Molecular identification of Bursaphelenchus cocophilus associated to oil palm (Elaeis guineensis) crops in Tibu (North Santander, Colombia)

In total, 24 lots were visited located in 12 farms of Tibu (Norte de Santander), each of about 10 ha; in each plantation, plants with abnormal aspect were identified, and previously selected by the health censors (expert persons). In each case, description of the external and internal symptoms was recorded when the palms were knocked down with a chainsaw at the base of the stem and then cutting in sections to observe the internal symptoms. Similarly, cuts were made in petioles, bunches, buds, and spear leaf.

Symptomatic tissue samples as petioles, base of spear leaf, inflorescences, peduncle of inflorescences and bunches and stem from eradicated palms, were collected and kept in plastic bags, properly identified and refrigerated until processing. To collect tissues samples in palms that were not eradicated, a manual drill was used to perforate the stem at a height of approximately 1.5 m from the base at a depth close to 30 cm.

Nematodes were extracted using the oxygenation-decantation method (Ravichandra, 2014). Five grams of tissue from each of the sampled organs were cutted into small portions and placed in a decantation sieve, with and without facial paper, rested on a decantation plate with enough water to cover the sample. After 24 h, the nematode suspension contained in the decantation plate was removed and concentrated at 20 mL with the 400-mesh sieve (Varón de Agudelo and Castillo, 2001).

To quantify the population of nematodes present in five grams of fresh tissue in each sample, three aliquots of 1 mL were taken and counting in chamber under a light microscope, Olympus PX40 and Olympus DP 73. For the morphological and morphometrical identification, the nematodes were killed with heat at 60°C for 4 min and fixated in 2% formalin. Then, semipermanent preparations were done and morphometric data were registered following Basil (1960), Brathwaite and Siddiqi (1975), Mai and Lyon (1975), Gerber et al. (1989). The morphometric data were taken using a compound microscope ZEISS Axio (China, microscope reference: 3136006171 Axio.A1).

DNA extraction was performed by the proteinase K method (Riascos-Ortiz et al., 2019). A specimen was divided into three parts with a sterile scalpel, transferring the sections to Eppendorf tubes with 15 µL lysis buffer (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). Subsequently, the tubes were incubated at −80°C (15 min), 65°C (1 h), and 95°C (15 min), centrifuged at 16,000 g (1 min) and stored at –20°C. The PCR amplification of the expansion segment D2-D3 of the large subunit of ribosomal DNA (28S) was performed with the forward D2A primers: (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and reverse D3B: (5′-TCCTCGGAAGGAACCAGCTACTA-3′) according to De Ley et al. (1999). The PCR conditions were initial denaturation during 2 min at 94°C followed by 40 cycles of 45 sec at 94°C, 45 sec at 55°C and 1 min at 72°C and final extension of 10 min at 72°C. The PCR products were sequenced in both directions by the company Bionner (South Korea).

The sequences obtained were edited using the Geneious software (Kearse et al., 2012). Once the sequences edition were carry out, their identity was confirmed using the software BLASTn (http://www.ncbi.nlm.nih.gov/BLAST). Subsequently, the sequences presented under the accession numbers in Table 1 were aligned and analyzed using the MUSCLE algorithm included in the program MEGA6 (Tamura et al., 2013). Based on the matrix obtained, the nucleotide substitution model was determined by taking into account the Bayesian information criterion (BIC) using ModelGenerator v.0.851 software (Keane et al., 2006). The phylogenetic relationship was determinate by the maximum likelihood (ML) method based on the general time reversible (GTR) model, and the Gamma distribution, which was used to model the differences in evolutionary speed between locations. The internal support of the nodes was carry out using the bootstrap method with 1,000 replicates. Aphelenchoides besseyi sequence was used as external group (AY508109).

During the sampling, 24 lots of 12 farms were visited, located in Punta de Palo, La Libertad, M14, M24, Oru L15, Playa Rica, Kilometer 15, Llano Grande, Refineria, and Campo Dos (Tibu, North Santander) for a total of 32 samples between stem, peduncle of bunches and inflorescences, petiolar base and spear leaf base. Palms with symptoms similar to those that have been associated with a red ring by many researchers were observed on all farms.

The symptoms observed in diseased palms of Tibu were varied; the most frequent being chlorosis of the young leaves, thin leaflets, collapsed, and dry bottom leaves which remain adhered to the stem, accumulation of spear leaf, and short leaves. As the disease progresses the chlorotic leaves turn brown and foliar drying is observed (Fig. 1). In this study, some palms had advanced symptoms. In cross sections of the stem, the initial symptoms were characterized by small reddish-brown necrotic spots distributed in the vascular bundles (Fig. 2A, B). In more advanced stages of the disease was possible to observe the complete reddish brown ring of a few centimeters wide and near the periphery of the stem (Fig. 2C, D). The upper third leaves were shorter, chlorotic, and sometimes with thin, and dry leaflets. Necrotic points and sometimes large areas affected with necrotized tissue were observed when cross-sectional and longitudinal cutting of the petioles (Fig. 2E-G). When the sample was collected with the drill it was possible to observe in some palms small pieces of necrotic reddish tissue indicating that the palm had formed the ring.

Figure 1:

Figure 2:

The Bursaphelenchus cocophilus nematode (Fig. 3) was recovered from all tissues evaluated: stem, petiolar bases, inflorescences, bunch peduncle, and spear leaf base in variable populations (Table 2). A range of population from 720 to 1,295 individuals per 5 grams of stem tissue and petiolar base was found, respectively (Table 3). The nematode was recovered in almost all the sampled plants except in two palms, one of them had the external symptoms similar to those of the red ring; however, the ring was not observed internally and the nematode was not recovered in the stem tissue. On the other hand, the bud of this plant was decomposed and when cut it presented an aqueous substance of white color and foul odor. In the case of the second palm, the sampling was done with the drill and by this method it was not possible to confirm the presence of the ring; however, external symptoms were observed. Regarding the extraction method, it was possible to obtain the nematode with and without facial paper; however, the highest absolute frequency was obtained when the samples were processed without facial paper presenting values of 71.4% when the tissue came from eradicated palms and 85.7% of the tissue taken with a drill (Table 3).

Figure 3:

From the samples processed in the present study, only were obtained juveniles and males from B. cocophilus. To morphological level the males were characterized to present appearance of thread with a thin stylet and sometimes with little visible basal knobs and a tail curved ventrally in a resting state. Because females were not observed, morphometric data were registered for the males. The values registered for the diagnostic characters recommended were similar to those reported in the original and reference descriptions of B. cocophilus (Table 4).

Four consensus sequences, with accession numbers MN612640 to MN612643, of segment D2-D3 were obtained, which presented a percentage of similarity between 99.5 and 100% with reference sequences of B. cocophilus previously deposited in GenBank (AY508076, KT156770, KT156772). The sequences of segment D2-D3 obtained in this study are the first of this amplicon reported for B. cocophilus in oil palm in Colombia. According to the phylogenetic tree of ML (Fig. 4), the B. cocophilus consensus sequences obtained in this study for D2-D3 grouped in the same clade with reference sequences of the same species recorded for coconut palm (KT156772 from Espírito Santo-Brazil, KT156775, KT156776 from Nariño-Colombia) with a 100% bootstrap support. Likewise, the clade of B. cocophilus is clearly separated from that of other species of the same genus.

Figure 4: