Characterization of Pterotylenchus cecidogenus in Desmodium ovalifolium cover crop from oil palm plantations in central Colombia

We visited 30 oil palm plantations (Elaeis guineensis) of between 3 and 6 years of establishment with Desmodium ovalifolium as a cover crop. To verify the health status of the cover crop and identify plants with abnormal aspects, one sample per lot was taken, each of which was composed of five subsamples (Table 1). The selected plantations were located in central Colombia (departments of Santander [26], Norte de Santander [1], Cesar [3]).

Symptomatic tissue samples as stems (with or without galls), leaves, pods, and inflorescences were collected and kept in labeled plastic bags and refrigerated until processing. Simultaneously, at locations where plants with symptoms were found, samples of young roots and rhizospheric soil were collected from close to the area with the highest concentration of roots in order to determine if the nematode affected the root system or survived in the ground.

Nematodes were extracted using the oxygenation-decantation method (Ravichandra, 2014). In brief, 1 g of fresh tissue from each of the sampled organs was cut into small portions and placed in a decantation sieve without a paper towel but with enough water to cover the sample. After 24 h, the decantation plate with the nematode suspension was removed and concentrated to 20 mL with a 400-mesh sieve (Varón de Agudelo and Castillo, 2001).

Soil nematodes were extracted by suspension, filtration, and decantation methods (Ravichandra, 2014). In brief, 100 cm³ of soil was placed in a container with water. After stirring for 2 min, the suspension was passed through a series of three sieves arranged from larger to smaller mesh diameters (reference No. 20 = 840  µm, 200 = 75  µm, 400 = 37  µm). The contents of the last two sieves were collected for decantation using a sieve previously arranged with a paper towel, and rested on a decantation plate with sufficient water. After 24 hr, the nematode suspension contained in the decantation plate was removed and concentrated to 20 mL with a 400-mesh sieve (Varón de Agudelo and Castillo, 2001).

For the morphological and morphometric identification of Pterotylenchus, 23 females were treated with heat at 60°C for 4 min and fixed in 2% formalin. Then, semipermanent preparations were performed, and morphometric data (Table 2) were registered following Siddiqi and Lenné (1984). The morphometric data were obtained using a compound microscope (ZEISS Axio A1, Suzhou, China).

DNA extraction was performed using the proteinase K method (Riascos-Ortiz et al., 2019). In brief, the nematodes were divided into three parts with a sterile scalpel and transferred to Eppendorf tubes with 15 µL lysis buffer (50 mM KCl, 10 mM Tris pH 8.0, 15 mM MgCl2, 0.5% Triton × 100, 4.5% Tween 20, and 0.09% Proteinase K). Subsequently, the tubes were incubated at ‒80°C for 15 min, 65 °C for 1 h, and 95°C for 15 min, centrifuged at 16,000 × g for 1 min, and stored at ‒20°C. The polymerase chain reaction (PCR) amplification of the expansion segment D2-D3 of the large subunit of ribosomal DNA (28S) was performed with the primers D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) forward and D3B (5′-TCCTCGGAAGGAACCAGCTACTA-3′) reverse, according to De Ley et al. (1999). In addition, the partial region of the internal transcribed spacer (ITS), which includes ITS1, 5.8S, and ITS2, was amplified using the primers TW81 forward (5′-GTTTCCGTAGGTGAACCTGC-3′) and AB28 reverse (5′-ATATGCTTAAGTTCAGCGGGT-3′), as proposed by Maafi et al. (2003). The PCR conditions for the amplification of both partial regions were initial denaturation for 2  min at 94°C, followed by 40 cycles of 45 s at 94°C, 45 s at 55°C, 1 min at 72°C, and a final extension of 10 min at 72°C. A total of 25 PCR products were sequenced in both directions by Bionner (South Korea).

The consensus sequences obtained (12 of D2–D3 and 13 of ITS) were edited using the Geneious software (Kearse et al., 2012). Once the sequence editions were carried out, their identities were confirmed using the BLASTn software (http://www.ncbi.nlm.nih.gov/BLAST). Subsequently, the sequences presented under the accession numbers in Table 3 were independently aligned and analyzed using the MUSCLE algorithm included in the MEGA6 program (Tamura et al., 2013). Based on both obtained matrices, the nucleotide substitution models were determined by taking into account the Bayesian information criterion (BIC) using the ModelGenerator v.0.851 software (Keane et al., 2006). The phylogenetic relationships based on D2–D3 and ITS were determined by the maximum likelihood (ML) method based on the Tamura-Nei model (Tamura et al., 2013), which was used to model the differences in evolutionary speed between locations. Internal support of the nodes was performed using the bootstrap method with 1000 replicates. The Cervidellus cervus sequence was used as an external group (HM452377) for D2-D3 and Radopholus similis (GQ281456) for ITS.

