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Intraspecific variation in Radopholus similis isolates assessed with restriction fragment length polymorphism and DNA sequencing of the internal transcribed spacer region of the ribosomal RNA cistron
International Journal for Parasitology 32 (2002) 199–205 www.parasitology-online.com
Intraspecific variation in Radopholus similis isolates assessed with restriction fragment length polymorphism and DNA sequencing of the internal transcribed spacer region of the ribosomal RNA cistron q Gamal A.A. Elbadri a, Paul De Ley b,*, Lieven Waeyenberge c, Andy Vierstraete d, Maurice Moens c,d, Jacques Vanfleteren d a
Department of Crop Protection, Agricultural Research Corporation, P.O. Box 126, Wad Medani, Sudan b Department of Nematology, University of California – Riverside, Riverside, CA 92521, USA c Centrum voor Landbouwkundig Onderzoek, Departement Gewasbescherming, Burgemeester Van Gansberghelaan 96, B-9820 Merelbeke, Belgium d Universiteit Gent, Vakgroep Biologie, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium Received 21 May 2001; received in revised form 27 September 2001; accepted 27 September 2001
Abstract Restriction fragment length polymorphism and direct sequencing of the internal transcribed spacer rDNA region of 19 isolates of Radopholus similis yielded significant diversity, both among isolates and within some individuals. Restriction fragment length polymorphism with HaeIII, AluI and Tru9I yielded two sets of patterns. Digestion with RsaI revealed one or two supernumerary bands in single nematodes from five isolates, and sequencing confirmed microheterogeneity in four of these. Phylogenetic analysis grouped most isolates closely together, except for the five isolates with additional bands for RsaI. Our data reveal more population structure than previously found and lend further support to the synonymy of R. similis and ‘Radopholus citrophilus’. q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Banana; Black pepper; Ornamentals; Polymerase chain reaction; Sequence polymorphism; Nematoda
1. Introduction The burrowing nematode Radopholus similis Thorne, 1949 belongs to the predominantly plant parasitic order, Tylenchida, and is a major pathogen of banana and plantain throughout the tropics. As a migratory endoparasite, it is capable of causing extensive necrosis of the root cortex, and may ultimately lead to toppling of entire plants (Gowen and Que´ne´herve´, 1990), thus resulting in dramatic yield losses (Fogain, 2000). However, different physiological races or biotypes of R. similis are known, and these exhibit widely different levels of pathogenicity (see Stoffelen et al., 2000). Knowledge of genetic diversity of the parasite is therefore essential to effective resistance breeding programs for the host (Hahn et al., 1994). In this paper, we explore intraspecific genetic variation among 19 R. similis isolates kept in continuous culture and morphologically characterised in previous work (Elbadri et q Nucleotide sequence data reported in this paper are available in the GenBank database under the accession numbers AF375348–AF375395. * Corresponding author. Tel.: 11-909-787-2280; fax: 11-909-787-3719. E-mail address: [email protected] (P. De Ley).
al., 1999). We amplified the internal transcribed spacer (ITS) rDNA region of each isolate for restriction fragment length polymorphism (RFLP) analysis and sequencing, and investigated the resulting implications for diagnosis, host preferences and phylogenetic relationships (see Dorris et al., 1999 for more information on rDNA and nematode phylogeny). Our observations and conclusions differ considerably from those reached recently in a similar study (Kaplan et al., 2000). 2. Materials and methods 2.1. Origin, culture and multiplication on banana of the nematodes Nineteen R. similis isolates were collected from locations around the world (Table 1), mostly from banana roots or surrounding soil. All isolates were maintained on carrot discs (Verdejo-Lucas and Pinochet, 1992). In vitro propagated plantlets of banana cv. ‘Grand Naine’ were transplanted to 13 cm pots with sterile soil and acclimatised for 3 weeks. For each isolate, 1,000 nematodes from carrot disk
0020-7519/02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(01)00319-8
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Table 1 Origin of Radopholus similis isolates used in this study, number of sequences obtained from one individual of each isolate, and number of distinct variants among these sequences Code
Locality, country
Host plant
Source
Number of sequences/ number of distinct variants
Anth Aus Bh Cal Che CR Cub Gh Gu Indo Kam Kar Pan Philo Ros Sen Tam Ug WDA
Leuven, Belgium Cairns, Australia Burgershall, S Africa Aalst, Belgium Chendi, Sudan La Estrella, Costa Rica Villa Clara, Cuba –, Ghana Balikoure, Guinea Bangka, Indonesia Kamlin, Sudan Karkoug, Sudan Changuinola, Panama Hamburg, Germany Roseris, Sudan Sennar, Sudan Tamis, Sudan Kampala, Uganda –, –
Anthurium andreanum Banana Banana Calathea makoyana Banana Banana Banana Banana Banana Piper nigrum Banana Banana Banana Philodendron sp. Banana Banana Banana Banana Banana
M. Moens J. Pinochet B. Meyers M. Moens G. Elbadri J. Pinochet J. Pinochet D. De Waele J. Pinochet N. Herdradjat G. Elbadri G. Elbadri J. Pinochet M. Moens G. Elbadri G. Elbadri G. Elbadri J. Pinochet J. Pinochet
1/1 1/1 1/1 5/3 13/8 1/1 1/1 1/1 1/1 1/1 1/1 9/4 1/1 1/1 1/1 6/4 1/1 1/1 1/1
culture were inoculated on each of eight plants, and all 152 plants were arranged in a complete randomised block design and grown for 12 weeks in the greenhouse in 3 l pots with sterilised sandy soil. Plant roots were then cut into 5 mm fragments, 10 g of these were macerated and put into a 1 l suspension, and 500 ml of each suspension was extracted with an automated apparatus (Hendrickx, 1995). The reproduction factor, Rf (the ratio of the extracted number of nematodes over the initial size of inoculum), was calculated for each replicate, and statistically compared with Dunnett’s test. 2.2. DNA extraction and PCR amplification Single nematodes were prepared for PCR as described by Waeyenberge et al. (2000), but using a five-fold dilution of their lysis buffer. The DNA amplification profile consisted of 4 min at 948C; 35 cycles of 1 min at 948C, 1.5 min at 508C, and 2 min at 728C; and a final 5 min extension at 728C. We used the two 21 bp PCR primers derived from the Xiphinema bricolensis sequence by Vrain et al. (1992). In some isolates, amplification with these primers was unsuccessful, and we therefore compared sequences from other isolates to derive and apply two modified primers: Vrain2 18S (.5 0 -CTTTGTACACACCGCCCGTCGCT-3 0 ) and Vrain2 26S (.5 0 TTTCACTCGCCGTTACTAAGGGAATC-3 0 ). 2.3. Restriction fragment length polymorphism analysis Five microlitres of each good PCR product was digested with 10 U of each of seven restriction enzymes: AluI, HaeIII, HinfI, NdeII, RsaI, TaqI and Tru9I, incubated overnight at 378C (AluI, HaeIII, HinfI, NdeII and RsaI) or 658C
