Morpho-molecular characterization of Colombian and Brazilian populations of Rotylenchulus associated with Musa spp

Soil and root samples of banana and plantain were collected from farms in Bolo and Rozo (Palmira, Valle del Cauca, Colombia) and Minas Gerais (Brazil) between 2016 and 2018. Composite soil and root samples of 1 kg were collected from each sampled farm from the root zone of 15 to 20 randomly selected plants ha⁻¹. Secondary and tertiary roots and soil were collected to a distance of 25 cm of the pseudostem and among 0 to 30 cm of profundity with aid of a spade, soil auger and knife. A modification of Cobb’s method was used to extract the nematodes from soil and root (Ravichandra, 2014). Nematodes were killed by heat at 65°C for 4 min and then fixed with 2% formalin (Rosa et al., 2014). Key morphometric measurements for the genus (Table 1) were taken according to Robinson et al. (1997), Van den Berg et al. (2015), and Palomares-Rius et al. (2018). Microphotographs were taken using a light microscope equipped with differential interference contrast-DIC (DM2500, Leica, Germany).

Morphometric data generated from this study and data sourced from the literature for other Rotylenchulus species (Van den Berg et al., 2003, 2015; Agudelo et al., 2005) were subjected to principal component analysis (PCA) using Community Analysis Package (PISCES Conservation Ltd, Lymington, UK) (Henderson and Seaby, 2014).

Nematode DNA extraction followed Múnera et al. (2009) with modifications. A single nematode was crushed with a sterile scalpel and transferred to an Eppendorf tube with 15 µl worm 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 tube was stored at −80°C for 15 min, incubated at 65°C for 1 hr and thereafter at 95°C for 15 min. Finally, the tube was centrifuged at 16,000 g for 1 min and stored at −20°C until further processing. The D2-D3 expansion region of the large subunit (LSU) of ribosomal DNA (28 S) was amplified using primers D2A (forward, 5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B (reverse, 5′-TCCTCGGAAGGAACCAGCTACTA-3′) (De Ley et al., 1999). Also, a partial region of the mitochondrial cytochrome oxidase subunit I (COI) was amplified using primers JB3 (forward, 5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) and JB4.5 (reverse, 5′-TAAAGAAAGAACATAATGAAAATG-3′) (Bowles et al., 1992). The PCR conditions were initial denaturation during 2 min at 94 ºC 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 ºC for the amplification of D2-D3; initial denaturation during 2 min at 94 ºC 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 ºC for the amplification of COI. PCR products were sequenced in both directions by BIONEER Korea.

Basic local alignment search tool (BLAST) at National Center for Biotechnology Information (NCBI) was used to confirm the species identity of the DNA sequences obtained in this study (Altschul et al., 1990). Consensus sequences were edited using Geneious software R6 (Biomatters; www.geneious.com) with multiple alignments performed in MAFFT v7 (Katoh et al., 2002) using sequences generated in this study and Rotylenchulus sequences obtained from GenBank. jModelTest v2.1.7 software was used to determine the nucleotide substitution model that was a best fit for each alignment based on the Akaike information criterion corrected for small sample sizes (Posada, 2008). Maximum likelihood (ML) and Bayesian inference (BI) were used to estimate phylogenies for the D2-D3 and COI regions. For ML, 250 bootstraps were used and the general time reversible model with allowance for a gamma distribution of rate variation (GTR + Γ) in RaxML v8 (Stamatakis, 2014). Inferred phylogenies by BI (MrBayes v3.2.6, Ronquist et al., 2012), used the general time reversible model with allowance for a gamma distribution of rate variation and a proportion of invariant sites (GTR + Γ + I) for LSU, and GTR + Γ for COI. Two independent metropolis-coupled Markov chain Monte Carlo (MCMCMC) searches for 2 million generations, sampled every 2,000 steps were used for both the D2-D3 and COI regions. Convergence was assessed in Tracer v1.5, using a burn in of 20%, and by examining the average standard deviation of split frequencies among parallel chains. A consensus tree was calculated for each region from the posterior distribution of 1,600 phylogenies. Hoplolaimus seinhorsti and Hoplolaimus magnistylus were used as outgroups for D2-D3 and COI, respectively, for the ML and Bayesian analyses (Miller et al., 2010).

The Colombian and Brazilian populations analyzed in this study were identified morphologically and morphometrically as R. reniformis (Table 1 and Fig. 1). Diagnostic characters and morphological characteristics for populations assessed in this study closely resembled those of type and topotype populations (Table 1 and Fig. 2A–F). Multivariate analysis showed that Colombian and Brazilian populations grouped closely with R. reniformis reference populations (from USA) but disparate to other valid species of Rotylenchulus, including R. borealis, R. clavicaudatus, R. leptus, R. macrodoratus, R. macrosoma, R. macrosomoides, and R. sacchari (according with measurements reported by Van den Berg et al., 2003, 2015; Agudelo et al., 2005). The Principal Components 1 and 2 had eigenvalues greater than or equal to 1 and explained 84% of variance. The first three principal components explained 94.6% of the variation recorded. The main influencing morphological/morphometric characters were L, a and stylet (PC1) and in PC2, c’, b, and V (Table 2).

Figure 1:

Figure 2:

Consensus sequences of the D2-D3 expansion region obtained for Colombian populations had a strong affinity (99% similarity) with a number of R. reniformis reference sequences (KP054126, KT003743, KP054077, KP054088, KT003744, KF999977, KF999978, and DQ328713). Similarly, COI sequences also had a strong affinity (99% similarity) with R. reniformis reference sequences (KT003727, KT003728, KT003729, KT003730, and KT003731). All sequences obtained in this study were deposited in NCBI under accession numbers MK879441-MK879450 (D2-D3) and MK908051-MK908060 (COI).

ML (Fig. 3) and Bayesian (Fig. 4) D2-D3 phylogenies clustered, with strong support, Colombian sequences of R. reniformis from this study with those of R. reniformis Type A. Irrespective of analytical method used (ML or BI), Colombian sequences of R. reniformis grouped with R. reniformis reference sequences associated with different host plants and geographic origin (KFF999978 of Podocarpus macrophyllus from Japan, HM131878, HM131868, HM131860, and GU120091 from China, and DQ328713 from Brazil) (Figs. 3-4).

Figure 3:

Figure 4:

All Colombian R. reniformis COI sequences clustered together with other R. reniformis populations (Figs. 5-6, BS = 100%, PP = 1). In both phylogenies, Colombian populations grouped with R. reniformis associated with different host plants and geographic origin (KT003727 from Florida, USA, KT003728 of cotton from Arkansas, USA, KT003729 of Sansevieria sp. From Florida, USA, KT003730 of Euphorbia sp. From Florida, USA and KT003731 of Yucca elephantipes from Florida, USA) (Figs. 5-6).

Figure 5:

Figure 6: