This work is licensed under the Creative Commons Attribution 4.0 International License.
Pratylenchus is a migratory endoparasitic nematode that limits the production of a wide range of crops in tropical and subtropical areas of the world (Sasser and Freckman, 1987; Castillo and Vovlas, 2007; Múnera et al., 2009). Crops affected by Pratylenchus include different species of the Musaceae family, including plantain (Musa paradisiaca [L.] AAB Simmonds cv. Dominico Hartón) and banana (Musa acuminata), in which the nematode damages the root system of the plant (Riascos-Ortiz et al., 2022). Symptoms induced by the nematode in Musaceae roots include initial internal yellow lesions that later turn purple and finally brown. Externally, the necrotic areas of the roots appear black due to the destruction of the cortical tissues (CABI, 2022).
In affected plantain and banana plants, the nematode reduces the root system and the ability to take up water and nutrients, which causes foliar chlorosis, growth retardation, decreased bunch weight, lengthening of the productive cycle, and returns or weak suckers (Múnera, 2008; De Luca et al., 2012). In fields severely infested with the nematode, plants suffer toppling and complete bunch loss (CABI, 2022). Production losses are approximately 15%, although they can be higher in plantations >3-yr old and with little agronomic management (Guzmán and Castaño, 2004).
In addition to the direct damage caused to plantain and banana roots, a high correlation has been recorded between Pratylenchus and infection by fungi such as Fusarium oxysporum, F. redolens, F. sambucium, Nigrospora musae, and Rhizoctonia solani, and also bacteria such as Xanthomonas campestris (Bridge et al., 1997).
The identification of these species is not an easy task due to their morphological and morphometric similarities (overlap of characteristic between species). This condition has led some of these species to be considered cryptic, including P. speijeri and P. coffeae, which are morphologically indistinguishable; and a clear separation is possible only through molecular analysis (De Luca et al., 2012). Other species can be separated using a few characteristics of diagnostic value; examples of this mode of distinguishing would be the case of P. araucensis that differs from P. speijeri by the length of the stylet (14.7– 15.9 μm vs. 16.5–18.0 μm), from P. coffeae by the position of the vulva (78% vs. 80%), and from sister species such as P. jaehni and P. loosi mainly by body length (462 μm vs. 488 μm and 522 μm) (Múnera et al., 2009; De Luca et al., 2012).
Pratylenchus araucensis was identified by integrative taxonomy attacking Musaceae in the eastern region of Colombia (Múnera et al., 2009). Although other works have registered Pratylenchus affecting Musaceae in the country, these reports lack morphometric and molecular information, restricting the report of the nematode to the genus level (Zuñiga et al., 1979; Guzmán and Castaño, 2004, 2007; Riascos-Oritiz et al., 2021). This indicates that there is a lack of knowledge of the species of Pratylenchus associated with Musaceae in Colombia, which could make it difficult to manage nematode populations in the main fruit-production areas in the country.
Therefore, it is necessary to expand at the species level the taxonomic knowledge of Pratylenchus populations associated with plantain and banana in Colombia, especially in the fruit-production areas of the center and southwest, where reports of the nematode have been developed up to the genus level. Against this background, the present study raised the following objectives: (i) to identify populations of Pratylenchus from the central and southwestern areas of Colombia through morphological, morphometric, and molecular analysis; (ii) to ascertain the intraspecific diversity of Pratylenchus species identified from molecular data; and (iii) to ascertain the evolutionary relationships of the analyzed populations based on the D2–D3 segment of LSU ribosomal DNA (rDNA) and COI mtDNA.
Materials and Methods
Sampling and morphological and morphometric identification
Composite samples of rhizosphere soil and roots were collected from plantain and banana crops located in the states of Valle del Cauca (municipality of Buenaventura [85 m.a.s.l., average temperature of 26°C, and annual rainfall of 9,000 mm]), Caldas (municipality of Palestina [1,050 m.a.s.l., average temperature of 18°C, and annual rainfall of 2,859 mm]), and Quindío (municipality of Calarcá [1,573 m.a.s.l., average temperature of 19°C, and annual rainfall of 2,500 mm]), Colombia, during the year 2018. Each sample of approximately 1 kg was composed of subsamples extracted from 15 to 20 plants per hectare, which were randomly selected. The samples were taken at 25 cm from the pseudostem and at a depth between 0 cm and 30 cm at three equidistant points.
The morphometric data recorded in this study and others taken from the literature (Inserra et al., 2001; Múnera, 2008; De Luca et al., 2012) were statistically analyzed using principal component analysis (PCA) to establish possible groupings and discriminant diagnostic characteristic that allow identifying the populations studied at the species level, using version 9.4 of the developed by the SAS institute statistical package.
Molecular analysis
For the DNA extraction, the protocol proposed by Riascos-Oritiz et al. (2019) was used. For this purpose, a single specimen was cut and transferred to a tube with 15 μl of lysis buffer (50 Mm KCl, 10 Mm Tris pH 8.3; 2.5 Mm MgCl2; 0.45% NP 40; 0.45% Tween 20; 60 μg/ml proteinase K). Subsequently, the tube was incubated at -80°C (15 min), 65ºC (1 hr), and 95°C (15 min). After this, the tube was centrifuged (1 min at 16,000 g) and stored at -20°C. Using PCR, the D2–D3 region of the large subunit (LSU) of rDNA (28S) was amplified using forward primer D2A (5´-ACAAGTACCGTGAGGGAAAGTTG-3´) and reverse primer D3B (5´-TCCTCGGAAGGAACCAGCTACTA-3´) (De Ley et al., 1999). The cytochrome oxidase subunit I region of mitochondrial DNA (COI) was amplified using forward primer JB3 (5´-TTTTTTGGGCATCCTGAGGTTTAT-3´) and reverse primer JB4.5 (5´-TAAAGAAAGAACATAATGAAAATG-3´) (Bowles et al., 1992). 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, 1 min at 72°C, and final extension of 10 min at 72°C for the D2–D3 region; and initial denaturation during 2 min at 94°C followed by 40 cycles of 45 sec at 94°C, 45 sec at 54°C, 1 min at 72°C, and final extension of 10 min at 72°C for COI. The PCR products were sequenced in both directions by Bioneer Corporation, Daejeon, South Korea.
Phylogenetic analysis
The consensus sequences were edited using the BioEdit 7.0.5.3 (Hall, 1999). Once the sequences were refined, their identity was confirmed by comparing them with the GenBank database, using the BLAST software (http://www.ncbi.nlm.nih.gov/BLAST). Subsequently, the sequences presented under the accession numbers in Table 1 were manually aligned using MEGA6 (Tamura et al., 2013). Based on the matrix obtained for the two genes used, it was possible to determine the nucleotide substitution model, taking into account the Bayesian information criterion (BIC) using the Model Generator v.0.851 software (Keane et al., 2006). The phylogenetic analysis was determined using the maximum likelihood (ML) method together with the Kimura 2-parameter model, and the internal reliability of the nodes was determined by using the method with 1,000 interactions. As an external group of the phylogenetic tree of the LSU partial region, the species Belonolaimus longicaudatus (KF963100) was used and for COI the species Mesocriconema xenoplax (MG422913).
Morphometric data of studied and reference populations of Pratylenchus araucensis.
Characteristic
Plantain, Zabaletas - Buenaventura
Plantain, Delfina - Buenaventura
Plantain, Caldas
Plantain, Calarcá - Quindío
Banana, Calarcá - Quíndio
Musa sp. Arauca, type population (Múnera et al., 2009)
n = 19
n = 14
n = 19
n = 15
n = 10
n = 40
L
456 ± 24.0
501.8 ±56.0
484.5 ± 24.7
549.2 ± 13.9
551.3 ± 13.5
462 ± 31
(401.2 - 496.3)
(449.3-668.7)
(444.4 - 538.0)
(518.4-578.3)
(534.7 - 570.4)
(376-511)
a
26.3 ±2.1
28.2 ± 1.9
29.9 ±2.0
25.2 ±1.3
24.8 ± 1.4
23.9 ±2.3
(20 - 29.8)
(23.5-31)
(23.0-32.8)
(21.6-26.9)
(21.6-26.3)
(19.6-29.2)
b
5.8 ±1.0
6.1 ±0.7
6.4 ±0.5
6.2 ±1.7
6.7 ±1.4
6.2 ±0.6
(4.7-7.0)
(5.1 -6.9)
(4.9 - 7.3)
(5.1 -7.4)
(5.5-7.7)
(4.8 - 7.8)
b'
4.4 ±0.6
4.7 ±0.2
4.3 ±0.5
4.1 ±0.8
4.5 ±1.0
4.7 ±0.4
(4.0-5.7)
(3.7-6.0)
(3.7-5.2)
(3.7-5.0)
(4.0 - 4.9)
(3.6 - 5.9)
c
17.3 ±1.2
18.2 ± 1.5
18.9± 1.3
18.3 ±1.0
17.5 ±0.4
17.6 ±1.4
(14.1 -22.3)
(14.9-21.8)
(15.2-23.4)
(16.7-20.2)
(16.6-18.0)
(14.5-21.1)
c'
2.3 ±0.5
2.5 ±0.4
2.6 ± .02
2.1 ±0.1
2.2 ±0.1
2.3 ±0.3
(1.4-2.8)
(1.7-3.0)
(1.9-3.1)
(2.0-2.3)
(2.1 -2.5)
(1.7-2.9)
V%
76.7 ±1.8
77.6 ± 1.1
80.1 ± 1.6
76.5 ± 1.95
74.7 ±0.6
78 ± 1.2
(73.9-80.1)
(75.7 - 79.4)
(76.5 - 83.6)
(72.9-81.4)
(74.0 - 75.9)
(75 - 80)
Stylet length
12.1 ±0.6
12.6± 1.1
11.9 ±0.6
14.4 ±0.4
14.7 ±0.4
15.3 ±0.4
(11.1 -13.1)
(10.7-14.0)
(10.6-12.9)
(13.5-14.9)
(14.2-15.6)
(14.7-15.9)
Tail length
26.3 ±2.0
27.5 ± 1.8
25.7 ± 1.8
30.1 ±2.0
31.5 ±0.8
26.3 ±2.5
(20.9-29.6)
(24.0-31.1)
(22.0-29.4)
(27.1 -34.6)
(30.2-32.7)
(21.5-31.9)
DGO
2.8 ±0.6
2.9 ±0.5
2.6 ±0.4
2.3 ±0.1
2.5 ±0.1
2.7 ±0.4
(2.3-4.1)
(2.1 -3.6)
(2.0-3.2)
(2.1 -2.5)
(2.3-2.6)
(1.8-3.1)
Maximum body
16.7 ±1.5
17.8 ±2.0
15.7 ± 1.2
21.8 ±1.4
22.3 ± 1.3
17.0 ±1.8
diameter
(13.7-19.7)
(14.0-20.5)
(12.3-17.2)
(20.5-26.0)
(21.2-24.7)
(13.5-20.9)
Anal body
11.1 ±0.8
11.6 ±0.9
10.2 ± 0.8
14.1 ±0.6
14.1 ± 0.6
11.4 ± 1.5
diameter
(9.7-12.5)
(10.0-13.2)
(9.0 - 11.5)
(13.1 -15.2)
(13.2-14.8)
(8.6-17.2)
Number of head
2.0 ±0.0
2.0 ±0.0
2.0 ±0.0
2.0 ±0.0
2.0 ±0.0
annuli
(2.0-2.0)
(2.0-2.0)
(2.0-2.0)
(2.0-2.0)
(2.0-2.0)
Lip region width
6.1 ±0.5
7.1 ±0.9
6.3 ±0.5
8.2 ±0.4
8.1 ±0.3
7.2 ±0.3
(4.7-6.8)
(5.6-9.1)
(5.5 - 7.3)
(7.7-8.9)
(7.5-8.5)
(6.7 - 7.4)
Lip region height
2.5 ±0.6
2.8 ±0.3
2.5 ±0.4
2.1 ±0.2
2.4 ±0.2
2.5 ±0.7
(1.5-3.6)
(2.3-3.2)
(1.8-3.1)
(1.8-2.4)
(1.9-2.6)
(2.0-4.0)
Pharyngeal gland
35.7 ±5.3
31.7 ± 1.4
32.8 ±4.7
28.6 ±3.6
40.5 ± 1.1
24.2 ± 7.7
length
(25.8-45.7)
(30.1 -34.4)
(24.0-39.2)
(24.1 -33.8)
(38.9-42.2)
(10.4-46.6)
PUS
18.5 ±5.5
31.2 ± 1.6
24.5 ±4.8
29.6 ±0.8
30.4 ± 1.2
18.1 ±4.0
(8.4-27.3)
(29.8-33.2)
(14.8-32.0)
(28.6 - 31.4)
(28.8-32.0)
(9.8-24.5)
Measurements in micrometers; mean ± s.d. (range).
Results
Morphological and morphometric identification
Five populations of Pratylenchus, four from plantain (two from Valle del Cauca, one from Caldas, and one from Quindío) and one from banana (from Quindío), were identified as P. araucensis. The morphological and morphometric data from the analyzed populations were similar to the data reported in the original description of P. araucensis (Table 1).
The populations identified in the present study as P. araucensis were morphologically characterized by the presence of a body that was slender and vermiform (Fig. 1A), flattened labial region with presence of two annuli, basal knobs of the stylet rounded to oblong (Figs. 1B,C), vulva located posteriorly (Figs. 1D–F), round spermatheca, the shape of the tail conoid to subcylindrical, end of the tail rounded to truncated (Figs. 1G–J), and presence of males (Fig. 1G).
Figure 1
Pratylenchus araucensis. (A–F): Female. (A). Body of a female, (B). Anterior body region, (C and D). Vulval region. (E and F). Posterior region. (G). Posterior region of the body of a male. St = Stylet, v = Vulva, an = Anus, spc = Spicule.
The multivariate statistical analysis showed that the populations identified as P. araucensis, from the states of Valle del Cauca, Caldas, and Quindío, had such morphological characteristics to be grouped in the same cluster with the type population of the same species previously recorded in the department of Arauca, Colombia, and to be grouped apart from other species reported in Musaceae (Fig. 2), such as P. coffeae and P. speijeri (Souza, 2008; Múnera et al., 2009; De Luca et al., 2012).
Figure 2
Biplot (separation) based on morphometric data of Pratylenchus araucensis populations. Populations of P. araucensis from Caldas, Quindío, and Valle del Cauca were assigned to their corresponding species based on morphometric data. The first two PCA axes are shown. Pa = Pratylenchus araucensis, Ara = Arauca, Cal = Caldas, CoQ = Quindío, ZaB = Zabaletas (Valle del Cauca [Buenaventura]), and DelB = Delfina (Valle del Cauca [Buenaventura]). PCA, principal component analysis.
According to PCA, the main components 1, 2, and 3 presented eigenvalues ≥1, explaining 87% of the variation. Thus, the characteristic with diagnostic values to delimit between the species P. araucensis, P. coffeae, P. jaehni, and P. loosi are: post-uterine sac length, total length, tail length, maximum width of the body, and width of the body at the level of the anus in the main component (CP)1; stylet length, vulva position, and index c at CP2; and width of the cephalic region and index a in CP3 (Table 2). These variables, according to the results of the multivariate analysis, are characteristic of diagnostic value that allow delimiting between the analyzed species.
Correlations between the first three main components and the morphometric parameters of P. araucensis females.
Variable
Main component
Prin1
Prin2
Prin3
Stylet length (μm)
0.298
0.440
-0.159
Vulvar position (%)
-0.288
0.431
0.178
Post-uterine sac length (μm)
0.315
0.176
0.407
Overall length
0.330
0.370
0.245
Tail length (μm)
0.379
-0.046
0.235
Maximum body width (μm)
0.396
0.005
-0.182
Width body anus (μm)
0.377
0.133
-0.188
Head region width (μm)
0.242
-0.345
0.447
Index a (μm)
-0.294
0.122
0.620
Index c (μm)
-0.182
0.547
-0.097
Numbers in bold format correspond to discriminant characteristic.
Molecular identification
A total of 33 DNA sequences was obtained in the present study, 16 corresponding to the D2–D3 segment of rDNA and 17 for COI of mitochondrial DNA (Table 3). All the sequences of the D2–D3 segment showed a similarity of 99.73% with reference sequences of P. araucensis (FJ463261, FJ463260, and FJ463258). The taxonomic identity of the analyzed populations could not be resolved based on sequences corresponding to COI, because the present study reports the first COI sequences for the species P. araucensis.
Information of D2–D3 rDNA and COI mtDNA regions downloaded from GenBank and obtained in the present study.
Based on the analysis of 16 partial sequences of the 746 bp D2–D3 segment, from three geographic regions, Valle del Cauca, Caldas, and Quindío, an intraspecific variation for P. araucensis of 0.27% (2 bp) was observed between populations of Valle del Cauca (Buenaventura) concerning the populations of Caldas, Quindío, and Arauca. Similarly, based on 17 partial sequences of the COI gene, from the same geographical areas, with a size of 393 bp, greater intraspecific variability was determined, with a value of 2.04% (8 bp) between the populations of Valle de Cauca (Buenaventura) and those of Caldas and Quindío (coffee region).
Phylogenetic analysis
The alignment based on the LSU region comprised a total of 50 taxa with 837 characters analyzed. For the COI region, it included 47 taxa, with a total of 470 characters. The phylogenetic analysis based on the D2–D3 segment allowed the grouping of the sequences obtained in the present study with the species P. araucensis with bootstrap support on the branch of 100% and apart from other morphologically similar species such as P. coffeae, P. speijeri and P. loosi (Fig. 3). The populations based on the phylogeny constructed for the COI gene were grouped in a clade apart from other Pratylenchus species and internally separated by geographic origin (Fig. 4).
Figure 3
Phylogenetic tree obtained by maximum likelihood of the D2–D3 (LSU) partial region of species of the genus Pratylenchus. The isolates corresponding to this work are marked in bold. The numbers above nodes indicate bootstrap values >70%. LSU, large subunit.
Figure 4
Phylogenetic tree obtained by maximum likelihood of the COI partial region of species of the genus Pratylenchus. The isolates corresponding to this work are marked in bold. The numbers above nodes indicate bootstrap values >70%.
Discussion
Species of Pratylenchus are difficult to separate from each other, due to their similarity in morphometrics and morphology, although the genus is easily recognizable (Castillo and Vovlas, 2007). However, based on an integrative taxonomy approach, including morphological, morphometric, and molecular characterization, the populations obtained in this study associated with Musa spp. crops were identified as P. araucensis. The morphological characteristics and morphometric data recorded for the analyzed populations are similar to those reported for the type population (Múnera et al., 2009).
The results indicated that P. araucensis, reported previously for the first time in the state of Arauca (eastern region of Colombia), is also present in the states of Quindío, Caldas (central region of Colombia), and Valle del Cauca (southwestern region of Colombia). The foregoing may be related to the movement of corms infested with the nematode between producing areas given the form of clonal propagation of plantain and banana crops (Riascos-Ortiz et al., 2022).
The presence of P. araucensis in plantain-producing areas with contrasting climatic conditions indicates that this species has a wide range of adaptation to temperature (from 18°C in Caldas and Quindío to 26°C in Valle del Cauca), rainfall (as confirmed by pluviometric measurements ranging from 2,500 mm/yr in Caldas and Quindío to 9,000 mm/yr in Valle del Cauca), and altitude (from 7 m.a.s.l in Valle del Cauca to 1,573 m.a.s.l in Caldas and Quindío) (Orión et al., 1979, 1984; Glazer and Orion, 1983; Talavera et al., 1998; Orión, 2000; Múnera et al., 2009; Bucki et al., 2020).
The populations analyzed in this research were identified through the use of morphological and morphometric characteristic. PCA separated the species identified in this study from others that are morphologically and morphometrically similar, showing the importance of morphological and morphometric data, in addition to multivariate statistical analysis for the identification of species of the genus Pratylenchus (Castillo and Vovlas, 2007; Múnera et al., 2009; Singh et al., 2018; Qing et al., 2019; Riascos-Oritiz et al., 2019).
The analysis of the main components indicated that among the diagnostic characteristic with the highest resolution to discriminate or delimit between some species reported in Musacaes are the length of the post-uterine sac, total length of the body, length of the tail, maximum width of the body, width of the body at the level of the anus, length of the stylet, position of the vulva, index c, width of the cephalic region, and index a, mainly. These variables, except the width of the cephalic region, are considered of diagnostic value in the separation of P. araucensis from P. coffeae, P. jaehni, and P. loosi (Múnera et al., 2009).
Based on the sequence of the D2–D3 segment of rRNA, it was confirmed that the populations analyzed belong to the species P. araucensis. According to the phylogenies constructed for both genes, and using molecular data generated in this study and others obtained from GenBank, P. araucensis is separated from different sister species of the same genus, including some reported in Musaceae. These results are consistent with those recorded for the morphological and morphometric analysis (Múnera et al., 2009). The phylogenetic tree generated by COI molecular data showed that the populations of P. araucensis analyzed in this research were grouped by geographic origin, indicating the existence of intraspecific diversity in this species.
Different research works have established the levels of intraspecific diversity for various species of Pratylenchus. For example, Kolombia et al. (2020) reported a low intraspecific variability of 0 bp to 8 bp (0%–1.93%) among populations of P. hexincisus from China, Italy, and the United States and a high intraspecific variability of 54 bp to 102 bp (19.1%–24.5%) among populations from the same countries. Mirghasemi et al. (2019) reported a low intraspecific variability of 0.0% to 0.96% (0–4 bp) between populations of P. loosi from Iran, China, and Japan. Based on the results of these investigations, the intraspecific variability recorded for P. araucensis in the present study can be considered low. However, the nucleotide differences between populations of P. araucensis from Valle del Cauca and those from Caldas and Quindío may be related to contrasting climatic differences between regions, specifically rainfall (Castillo and Vovlas, 2007; Mirghasemi et al., 2019).