(TaqI and Tru9I). The digested DNA was separated by electrophoresis in 1.5% agarose for 3 h, and visualised with ethidium bromide. The procedure was repeated until RFLP patterns were obtained from three individual nematodes of each isolate. 2.4. Nucleotide sequencing A PCR product from a single nematode from each isolate was prepared for sequencing by adding 0.6 ml exonuclease I (10 U/ml), 0.6 ml shrimp alkaline phosphatase (2 U/ml), and 15 ml mineral oil to 3 ml of each PCR product, centrifuging the mixture, and incubating it first for 15 min at 378C, and then for 15 min at 808C. Next, each sample was treated with BigDyee Terminator Ready Reaction mix according to the manufacturer’s protocol (DNA sequencing Kit, PE Biosystems) using primer Vrain2 18S or Vrain2 26S, and loaded on an automated sequencer (ABI Prism 377 DNA Sequencer, PE Biosystems). Direct sequencing of the Calathea, Chendi, Sennar and Karkoug isolates produced electropherograms with numerous multiple peaks, and the PCR products obtained for these isolates were therefore cloned using pGEM-T Vector and JM109 Competent Cells (pGEM w-T Vector System II, Promega). Ligation and transformation reactions were set up as described by the Promega protocol (Protocols and applications guide, 3rd Edition, 1996, Promega). Clones were screened with PCR and RFLP to verify whether the cloning procedure was successful. Appropriate-sized PCR products were then sequenced from individual colonies of each of the four cloned isolates, until at least three distinct sequence variants were obtained/isolate (Table 1).
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2.5. Sequence alignment and phylogenetic inference All obtained sequences were aligned automatically using ClustalX (Thompson et al., 1997), along with two ITS region sequences published by Kaplan (1994) for R. similis and Radopholus citrophilus Huettel et al., 1984, and verified by eye using GeneDoc 2.5 (Nicholas and Nicholas, 1997). For each group of identical sequences, all except one were removed in order to speed up calculation times for subsequent analyses. Alignment presented no problems, except for one ambiguous trio of positions. A second alignment was therefore produced, differing only from the first one in that it represented the alternate homology pattern for these three positions. Both alignments were analysed with PAUP* 4.0b4a (Swofford, D.L., 1998. PAUP* – Phylogenetic Analysis using Parsimony (* and other methods), Version 4, Sinauer), using the unweighted pair group method with arithmetic mean (UPGMA), neighbour-joining, maximum parsimony and maximum likelihood algorithms. In the absence of a suitably close outgroup taxon, all trees were calculated without rooting. UPGMA was applied with uncorrected distance measure, while neighbour-joining was implemented with LogDet/paralinear distances (Lockhart et al., 1994). Heuristic searches were completed with maximum parsimony (characters unordered and of equal weight, gaps treated as missing data, random addition sequence with 1,000 replicates, multiple trees threshold set to 500, without steepest descent) and maximum likelihood (nucleotide frequencies set to empirical values, two substitution types, HKY-
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model (Hasegawa, Kishino and Yano), transition/transversion ratio set to 2). Furthermore, bootstrap analyses were performed for UPGMA, neighbour-joining and maximum parsimony using 3,000 replicates and similar settings as above (except for maximum parsimony now being set to simple addition sequence). Finally, one million random trees were generated and compared with maximum parsimony in order to generate the g1 shape parameter of tree length distribution, which assesses the phylogenetic signalto-noise ratio of the alignment (Hillis and Huelsenbeck, 1992). Absolute distances and pairwise character distances were calculated for all pairwise combinations of operational taxonomic units (OTUs).
3. Results 3.1. Restriction fragment length polymorphism patterns Amplification of the ITS region of nematodes from all isolates yielded a 920 bp fragment. The restriction pattern obtained by AluI (Fig. 1A), divided the isolates into two groups. A first one contained 14 isolates showing four bands of about 380, 270, 150 and 120 bp. The second group was composed of the Karkoug, Sennar, Chendi, Calathea and Indonesia isolates, displaying three bands of about 390, 380 and 150 bp. Tru9I restriction (Fig. 1B) yielded the same two groups: the first with five bands of about 300, 280, 170, 120 and 50 bp, and the second with six bands of about 260, 230, 180, 150, 100 and 50 bp.
Fig. 1. (A) AluI; and (B), Tru9I. Restriction fragments of amplified internal transcribed spacer of Radopholus similis isolates. (L) DNA size marker (100 bp). (1) Costa Rica; (2), Uganda; (3), Australia; (4), Panama; (5), Guinea; (6), Cuba; (7), Burgershall; (8), WDA; (9), Kamlin; (10), Karkoug; (11), Sennar; (12), Roseris; (13), Chendi; (14), Tamis; (15), Philodendron; (16), Anthurium; (17), Calathea; (18), Indonesia; (19), Ghana.
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After restriction with RsaI (data not shown), we always obtained two strong bands of about 660 and 260 bp, but PCR products of the Karkoug, Sennar, Chendi, Calathea and Indonesia isolates also displayed a band of about 190 bp. HaeIII only digested PCR products from the Panama, Guinea, Cuba, WDA, Roseris, Tamis and Ghana isolates, resulting in 670 and 250 bp fragments. HinfI cut all PCR products into five bands of about 300, 270, 180, 90 and 60 bp, while NdeI cut all PCR products into four bands of about 420, 310, 120 and 70 bp. TaqI restriction yielded two main bands of about 450 and 230 bp for all isolates, and two faint bands of 100 and 70 bp.
pairwise distances within the heterogeneous group varied between 0 and 16 units, and translated into longer branch lengths in all of the best trees. In particular, the internal branch separating the Indonesian subgroup from the Sennar subgroup (branch II) was of similar length to branch I. At least one sequence variant from each of the four cloned PCR products was more similar to variants obtained from an individual from a different isolate, than to other variants from the same nematode. The most striking example was Chendi clone 9, which was part of the Indonesian subgroup and differed by 13–14 substitutions from most of the other Chendi clones, while being identical to Calathea clone 0.
3.2. Nucleotide sequencing A total of 25 sequences were included in our analysis: eight sequence variants of the Chendi isolate, four variants each of the Karkoug and Sennar isolates, three of the Calathea isolate, four invariant sequences representing the remaining 15 isolates (most of which had identical sequences), and two published sequences (Kaplan, 1994). The length of the entire ITS region varied between 583 and 585 nucleotides. Both alignments were only 533 positions wide due to slightly incomplete sequence stretches obtained for seven of the 44 sequenced clones. Up to 20 and 21 substitutions were assumed in alignments 1 and 2, respectively, and both alignments contained only 22 parsimony-informative positions. The values obtained for the tree length distribution shape parameter, g1, were highly significant for both alignments (20.885 and 20.926, respectively), indicating that the data were non-random with probabilities exceeding 99% (Hillis and Huelsenbeck, 1992).
3.4. Population growth on banana cv. ‘Grand Naine’ Multiplication varied extensively among replicates and
3.3. Phylogenetic analysis: segregation into three clusters All tree-construction methods applied to both alignments yielded trees with identical basic branching orders, with very high bootstrap values for two major internal branches (Figs. 2 and 3). Variations occurred only in local placement of terminal OTUs. All trees yielded a homogeneous cluster representing 14 of our isolates, as well as the two sequences of Kaplan (1994). Absolute distances within this cluster equalled 0–2 units, and it was always resolved as a convex group, i.e. depending on root placement the group might be monoor paraphyletic, but not polyphyletic. Sequences of the isolates from Sennar, Chendi, Karkoug, Calathea and Indonesia always constituted a second convex group, that was much more heterogeneous and separated from the homogeneous group by absolute distances of 7–21 units. Within the heterogeneous group, sequences from the Indonesian isolate and from the clones Calathea 0, Calathea 11, Chendi 9, Karkoug 1, Karkoug 7, and Karkoug 8 always resolved as a convex internal subgroup (hereafter referred to as the Indonesian subgroup) clearly separated from the remaining OTUs (hereafter referred to as the Sennar subgroup). The internal branch between the homogeneous and the heterogeneous group (branch I) was always clearly longer than any branches within the homogeneous group. Absolute
Fig. 2. Unrooted best tree obtained with maximum parsimony analysis (heuristic search, characters unordered and of equal weight, gaps treated as missing data, random addition sequence with 1,000 replicates, multiple trees threshold set to 500, without steepest descent) of alignment 1 of internal transcribed spacer region sequences obtained from 19 isolates of Radopholus similis. Branch lengths are absolute (scale bar represents one parsimony-informative substitution). Depicted bootstrap values were taken from corresponding branches in the consensus tree obtained with bootstrap analysis (3,000 bootstrap replicates, maximum parsimony criterion, heuristic search, characters unordered and of equal weight, gaps treated as missing data, random addition sequence with 1,000 replicates, multiple trees threshold set to 500, with steepest descent). Philo-Anth represents identical sequences obtained from the following isolates: Australia, Burgershall, Costa Rica, Kamlin, Ghana, Panama, Roseris, Uganda, Anthurium, Philodendron.
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Fig. 3. Consensus of the unrooted best trees obtained from phylogenetic analysis of two alignments of internal transcribed spacer region sequences from 19 isolates of Radopholus similis (see text for details on algorithm settings). Branch lengths are not to scale. The homogeneous group (upper right) is always clearly separated from all other operational taxonomic units by long internal branch I. Box I lists bootstrap values (B) calculated for this branch from bootstrap trees and branch lengths (L) obtained from best trees for each algorithm (unweighted pair group method with arithmetic mean, neighbour-joining, maximum parsimony, maximum likelihood) and each alignment (1 and 2). Within the heterogeneous group, the Indonesian subgroup (lower left) is always clearly separated by long internal branch II, and box II lists the corresponding bootstrap and branch length values for all methods and alignments. For neighbour-joining and maximum likelihood, branch length values are proportional to half the distance between reference operational taxonomic units, citroK and similK. For unweighted pair group method with arithmetic mean UP and maximum parsimony, branch lengths are absolute, and the distance between citroK and similK equals 2. Abbreviations are as in Fig. 2.
among isolates (Fig. 4). Only three isolates differed significantly from null growth according to the Dunnett test: the Uganda and Cuba isolates with P . 0:99, and the Tamis isolate with P . 0:95. Isolates with the lowest average Rf included those forming the heterogeneous group in our phylogenetic analysis (on the right in Fig. 4), and/or isolates originally sampled from hosts other than banana (marked with asterisks in Fig. 4).
4. Discussion 4.1. Evidence for genetic diversity in R. similis Fallas et al. (1996) previously detected variations
between 10 isolates of R. similis, using AluI and HaeIII digestion patterns. In our study, AluI, RsaI and Tru9I digestion separated the Karkoug, Sennar, Chendi, Indonesia and Calathea isolates more or less clearly from the remainder. For each of our isolates and for most of the applied enzymes, the sum of the sizes of the restriction fragments was equal to the size of the PCR product. However, for TaqI, the sum was 850 bp, and for Tru9I, we obtained a sum of 920 bp for one group, versus 970 for the other group. This suggests microheterogeneity of the ITS region repeats within individuals, a phenomenon documented in several other plant parasitic nematodes (e.g. Zijlstra et al., 1995; Hugall et al., 1999; Waeyenberge et al., 2000). In our case, sequence data clearly support the separation of the five isolates with aberrant RFLP patterns from the rest
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Fig. 4. Reproduction factor Rf (the ratio of extracted number of nematodes over initial size of inoculum) of 19 R. similis isolates on banana cv. ‘Grand Naine’. White crossbars represent averages of eight replicates, white vertical bars are standard deviations, black vertical lines are ranges. Isolates are presented in order of appearance from upper right to lower left in Fig. 3; those marked with asterisks were originally isolated from hosts other than banana.
of our material, and also confirm the presence of multiple sequence variants within individuals from four of these five isolates. Interestingly, the same five isolates also display a low to very low average reproduction on banana (Fig. 4). RFLP of the ITS region with AluI and Tru9I therefore merits further investigation as a possible marker for pathogenicity of R. similis to banana, and it might even be possible to develop a PCR-based diagnostic test by designing a primer for the region corresponding to positions 490–510 of our alignments. Although it is commonly stated that R. similis has a high degree of genomic conservation (e.g. Kaplan, 1994), with random amplification of polymorphic DNA (RAPD), genetic similarity between certain R. similis isolates was found to be as low as 75% (Hahn et al., 1994), or even 67% (Fallas et al., 1996). Sequence divergence among our isolates is congruent with the quoted RAPD data, and contradicts the homogeneity reported for ITS1 sequences of 55 R. similis isolates (Kaplan et al., 2000). Most of the phylogenetically-informative divergence observed in our material actually pertains to differences in ITS2: up to 12 or 13 parsimony-informative substitutions in our two alignments occur in ITS2, versus only up to five in ITS1, respectively. The discrepancy between Kaplan et al. (2000) and our findings may therefore be due to a combination of different isolates sampled, and their choice of sequencing ITS1 alone. 4.2. Taxonomical interpretations One might be tempted to translate the differences observed here into formal taxonomic segregation, since they exceed those occurring between the sequences previously determined by Kaplan (1994) for R. similis and R. citrophilus (two substitutions between the latter pair versus up to 21 substitutions in pairwise comparison of
our new sequences). They are also larger than those occurring between, for example, the cryptic species Meloidogyne fallax Karssen, 1996 and Meloidogyne chitwoodi Golden, O’Bannon, Santo and Finley, 1980 (only nine substitutions out of 510 bp; I.T. De Ley, personal communication). However, for a number of reasons, we do not suggest taxonomic subdivision, but instead consider our data to add further support to the proposed synonymy of R. similis and R. citrophilus (Valette et al., 1998; Elbadri et al., 1999; Kaplan et al., 2000). Firstly, our isolates display no morphological correlates for such a subdivision (Elbadri et al., 1999). Secondly, the low reproduction of some isolates on banana is interesting, but neither statistically significant nor fully consistent with molecular relationships, and above all, we should be careful not to overemphasise host specificity as a taxonomic character — exactly such an emphasis led to the original proposal of R. citrophilus. Thirdly, the divergence within our heterogeneous group of five isolates is on a similar scale of magnitude to the divergence between isolates of both groups, and it is therefore more prudent to assume that the aberrant isolates are divergent members of one and the same species. Fourthly, and perhaps most intriguingly, we should keep in mind the possibility that the observed pattern of sequence microheterogeneity reflects genetic exchange between some of our sampled isolates (Hugall et al., 1999). Contrary to Kaplan et al. (2000), we detected small but significant levels of genetic divergence between R. similis isolates, of up to 4% substitutions in the ITS region. This divergence is reflected in restriction patterns obtained with the enzymes AluI and Tru9I, which separate the five most divergent isolates from all others. Furthermore, significant variation occurs within individual genomes from these divergent isolates, and some variants are more similar to those found in other isolates, than to other variants from the same individual. These results suggest that genetic
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diversity of rDNA loci in R. similis may indeed be relevant to host preferences, but also that identification and diagnosis on the basis of the ITS region is less straightforward than would appear from previous studies. Both these aspects are directly relevant to the control of R. similis, and clearly merit further investigation. Acknowledgements The first author would like to thank the Islamic Development Bank for financial support for this study. J.V. and P.D.L. acknowledge support through grant G.00292.00 from the Fund for Scientific Research-Flanders. The authors thank Dr J. Pinochet, Professor B. Meyer and Mr N. Herdradjat for providing them with the studied isolates. References Dorris, M., De Ley, P., Blaxter, M.L., 1999. Molecular analysis of nematode diversity and the evolution of parasitism. Parasitol. Today 15, 188– 93. Elbadri, G.A.A., Geraert, E., Moens, M., 1999. Morphological differences among Radopholus similis (Cobb, 1893) Thorne, 1949 populations. Russ. J. Nematol. 7, 139–53. Fallas, G.A., Hahn, M.L., Fargette, M., Burrows, P.R., Sarah, J.-L., 1996. Molecular and biochemical diversity among isolates of Radopholus spp. from different areas of the world. J. Nematol. 28, 422–30. Fogain, R., 2000. Effect of Radopholus similis on plant growth and yield of plantains (Musa, AAB). Nematology 2, 129–33. Gowen, S., Que´ ne´ herve´ , P., 1990. Nematode parasites of bananas, plantains and abaca. In: Luc, M., Sikora, R.A., Bridge, J. (Eds.). Plant parasitic nematodes in subtropical and tropical agriculture. CAB International, Wallingford, UK. pp. 431–460. Hahn, M.L., Burrows, P.R., Nalini, C.G., Bridge, J., Vines, N.J., Wright, D.J., 1994. Molecular diversity amongst Radopholus similis populations from Sri Lanka detected by RAPD analysis. Fundam. Appl. Nematol. 17, 275–81. Hendrickx, G., 1995. Automatic apparatus for extracting free-living nematode stages from soil. Nematologica 41, 30.
