar symptoms in the foliage (Dean, 1979). ring had been eliminated, to prevent their spread. The most frequent symptoms are young leaf chlorosis, However, palm growers have observed that some drying of lower leaves, breaking of petioles, and leaf plants remain diseased for a longer time without folding. Internally in the petioles and spear leaf, brown showing the severity of the disease, so there is doubt or reddish brown spots may be observed, and in the about the diagnosis and identity of the causal agent, stem continuous or discontinuous, brown or reddish especially because sometimes the continuous ring is brown rings (Brathwaite and Siddiqi, 1975; Chinchilla, not observed or because the nematode is not found 1992; Calvache et al., 1995; Brammer and Crow, in sampling surveys. Due to the observations made by 2002b). The final state of the disease is the death of the palm growers, this research had as main objective plants (Thorne, 1961; Giblin-Davis, 1990; Sáenz, 2005; the diagnostic of the disease by the verification of Giblin-Davis et al., 2010). the symptoms, the identification of the nematode Bursaphelenchus cocophilus can be found and confirmation of its identity at the morphometrical infecting the parenchymal tissues of the stem, and molecular levels using diagnostic characters petioles and sometimes in roots, the intercellular recommended and the amplification of the large spaces, and in an advanced state of the disease, they subunit D2-D3 of the 28S. 2 JOURNAL OF NEMATOLOGY Materials and methods divided into three parts with a sterile scalpel, transferring the sections to Eppendorf tubes with 15 µL lysis buffer Symptoms observation and sampling (50 mM KCl, 10 mM Tris pH 8.0, 15 mM MgCl2, 0.5% Triton x – 100, 4.5% Tween – 20, 0.09% Proteinase K). In total, 24 lots were visited located in 12 farms of Subsequently, the tubes were incubated at −80°C (15 min), Tibu (Norte de Santander), each of about 10 ha; in 65°C (1 h), and 95°C (15 min), centrifuged at 16,000 g each plantation, plants with abnormal aspect were (1 min) and stored at –20°C. The PCR amplification of identified, and previously selected by the health the expansion segment D2-D3 of the large subunit of censors (expert persons). In each case, description ribosomal DNA (28S) was performed with the forward D2A of the external and internal symptoms was recorded primers: (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and when the palms were knocked down with a chainsaw reverse D3B: (5′-TCCTCGGAAGGAACCAGCTACTA-3′) at the base of the stem and then cutting in sections according to De Ley et al.
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