To quantify the nematode populations present in 1 gram of fresh tissue in each sample, three aliquots of 1  mL were taken and counted in a chamber under a light microscope (Olympus PX40, Allentown, PA) and an adapted camera (Olympus DP 73, Allentown, PA). The same methodology was used to quantify the nematode population in 100 cm³ of soil from each sample. For the analysis of the populations of nematodes present in the soil and tissue of Desmodium plants, parasitic and ecological parameters such as frequencies and prominence values were taken into consideration, including (Norton, 1978; Volcy, 1998):

Absolute frequency = (number of samples in which a genus appears/total samples evaluated) * 100;

Relative frequency = (absolute frequency of the nematode/sum of absolute frequencies) * 100;

Absolute density = number average of individuals per 100 g of soil or number of individuals per 1 g of tissue;

Relative density = (absolute density of the nematode genus/sum of the absolute densities of all genera) × 100;

Prominence value = absolute density * √ absolute frequency;

Relative prominence value = (genus prominence value/sum of the prominence value of all genera) × 100.

We collected 30 samples from 30 plots located on 28 palm oil plantations in the departments of Cesar (26), Santander (3), and Norte de Santander (1), all of which utilized Desmodium ovalifolium as the cover crop (Table 1).

The symptoms observed in diseased plants of D. ovalifolium were characterized by yellowing, chlorosis, drying of leaves, wilting of shoots, and death of branches and plants (Fig. 1). In the stem, galls were observed in the nodes, more frequently in the basal part of the stems, and in some cases in the upper nodes. In the initial stages, galls were light brown in color; as they became older, galls became dark brown. The oldest galls presented a cracked suberous, which, when dried, acquired a dark, almost black color. The galls were an integral part of the stem, and it was not possible to separate them without damaging the tissue (Fig. 1E).

Figure 1:

The nematodes extracted from D. ovalifolium stems with gall symptoms presented morphological and morphometric characteristics similar to those reported for the nematode P. cecidogenus. Females were morphologically distinguished by the following features: a post-mortem habitus that was straight or slightly ventrally arcuate, a vulva covered by large flaps, and a that was tail elongate-conoid to a sharply pointed tip; no males were identified (Table 2; Fig. 2).

Figure 2:

The amplification of the segment D2-D3 and ITS from the ribosomal DNA region yielded amplicons of 728 and 1000 bp, respectively. The comparison of the sequences against the GenBank database did not present percentages of similarity equal to or greater than 99% with other reference sequences previously reported. However, the sequences of segment D2-D3 were very similar to those of KT192617 and KT192618 (identity levels of 81.06% and 81.14%, respectively; E-value: 0.0) of the species Ditylenchus phyllobius (Sinm. Orrina phyllobia). Similar results were obtained with the partial sequences of ITS, with 88% similarity to D. phyllobius (KT192616.1; E-value of 0.0). The partial sequences obtained in this study are the first reported for P. cecidogenus and were deposited for consultation in the NCBI database (Table 3).

Phylogenetic analysis based on the use of segment D2-D3 comprised a total of 36 taxa and 824 characters, including gaps, of which 166 were conserved, 636 were variable, and 363 were informative parsimonious sites. Phylogenetic analysis based on the ITS region included 35 taxa and 1499 characters, including gaps, of which 717 were conserved, 736 were variable, and 645 were informative parsimonious sites. In both analyses, the maximum likelihood algorithm grouped P. cecidogenus consensus sequences of this study with high bootstrap values of 100%, in a clade separated from other species of the Anguinidae family but close to the clade of Ditylenchus phyllobius (Sinm. Orrina phyllobia) with 100% bootstrap support (Figs. 3, 4).

Figure 3:

Figure 4:

The juvenile stages and females of P. cecidogenus were recovered in 56.7% of the tissue samples with galls, with an average population of 1,768 individuals per gram of fresh tissue. In addition, the juvenile and female stages were present in 10% of the node samples, with an average population of 23.8 individuals per gram of fresh tissue. Pterotylenchus was not detected in leaf samples. Only three inflorescences were analyzed because our sampling period did not match the flowering season.

Analysis of the parasitic and ecological parameters in roots showed that the genera of parasitic nematodes of plants with the highest relative importance in Desmodium roots were Meloidogyne (second-stage juveniles) followed by Pterotylenchus, Helicotylenchus, and Pratylenchus (juveniles and females). Xiphinema is a plant-parasitic nematode that is less important (Table 4). In relation to other genera, Pterotylenchus presented a prominence value of 46.7, with a low distribution (13.3) and population level (12), and eight individuals per gram of fresh root. Differences in the genera Pterotylenchus were associated with rhizospheric soil and Desmodium roots; however, their parasitic activity was not determined.

In the rhizospheric soil of Desmodium, the genera of parasitic nematodes of plants of greater relative importance were Helicotylenchus, followed by Tylenchorhynchus (juveniles and females). Xiphinema (juveniles), Meloidogyne (second-stage juveniles), and Pterotylenchus (juveniles and females) were of intermediate importance. Pratylenchus (juveniles and females), Criconemella, and Trichodorus (juveniles) were recorded in the soil samples, but their distribution and population levels were low (Table 5). Pterotylenchus had a prominence value of 11.1, with a low distribution (10) and population level (3.5 individuals per 100 cc of soil).