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Hillis, D.M., Huelsenbeck, J.P., 1992. Signal, noise, and reliability in molecular phylogenetic analyses. J. Hered. 83, 189–95. Hugall, A., Stanton, J., Moritz, C., 1999. Reticulate evolution and the origins of ribosomal internal transcribed spacer diversity in apomictic Meloidogyne. Mol. Biol. Evol. 16, 157–64. Kaplan, D.T., 1994. Molecular characterisation of the burrowing nematodes sibling species, R. citrophilus and R. similis. In: Lamberti, F., De Giorgi, C., Bird, D.M. (Eds.). Advances in Molecular Plant Nematology, Plenum Press, New York, pp. 77–83. Kaplan, D.T., Thomas, W.K., Frisse, L.M., Sarah, J.-L., Stanton, J.M., Speijer, P.R., Marin, D.H., Opperman, C.H., 2000. Phylogenetic analysis of geographically diverse Radopholus similis via rDNA sequence reveals a monomorphic motif. J. Nematol. 32, 134–42. Lockhart, P.J., Steel, M.A., Hendy, M.D., Penny, D., 1994. Recovering evolutionary trees under a more realistic model of sequence evolution. Mol. Biol. Evol. 11, 605–12. Nicholas, K.B., Nicholas Jr., H.B., 1997. GeneDoc: A Tool for Editing and Annotating Multiple Sequence Alignments, Distributed by the author. Stoffelen, R., Verlinden, R., Xuyen, N.T., Swennen, R., De Waele, D., 2000. Host plant response of Eumusa and Australimusa bananas (Musa spp.) to migratory endoparasitic and root-knot nematodes. Nematology 2, 907–16. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24, 4876–82. Valette, C., Mounport, D., Nicole, M., Sarah, J.-L., Baujard, P., 1998. Scanning electron microscopy study of two African populations of Radopholus similis (Nematoda: Pratylenchidae) and proposal of R. citrophilus as a junior synonym of R. similis. Fundam. Appl. Nematol. 21, 139–46. Verdejo-Lucas, S., Pinochet, J., 1992. Population densities for five migratory endoparasitic nematodes on carrot disk culture. J. Nematol. 24, 96– 98. Vrain, T.C., Wakarchuk, D.A., Levesque, A.C., Hamilton, R.I., 1992. Intraspecific rDNA restriction fragment length polymorphism in the Xiphinema americanum group. Fundam. Appl. Nematol. 15, 563–73. Waeyenberge, L., Ryss, A., Moens, M., Pinochet, J., Vrain, T.C., 2000. Molecular characterization of 18 Pratylenchus species using rDNA restriction fragment length polymorphism. Nematology 2, 135–42. Zijlstra, C., Lever, A.E., Uenk, B.J., Van Silfhout, C.H., 1995. Differences between ITS regions of isolates of root-knot nematodes Meloidogyne hapla and M. chitwoodi. Phytopathology 85, 1231–1237.
Intraspecific variation in Radopholus similis isolates assessed with restriction fragment length polymorphism and DNA sequencing of the internal transcribed spacer region of the ribosomal RNA cistron q Gamal A.A. Elbadri a, Paul De Ley b,*, Lieven Waeyenberge c, Andy Vierstraete d, Maurice Moens c,d, Jacques Vanfleteren d a
Department of Crop Protection, Agricultural Research Corporation, P.O. Box 126, Wad Medani, Sudan b Department of Nematology, University of California – Riverside, Riverside, CA 92521, USA c Centrum voor Landbouwkundig Onderzoek, Departement Gewasbescherming, Burgemeester Van Gansberghelaan 96, B-9820 Merelbeke, Belgium d Universiteit Gent, Vakgroep Biologie, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium Received 21 May 2001; received in revised form 27 September 2001; accepted 27 September 2001
Abstract Restriction fragment length polymorphism and direct sequencing of the internal transcribed spacer rDNA region of 19 isolates of Radopholus similis yielded significant diversity, both among isolates and within some individuals. Restriction fragment length polymorphism with HaeIII, AluI and Tru9I yielded two sets of patterns. Digestion with RsaI revealed one or two supernumerary bands in single nematodes from five isolates, and sequencing confirmed microheterogeneity in four of these. Phylogenetic analysis grouped most isolates closely together, except for the five isolates with additional bands for RsaI. Our data reveal more population structure than previously found and lend further support to the synonymy of R. similis and ‘Radopholus citrophilus’. q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Banana; Black pepper; Ornamentals; Polymerase chain reaction; Sequence polymorphism; Nematoda
1. Introduction The burrowing nematode Radopholus similis Thorne, 1949 belongs to the predominantly plant parasitic order, Tylenchida, and is a major pathogen of banana and plantain throughout the tropics. As a migratory endoparasite, it is capable of causing extensive necrosis of the root cortex, and may ultimately lead to toppling of entire plants (Gowen and Que´ne´herve´, 1990), thus resulting in dramatic yield losses (Fogain, 2000). However, different physiological races or biotypes of R. similis are known, and these exhibit widely different levels of pathogenicity (see Stoffelen et al., 2000). Knowledge of genetic diversity of the parasite is therefore essential to effective resistance breeding programs for the host (Hahn et al., 1994). In this paper, we explore intraspecific genetic variation among 19 R. similis isolates kept in continuous culture and morphologically characterised in previous work (Elbadri et q Nucleotide sequence data reported in this paper are available in the GenBank database under the accession numbers AF375348–AF375395. * Corresponding author. Tel.: 11-909-787-2280; fax: 11-909-787-3719. E-mail address: [email protected] (P. De Ley).
al., 1999). We amplified the internal transcribed spacer (ITS) rDNA region of each isolate for restriction fragment length polymorphism (RFLP) analysis and sequencing, and investigated the resulting implications for diagnosis, host preferences and phylogenetic relationships (see Dorris et al., 1999 for more information on rDNA and nematode phylogeny). Our observations and conclusions differ considerably from those reached recently in a similar study (Kaplan et al., 2000). 2. Materials and methods 2.1. Origin, culture and multiplication on banana of the nematodes Nineteen R. similis isolates were collected from locations around the world (Table 1), mostly from banana roots or surrounding soil. All isolates were maintained on carrot discs (Verdejo-Lucas and Pinochet, 1992). In vitro propagated plantlets of banana cv. ‘Grand Naine’ were transplanted to 13 cm pots with sterile soil and acclimatised for 3 weeks. For each isolate, 1,000 nematodes from carrot disk
0020-7519/02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(01)00319-8
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Table 1 Origin of Radopholus similis isolates used in this study, number of sequences obtained from one individual of each isolate, and number of distinct variants among these sequences Code
Locality, country
Host plant
Source
Number of sequences/ number of distinct variants
Anth Aus Bh Cal Che CR Cub Gh Gu Indo Kam Kar Pan Philo Ros Sen Tam Ug WDA
