The oil palm industry is one of the most important in the Colombian agricultural sector. Currently, Colombia is the fourth producer of palm oil and the first in Latin America, with more than 535,000 hectares planted in 112 towns of 20 states, placing it as one of the main agricultural lines of the country (Fedepalma, 2018; Sistema de información estadística del sector palmero-SISPA, 2019). One of the most important aspects of Colombian palm production is related to phytopathological problems. These diseases are considered as the main threat and have been responsible for crop losses, including the so-called lethal ones such as sudden wilt (
The red ring disease has been reported in Central America (Guatemala, Nicaragua, Belize, Costa Rica, El Salvador, Honduras, Mexico, Panama), Latin America (Brazil, Ecuador, Guyana, French Guyana, Peru, Venezuela, and Colombia), and in the southern Caribbean (Granada, San Vicente, Tobago, Suriname, Trinidad, Dominican Republic) (Kraaijenga and den Ouden, 1966; Kastelein, 1987; Giblin-Davis, 2001; Sánchez and Cerda, 2002; Brammer and Crow, 2002a).
In Colombia, there is a history of the red ring since 1960s. In 1967, more than 10,000 ha of coconut palm disappeared on the Pacific coast. In 1986, red ring disease was detected in oil palm in Palmeras de la Costa plantation (El Copey, state of Cesar) and currently, in the Eastern Plains and the state of North Santander, causing in addition to the red ring, the short leaf syndrome (Varón de Agudelo and Granada, 1986; Chinchilla, 1992; Calvache et al., 1995; Aldana et al., 2015).
The red ring disease has caused losses that reached 8 million dollars between 1990 and 2002 in the North and Eastern production areas (Aldana et al., 2015). It has been registered mainly in crops from North, Central, and Eastern zone of Colombia, where management strategies are mainly preventive through frequent monitoring and trapping of the
The red ring symptoms in oil palm are varied in Colombia, in the case of the Eastern Plains the main symptom is the shortening of leaves. In the North Zone, there is shortening and copper coloration of the leaflets. In some cases, there is accumulation of spear leaf and when they open are very short and chlorotic (Chinchilla, 1992; Calvache et al., 1995). However, external symptoms are not considered a diagnostic method of the disease because there are other pathogens that induce similar symptoms in the foliage (Dean, 1979). The most frequent symptoms are young leaf chlorosis, drying of lower leaves, breaking of petioles, and leaf folding. Internally in the petioles and spear leaf, brown or reddish brown spots may be observed, and in the stem continuous or discontinuous, brown or reddish brown rings (Brathwaite and Siddiqi, 1975; Chinchilla, 1992; Calvache et al., 1995; Brammer and Crow, 2002b). The final state of the disease is the death of plants (Thorne, 1961; Giblin-Davis, 1990; Sáenz, 2005; Giblin-Davis et al., 2010).
Morphologically, males of
Currently, DNA sequences of segment D2-D3, Internal Transcribed Spacer-(ITS) of ribosomal RNA and Cytochrome oxidase subunit I-COI of mitochondrial DNA have been obtained for
In Tibu, oil palm plants with symptoms similar to those induced by the disease known as the red ring had been eliminated, to prevent their spread. However, palm growers have observed that some plants remain diseased for a longer time without showing the severity of the disease, so there is doubt about the diagnosis and identity of the causal agent, especially because sometimes the continuous ring is not observed or because the nematode is not found in sampling surveys. Due to the observations made by the palm growers, this research had as main objective the diagnostic of the disease by the verification of the symptoms, the identification of the nematode and confirmation of its identity at the morphometrical and molecular levels using diagnostic characters recommended and the amplification of the large subunit D2-D3 of the 28S.
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 (
Information of partial sequences D2-D3 of ribosomal DNA downloaded from GenBank and obtained in the present study for
Isolate | Species name | Location | Plant-host | Insect host | GenBank accession number | Reference or source |
---|---|---|---|---|---|---|
1 |
|
Tibu, Norte de Santander, Colombia |
|
|
MN612640 | Present study |
2 |
|
Tibu, Norte de Santander, Colombia |
|
|
MN612641 | Present study |
3 |
|
Tibu, Norte de Santander, Colombia |
|
|
MN612642 | Present study |
4 |
|
Tibu, Norte de Santander, Colombia |
|
|
MN612643 | Present study |
136 |
|
USA | None |
|
AY508073 | Ye et al. (2007) |
137 |
|
Austria |
|
|
AY508074 | Ye et al. (2007) |
170 |
|
Turkey |
|
None | AY508093 | Ye et al. (2007) |
S12 |
|
Brazil |
|
|
KT156772 | Silva et al. (2016) |
NT25 |
|
Colombia |
|
|
KT156775 | Silva et al. (2016) |
NT26 |
|
Colombia |
|
|
KT156776 | Silva et al. (2016) |
153 |
|
Germany | Greenhouse soil | Unknown | AY508082 | Ye et al. (2007) |
154 |
|
Greece |
|
|
AY508083 | Ye et al. (2007) |
168 |
|
Germany |
|
|
AY508091 | Ye et al. (2007) |
173 |
|
Germany |
|
|
AY508096 | Ye et al. (2007) |
171 |
|
USA | None |
|
AY508094 | Ye et al. (2007) |
174 |
|
USA | None |
|
AY508097 | Ye et al. (2007) |
176 |
|
USA | None |
|
AY508099 | Ye et al. (2007) |
180 |
|
Italy |
|
Unknow | AY508103 | Ye et al. (2007) |
1057 J |
|
Poland |
|
|
MF422699 | Tomalak et al. (2017) |
98 |
|
USA |
|
– | AY508109 | Ye et al. (2007) |
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.
