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

Abstract Three populations, two from Colombia and one from Brazil, of Rotylenchulus reniformis associated with banana and plantain, were characterized using morphological, morphometric, and molecular methods. Morphometric data from these populations were similar to type and reference populations of R. reniformis. Partial sequences of both D2-D3 rDNA and mitochondrial cytochrome oxidase subunit I (COI) regions had a strong affinity (99% similarity) to previously published sequences of R. reniformis. Phylogenetic analyses (maximum likelihood and Bayesian inference) suggested that the Colombian populations of R. reniformis corresponded to the previously described Type A of the species. This is the definitive first report in Colombia of R. reniformis associated with banana and plantain crops.

While numerous reports of Rotylenchulus associated with Musa spp. in Colombia exist (Zuñiga et al., 1979;Barriga and Cubillos, 1980;Curiel and Ospino, 2001;Gómez, 2001;Guzmán et al., 2012), detailed morphological, morphometric, and molecular data were not included. Thus, in Colombia, there is limited knowledge as to which species of Rotylenchulus are associated with Musa spp. which impedes the deployment of effective management strategies to control the species. To address this knowledge gap, the present study aims to: identify by morphological, morphometric, and molecular analysis the species of Rotylenchulus associated with Musa spp. in Colombia, and analyze the phylogenetic relationship of Rotylenchulus species.

Materials and methods
Sampling, extraction, morphological, and morphometric analyses of nematodes 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 pseu-dostem 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).

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

Phylogenetic analysis
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 (MC-MCMC) 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).

Morphological and morphometric identification
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 (Fig. 3) and Bayesian (Fig. 4)

Discussion
Nematodes associated with plantain and banana analyzed in the present study were identified as R. reniformis by morphological, morphometric, and molecular methods. With regard to the morphometric analysis, measurements closely resembled those reported for type and topotype populations of R. reniformis and published dichotomous keys (Linford and Oliveira, 1940;Dasgupta et al., 1968;Robinson et al., 1997;Agudelo et al., 2005;Palomares-Rius et al., 2018). However, differences were noted for some diagnostic characters between the studied and reference populations suggesting intraspecific variation. Such variation is reported to be driven by temperature, nutrients and growth conditions of the host plant (Evans and Fisher, 1970;Nakasono, 2004;Nyaku et al., 2013Nyaku et al., , 2016. Morphological identification of Rotyl Peter Ramley and Roy enchulus species is considered problematic due to a high degree of intraspecific variation (Dasgupta et al., 1968;Germani, 1978;Robinson et al., 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).

Molecular identification
Consensus sequences of the D2-D3 expansion region obtained for Colombian populations had a strong affin- 1997). Notwithstanding, the intraspecific variation encountered in this study, key discriminatory diagnostic characters (L, stylet, b, c, c', and V) were identified through the use of multivariate analysis that supported robust identification of R. reniformis and separated the species from the other valid Rotylenchulus species (Linford and Oliveira, 1940;Dasgupta et al., 1968;Robinson et al., 1997;Van den Berg et al., 2015). Rotylenchulus borealis is a species reported in the banana crops of Cameroon, Kenya, South Africa, and Rwanda. However, literature revised show marked morphometric differences between R. borealis and R. reniformis populations analyzed in this study (Van den Berg et al., 2003). With regard to measurements of immature females, the principal differences between both species were body length (L), dorsal gland orifice (DGO), pharynx length, excretory pore, lip region height, and tail length, with higher values attributed to R. borealis (Van den Berg et al., 2003;Gaidashova et al., 2004).
Assessment of the D2-D3 and COI regions of the nematodes in our study had a strong affinity to previously published sequences attributed to R. reniformis. This was consistent with the morphometric and morphological data generated in this study. Tree topologies generated by ML and BI methods were similar. Our present study confirmed the results of Van den Berg et al. (2015) who found two distinct types of D2-D3 28 S rRNA in the R. reniformis genome. Type A, including all the studied Colombian populations, formed a well-supported group with Brazil, China, Japan, Spain, and USA populations and Type B which was disparate from Type A. However, the relation between type and pathogenicity or virulence is unknown. R. reniformis Type A has been reported associated with a range of economically important crops, including cotton (KY992808) ( Van den Berg et al., 2015;Palomares-Rius et al., 2018).
Based on PCA, the single Brazilian population studied grouped with Colombian populations identified morphometrically and molecularly as R. reniformis. This species has previously been reported associated with different crops in Brazil such as: Lycopersicum esculentum Mill., Gossypium hirsutum L., Carica papaya L., Glycine max (L.) Merril., Phaseolus vulgaris L., Passiflora edulis Sims., and Ananas comosus (L.) Merr. (Soares et al., 2003). It has also been reported to be associated with banana production in the Brazilian states of Bahia, Ceará, Paraíba, Rio de Janeiro, and Espirito Santo (Costa Manso et al., 1994).
The identification of R. reniformis in plantain and banana crops of Colombia and Brazil in the present study is consistent with previous reports of this nematode with Musa spp. from across the world (Fargette and Quénéhervé, 1988;Ngoc Chau et al., 1997;Khan and Hasan, 2010;Kamira et al., 2013;Daneel et al., 2015). This is the first report of R. reniformis in plantain and banana for Colombia through integrative taxonomy, contributing to the knowledge of the parasitic nematode community of this country, and is essential information for the future design of integrated Note: Key diagnostics for discriminating Rotylenchulus species are denoted in italic.    . The phylogeny is a consensus tree from a posterior distribution of 1,600 trees that were inferred in MrBayes. The outgroup (Hoplolaimus magnystilus) is shown in gray font; the sequences that were obtained in this study appear in bold typeface. Values at the nodes represent the posterior probability. The scale represents the number of substitutions per site. management programs for R. reniformis associated with Musa spp. (Robinson et al., 1997;Crozzoli et al., 2004).

Acknowledgments
The authors would like to thank Colciencias (Departamento Administrativo de Ciencias, Tecnología e Innovación) for financial support to the first author for his Doctorate studies, internship and research (announcement 727-2015). The authors would like to acknowledge the project named "Tecnologías innovadoras para el Manejo Integrado de plagas y enfermedades limitantes de plátano y banana en el Valle del Cauca", supported by the Sistema General de Regalías-SGR of Colombia, for financial support to enable collection of roots and soil samples from farms and acquisition of reagents. The authors would also like to thank the Molecular Biology Laboratory of the Universidad Nacional de Colombia in Palmira, Valle del Cauca-Colombia and Nematology Laboratory of Instituto Biológico in Campinas, São Paulo-Brazil for their scientific support and Plant-Pathology Laboratory of the Universidad del Pacifíco in Buenaventura, Colombia, where various root and soil samples of plantain and banana crops were processed. Lastly, the authors would like to thank Danny Rojas, Ángel Vale, Peter Ramley and Roy Neilson Neilson for their comments on the English language of the manuscript and the molecular data interpretation (bioinformatic analysis).