Leuven, Belgium Cairns, Australia Burgershall, S Africa Aalst, Belgium Chendi, Sudan La Estrella, Costa Rica Villa Clara, Cuba –, Ghana Balikoure, Guinea Bangka, Indonesia Kamlin, Sudan Karkoug, Sudan Changuinola, Panama Hamburg, Germany Roseris, Sudan Sennar, Sudan Tamis, Sudan Kampala, Uganda –, –
Anthurium andreanum Banana Banana Calathea makoyana Banana Banana Banana Banana Banana Piper nigrum Banana Banana Banana Philodendron sp. Banana Banana Banana Banana Banana
M. Moens J. Pinochet B. Meyers M. Moens G. Elbadri J. Pinochet J. Pinochet D. De Waele J. Pinochet N. Herdradjat G. Elbadri G. Elbadri J. Pinochet M. Moens G. Elbadri G. Elbadri G. Elbadri J. Pinochet J. Pinochet
1/1 1/1 1/1 5/3 13/8 1/1 1/1 1/1 1/1 1/1 1/1 9/4 1/1 1/1 1/1 6/4 1/1 1/1 1/1
culture were inoculated on each of eight plants, and all 152 plants were arranged in a complete randomised block design and grown for 12 weeks in the greenhouse in 3 l pots with sterilised sandy soil. Plant roots were then cut into 5 mm fragments, 10 g of these were macerated and put into a 1 l suspension, and 500 ml of each suspension was extracted with an automated apparatus (Hendrickx, 1995). The reproduction factor, Rf (the ratio of the extracted number of nematodes over the initial size of inoculum), was calculated for each replicate, and statistically compared with Dunnett’s test. 2.2. DNA extraction and PCR amplification Single nematodes were prepared for PCR as described by Waeyenberge et al. (2000), but using a five-fold dilution of their lysis buffer. The DNA amplification profile consisted of 4 min at 948C; 35 cycles of 1 min at 948C, 1.5 min at 508C, and 2 min at 728C; and a final 5 min extension at 728C. We used the two 21 bp PCR primers derived from the Xiphinema bricolensis sequence by Vrain et al. (1992). In some isolates, amplification with these primers was unsuccessful, and we therefore compared sequences from other isolates to derive and apply two modified primers: Vrain2 18S (.5 0 -CTTTGTACACACCGCCCGTCGCT-3 0 ) and Vrain2 26S (.5 0 TTTCACTCGCCGTTACTAAGGGAATC-3 0 ). 2.3. Restriction fragment length polymorphism analysis Five microlitres of each good PCR product was digested with 10 U of each of seven restriction enzymes: AluI, HaeIII, HinfI, NdeII, RsaI, TaqI and Tru9I, incubated overnight at 378C (AluI, HaeIII, HinfI, NdeII and RsaI) or 658C
(TaqI and Tru9I). The digested DNA was separated by electrophoresis in 1.5% agarose for 3 h, and visualised with ethidium bromide. The procedure was repeated until RFLP patterns were obtained from three individual nematodes of each isolate. 2.4. Nucleotide sequencing A PCR product from a single nematode from each isolate was prepared for sequencing by adding 0.6 ml exonuclease I (10 U/ml), 0.6 ml shrimp alkaline phosphatase (2 U/ml), and 15 ml mineral oil to 3 ml of each PCR product, centrifuging the mixture, and incubating it first for 15 min at 378C, and then for 15 min at 808C. Next, each sample was treated with BigDyee Terminator Ready Reaction mix according to the manufacturer’s protocol (DNA sequencing Kit, PE Biosystems) using primer Vrain2 18S or Vrain2 26S, and loaded on an automated sequencer (ABI Prism 377 DNA Sequencer, PE Biosystems). Direct sequencing of the Calathea, Chendi, Sennar and Karkoug isolates produced electropherograms with numerous multiple peaks, and the PCR products obtained for these isolates were therefore cloned using pGEM-T Vector and JM109 Competent Cells (pGEM w-T Vector System II, Promega). Ligation and transformation reactions were set up as described by the Promega protocol (Protocols and applications guide, 3rd Edition, 1996, Promega). Clones were screened with PCR and RFLP to verify whether the cloning procedure was successful. Appropriate-sized PCR products were then sequenced from individual colonies of each of the four cloned isolates, until at least three distinct sequence variants were obtained/isolate (Table 1).
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2.5. Sequence alignment and phylogenetic inference All obtained sequences were aligned automatically using ClustalX (Thompson et al., 1997), along with two ITS region sequences published by Kaplan (1994) for R. similis and Radopholus citrophilus Huettel et al., 1984, and verified by eye using GeneDoc 2.5 (Nicholas and Nicholas, 1997). For each group of identical sequences, all except one were removed in order to speed up calculation times for subsequent analyses. Alignment presented no problems, except for one ambiguous trio of positions. A second alignment was therefore produced, differing only from the first one in that it represented the alternate homology pattern for these three positions. Both alignments were analysed with PAUP* 4.0b4a (Swofford, D.L., 1998. PAUP* – Phylogenetic Analysis using Parsimony (* and other methods), Version 4, Sinauer), using the unweighted pair group method with arithmetic mean (UPGMA), neighbour-joining, maximum parsimony and maximum likelihood algorithms. In the absence of a suitably close outgroup taxon, all trees were calculated without rooting. UPGMA was applied with uncorrected distance measure, while neighbour-joining was implemented with LogDet/paralinear distances (Lockhart et al., 1994). Heuristic searches were completed with maximum parsimony (characters unordered and of equal weight, gaps treated as missing data, random addition sequence with 1,000 replicates, multiple trees threshold set to 500, without steepest descent) and maximum likelihood (nucleotide frequencies set to empirical values, two substitution types, HKY-
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model (Hasegawa, Kishino and Yano), transition/transversion ratio set to 2). Furthermore, bootstrap analyses were performed for UPGMA, neighbour-joining and maximum parsimony using 3,000 replicates and similar settings as above (except for maximum parsimony now being set to simple addition sequence). Finally, one million random trees were generated and compared with maximum parsimony in order to generate the g1 shape parameter of tree length distribution, which assesses the phylogenetic signalto-noise ratio of the alignment (Hillis and Huelsenbeck, 1992). Absolute distances and pairwise character distances were calculated for all pairwise combinations of operational taxonomic units (OTUs).
3. Results 3.1. Restriction fragment length polymorphism patterns Amplification of the ITS region of nematodes from all isolates yielded a 920 bp fragment. The restriction pattern obtained by AluI (Fig. 1A), divided the isolates into two groups. A first one contained 14 isolates showing four bands of about 380, 270, 150 and 120 bp. The second group was composed of the Karkoug, Sennar, Chendi, Calathea and Indonesia isolates, displaying three bands of about 390, 380 and 150 bp. Tru9I restriction (Fig. 1B) yielded the same two groups: the first with five bands of about 300, 280, 170, 120 and 50 bp, and the second with six bands of about 260, 230, 180, 150, 100 and 50 bp.
Fig. 1. (A) AluI; and (B), Tru9I. Restriction fragments of amplified internal transcribed spacer of Radopholus similis isolates. (L) DNA size marker (100 bp). (1) Costa Rica; (2), Uganda; (3), Australia; (4), Panama; (5), Guinea; (6), Cuba; (7), Burgershall; (8), WDA; (9), Kamlin; (10), Karkoug; (11), Sennar; (12), Roseris; (13), Chendi; (14), Tamis; (15), Philodendron; (16), Anthurium; (17), Calathea; (18), Indonesia; (19), Ghana.
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After restriction with RsaI (data not shown), we always obtained two strong bands of about 660 and 260 bp, but PCR products of the Karkoug, Sennar, Chendi, Calathea and Indonesia isolates also displayed a band of about 190 bp. HaeIII only digested PCR products from the Panama, Guinea, Cuba, WDA, Roseris, Tamis and Ghana isolates, resulting in 670 and 250 bp fragments. HinfI cut all PCR products into five bands of about 300, 270, 180, 90 and 60 bp, while NdeI cut all PCR products into four bands of about 420, 310, 120 and 70 bp. TaqI restriction yielded two main bands of about 450 and 230 bp for all isolates, and two faint bands of 100 and 70 bp.
pairwise distances within the heterogeneous group varied between 0 and 16 units, and translated into longer branch lengths in all of the best trees. In particular, the internal branch separating the Indonesian subgroup from the Sennar subgroup (branch II) was of similar length to branch I. At least one sequence variant from each of the four cloned PCR products was more similar to variants obtained from an individual from a different isolate, than to other variants from the same nematode. The most striking example was Chendi clone 9, which was part of the Indonesian subgroup and differed by 13–14 substitutions from most of the other Chendi clones, while being identical to Calathea clone 0.
3.2. Nucleotide sequencing A total of 25 sequences were included in our analysis: eight sequence variants of the Chendi isolate, four variants each of the Karkoug and Sennar isolates, three of the Calathea isolate, four invariant sequences representing the remaining 15 isolates (most of which had identical sequences), and two published sequences (Kaplan, 1994). The length of the entire ITS region varied between 583 and 585 nucleotides. Both alignments were only 533 positions wide due to slightly incomplete sequence stretches obtained for seven of the 44 sequenced clones. Up to 20 and 21 substitutions were assumed in alignments 1 and 2, respectively, and both alignments contained only 22 parsimony-informative positions. The values obtained for the tree length distribution shape parameter, g1, were highly significant for both alignments (20.885 and 20.926, respectively), indicating that the data were non-random with probabilities exceeding 99% (Hillis and Huelsenbeck, 1992).
3.4. Population growth on banana cv. ‘Grand Naine’ Multiplication varied extensively among replicates and
3.3. Phylogenetic analysis: segregation into three clusters All tree-construction methods applied to both alignments yielded trees with identical basic branching orders, with very high bootstrap values for two major internal branches (Figs. 2 and 3). Variations occurred only in local placement of terminal OTUs. All trees yielded a homogeneous cluster representing 14 of our isolates, as well as the two sequences of Kaplan (1994). Absolute distances within this cluster equalled 0–2 units, and it was always resolved as a convex group, i.e. depending on root placement the group might be monoor paraphyletic, but not polyphyletic. Sequences of the isolates from Sennar, Chendi, Karkoug, Calathea and Indonesia always constituted a second convex group, that was much more heterogeneous and separated from the homogeneous group by absolute distances of 7–21 units. Within the heterogeneous group, sequences from the Indonesian isolate and from the clones Calathea 0, Calathea 11, Chendi 9, Karkoug 1, Karkoug 7, and Karkoug 8 always resolved as a convex internal subgroup (hereafter referred to as the Indonesian subgroup) clearly separated from the remaining OTUs (hereafter referred to as the Sennar subgroup). The internal branch between the homogeneous and the heterogeneous group (branch I) was always clearly longer than any branches within the homogeneous group. Absolute
Fig. 2. Unrooted best tree obtained with maximum parsimony analysis (heuristic search, characters unordered and of equal weight, gaps treated as missing data, random addition sequence with 1,000 replicates, multiple trees threshold set to 500, without steepest descent) of alignment 1 of internal transcribed spacer region sequences obtained from 19 isolates of Radopholus similis. Branch lengths are absolute (scale bar represents one parsimony-informative substitution). Depicted bootstrap values were taken from corresponding branches in the consensus tree obtained with bootstrap analysis (3,000 bootstrap replicates, maximum parsimony criterion, heuristic search, characters unordered and of equal weight, gaps treated as missing data, random addition sequence with 1,000 replicates, multiple trees threshold set to 500, with steepest descent). Philo-Anth represents identical sequences obtained from the following isolates: Australia, Burgershall, Costa Rica, Kamlin, Ghana, Panama, Roseris, Uganda, Anthurium, Philodendron.
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Fig. 3. Consensus of the unrooted best trees obtained from phylogenetic analysis of two alignments of internal transcribed spacer region sequences from 19 isolates of Radopholus similis (see text for details on algorithm settings). Branch lengths are not to scale. The homogeneous group (upper right) is always clearly separated from all other operational taxonomic units by long internal branch I. Box I lists bootstrap values (B) calculated for this branch from bootstrap trees and branch lengths (L) obtained from best trees for each algorithm (unweighted pair group method with arithmetic mean, neighbour-joining, maximum parsimony, maximum likelihood) and each alignment (1 and 2). Within the heterogeneous group, the Indonesian subgroup (lower left) is always clearly separated by long internal branch II, and box II lists the corresponding bootstrap and branch length values for all methods and alignments. For neighbour-joining and maximum likelihood, branch length values are proportional to half the distance between reference operational taxonomic units, citroK and similK. For unweighted pair group method with arithmetic mean UP and maximum parsimony, branch lengths are absolute, and the distance between citroK and similK equals 2. Abbreviations are as in Fig. 2.
among isolates (Fig. 4). Only three isolates differed significantly from null growth according to the Dunnett test: the Uganda and Cuba isolates with P . 0:99, and the Tamis isolate with P . 0:95. Isolates with the lowest average Rf included those forming the heterogeneous group in our phylogenetic analysis (on the right in Fig. 4), and/or isolates originally sampled from hosts other than banana (marked with asterisks in Fig. 4).