The
Average number of nematodes (
Extraction method (Decantation) | Sampling method | |||
---|---|---|---|---|
Tissue class | With facial paper | Without facial paper | Palms knocked down | Palms standing (Drill) |
Peduncle | 10 | 8 | X | |
Petiolar Base | 16 | 501 | X | |
Spear leaf base | 5 | 0 | X | |
Inflorescences | 110 | – | X | |
Stem | 469 | 51 | X | |
Stem | 1 |
|
|
Population of
Extraction method (Decantation) | Sampling method | |||
---|---|---|---|---|
With facial paper | Without facial paper | Palms knocked down | Palms standing (Drill) | |
Maximum Population | 1,295 | 720 | X | |
Maximum population | 2 | 5 | X | |
Absolute density | 150 | 150 | X | |
Absolute density | 0.86 | 4 | X | |
Absolute frequency | 58.8 | 71.4 | X | |
Absolute frequency | 42.9 | 85.7 | X |
From the samples processed in the present study, only were obtained juveniles and males from
Morphometric data of males of a
Locality/Publication | Tibu-North Santander, Colombia oil palm (Present study) |
Brazil Coconut Red Ring Disease After (Lordello and Zamith, 1954) |
Trinidad coconut Red Ring Disease (Basil, 1960) |
Venezuela oil palm Red Ring Disease (Gerber et al., 1989) |
Venezuela oil palm Little leaf (Gerber et al., 1989) |
---|---|---|---|---|---|
Body length | 964.67±70.07 (807.09-1062.67) | 820-1420 | 1020 (840-1160) | 1017±77 (841-1111) | 866±71 (789-965) |
|
83.89±10.68 (69.53-99.78) | 92-143 | 120 (100-179) | 129.7±13.3 (113.8-150.0) | 99.3±11.8 (78.9-116.1) |
|
29.33±1.72 (27.02-32.25) | 22-47 | 28 (24-35) | 26.1±2.2 (22.5-29.2) | 22.4±2.8 (18.8-26.5) |
|
3.70±0.31 (3.19-4.31) | – | – | 5.6±0.5 (4.8-6.4) | 5.4±0.8 (4.7-6.8) |
|
96.58±0.20 (96.30-96.90) | – | – | – | – |
Max. Body diam. | 11.62±1.34 (9.97-14.22) | – | – | 8±1.0 (7.0-9.0) | 9.0±1.0 (7.0-10.0) |
Stylet | 10.90±0.88 (9.07-12.12) | 10.7-13.8 | 12-13 | 11±1.0 (11-12) | 12.0±1.0 (11.0-12.0) |
Lip region width | 4.61±0.52 (3.81-5.22) | – | 5.5 | – | – |
Lip region height | 3.13±0.43 (2.43-365) | – | 3.0 | – | – |
Median bulb length | 12.16±0.79 (10.75-13.04) | – | – | – | – |
Median bulb diam. | 5.99±0.84 (4.66-7.64) | – | – | – | – |
Tail length | 32.83±2.35 (28.94-37.90) | – | – | 39.0±4.0 (32.0-46.0) | 39.0±4.0 (33.0-46.0) |
Cloacal or anal body diam. | 8.90±0.71 (7.75-10.16) | – | – | 7.0±1.0 (5.0-8.0) | 7.0±1.0 (6.0-9.0) |
Spicule length | 15.21±1.41 (13.29-17.32) | – | – | 12.0±1.0 (11.0-13.0) | 11±1.0 (10.0-13.0) |
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
The symptoms observed in diseased oil palms of Tibu, included the syndrome of short leaf (recognized in Central America in 1986 and associated with
The nematode was recovered in almost all the sampled plants except in two palms. Several authors indicate that external symptoms are not enough to diagnose the disease, due to it may be caused by other pathogens or factors such as nutritional deficiencies or mechanical damage, so it is necessary to obtain samples and verify the presence of the nematode (Dean, 1979; Chinchilla, 1991). It is important to consider that when the palm is knocked down it is possible to observe internal symptoms in detail and take tissue samples at the different sites of the plant. Through of the drill method was not possible to confirm the presence of the ring in plants with external symptoms and the nematode only was recovered in samples taken in the stem in a low density, possibly because a very low population of the nematode or because the tissue was in an advanced state of necrosis. The drilling method is faster, but the ring is not always observed, therefore, the use of this technique may lead to underestimating the population level or absolute density of the nematode. In coconut palm, the punched borehole methodology was used by the Colombian Agricultural Institute (ICA), for the diagnostic and eradication of palms with a red ring during the 70s and 80s years (Victoria et al., 1970).
According with the extraction method, the nematode presented the highest absolute frequency when the samples were processed without and with facial paper with values of 71.4 and 85.7%, respectively. The advantage of facial paper is that it allows obtaining a cleaner sample with few tissue residues, which facilitates the microscope nematode observation.
The maximum population of nematode was found in the stem and the morphological and morphometrical characteristics match with those recorded for the species
In conclusion, the red ring disease of the oil palm in Tibu (North Santander) presents the same external and internal symptoms described in Colombia and other countries where the disease has been registered, even when they were very varied. It was possible to extract and identify the