4. Discussion 4.1. Evidence for genetic diversity in R. similis Fallas et al. (1996) previously detected variations
between 10 isolates of R. similis, using AluI and HaeIII digestion patterns. In our study, AluI, RsaI and Tru9I digestion separated the Karkoug, Sennar, Chendi, Indonesia and Calathea isolates more or less clearly from the remainder. For each of our isolates and for most of the applied enzymes, the sum of the sizes of the restriction fragments was equal to the size of the PCR product. However, for TaqI, the sum was 850 bp, and for Tru9I, we obtained a sum of 920 bp for one group, versus 970 for the other group. This suggests microheterogeneity of the ITS region repeats within individuals, a phenomenon documented in several other plant parasitic nematodes (e.g. Zijlstra et al., 1995; Hugall et al., 1999; Waeyenberge et al., 2000). In our case, sequence data clearly support the separation of the five isolates with aberrant RFLP patterns from the rest
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Fig. 4. Reproduction factor Rf (the ratio of extracted number of nematodes over initial size of inoculum) of 19 R. similis isolates on banana cv. ‘Grand Naine’. White crossbars represent averages of eight replicates, white vertical bars are standard deviations, black vertical lines are ranges. Isolates are presented in order of appearance from upper right to lower left in Fig. 3; those marked with asterisks were originally isolated from hosts other than banana.
of our material, and also confirm the presence of multiple sequence variants within individuals from four of these five isolates. Interestingly, the same five isolates also display a low to very low average reproduction on banana (Fig. 4). RFLP of the ITS region with AluI and Tru9I therefore merits further investigation as a possible marker for pathogenicity of R. similis to banana, and it might even be possible to develop a PCR-based diagnostic test by designing a primer for the region corresponding to positions 490–510 of our alignments. Although it is commonly stated that R. similis has a high degree of genomic conservation (e.g. Kaplan, 1994), with random amplification of polymorphic DNA (RAPD), genetic similarity between certain R. similis isolates was found to be as low as 75% (Hahn et al., 1994), or even 67% (Fallas et al., 1996). Sequence divergence among our isolates is congruent with the quoted RAPD data, and contradicts the homogeneity reported for ITS1 sequences of 55 R. similis isolates (Kaplan et al., 2000). Most of the phylogenetically-informative divergence observed in our material actually pertains to differences in ITS2: up to 12 or 13 parsimony-informative substitutions in our two alignments occur in ITS2, versus only up to five in ITS1, respectively. The discrepancy between Kaplan et al. (2000) and our findings may therefore be due to a combination of different isolates sampled, and their choice of sequencing ITS1 alone. 4.2. Taxonomical interpretations One might be tempted to translate the differences observed here into formal taxonomic segregation, since they exceed those occurring between the sequences previously determined by Kaplan (1994) for R. similis and R. citrophilus (two substitutions between the latter pair versus up to 21 substitutions in pairwise comparison of
our new sequences). They are also larger than those occurring between, for example, the cryptic species Meloidogyne fallax Karssen, 1996 and Meloidogyne chitwoodi Golden, O’Bannon, Santo and Finley, 1980 (only nine substitutions out of 510 bp; I.T. De Ley, personal communication). However, for a number of reasons, we do not suggest taxonomic subdivision, but instead consider our data to add further support to the proposed synonymy of R. similis and R. citrophilus (Valette et al., 1998; Elbadri et al., 1999; Kaplan et al., 2000). Firstly, our isolates display no morphological correlates for such a subdivision (Elbadri et al., 1999). Secondly, the low reproduction of some isolates on banana is interesting, but neither statistically significant nor fully consistent with molecular relationships, and above all, we should be careful not to overemphasise host specificity as a taxonomic character — exactly such an emphasis led to the original proposal of R. citrophilus. Thirdly, the divergence within our heterogeneous group of five isolates is on a similar scale of magnitude to the divergence between isolates of both groups, and it is therefore more prudent to assume that the aberrant isolates are divergent members of one and the same species. Fourthly, and perhaps most intriguingly, we should keep in mind the possibility that the observed pattern of sequence microheterogeneity reflects genetic exchange between some of our sampled isolates (Hugall et al., 1999). Contrary to Kaplan et al. (2000), we detected small but significant levels of genetic divergence between R. similis isolates, of up to 4% substitutions in the ITS region. This divergence is reflected in restriction patterns obtained with the enzymes AluI and Tru9I, which separate the five most divergent isolates from all others. Furthermore, significant variation occurs within individual genomes from these divergent isolates, and some variants are more similar to those found in other isolates, than to other variants from the same individual. These results suggest that genetic
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diversity of rDNA loci in R. similis may indeed be relevant to host preferences, but also that identification and diagnosis on the basis of the ITS region is less straightforward than would appear from previous studies. Both these aspects are directly relevant to the control of R. similis, and clearly merit further investigation. Acknowledgements The first author would like to thank the Islamic Development Bank for financial support for this study. J.V. and P.D.L. acknowledge support through grant G.00292.00 from the Fund for Scientific Research-Flanders. The authors thank Dr J. Pinochet, Professor B. Meyer and Mr N. Herdradjat for providing them with the studied isolates. References Dorris, M., De Ley, P., Blaxter, M.L., 1999. Molecular analysis of nematode diversity and the evolution of parasitism. Parasitol. Today 15, 188– 93. Elbadri, G.A.A., Geraert, E., Moens, M., 1999. Morphological differences among Radopholus similis (Cobb, 1893) Thorne, 1949 populations. Russ. J. Nematol. 7, 139–53. Fallas, G.A., Hahn, M.L., Fargette, M., Burrows, P.R., Sarah, J.-L., 1996. Molecular and biochemical diversity among isolates of Radopholus spp. from different areas of the world. J. Nematol. 28, 422–30. Fogain, R., 2000. Effect of Radopholus similis on plant growth and yield of plantains (Musa, AAB). Nematology 2, 129–33. Gowen, S., Que´ ne´ herve´ , P., 1990. Nematode parasites of bananas, plantains and abaca. In: Luc, M., Sikora, R.A., Bridge, J. (Eds.). Plant parasitic nematodes in subtropical and tropical agriculture. CAB International, Wallingford, UK. pp. 431–460. Hahn, M.L., Burrows, P.R., Nalini, C.G., Bridge, J., Vines, N.J., Wright, D.J., 1994. Molecular diversity amongst Radopholus similis populations from Sri Lanka detected by RAPD analysis. Fundam. Appl. Nematol. 17, 275–81. Hendrickx, G., 1995. Automatic apparatus for extracting free-living nematode stages from soil. Nematologica 41, 30.
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