Pratylenchus speijeri n. sp. (Nematoda: Pratylenchidae),a new root-lesion nematode pest of plantain in West Africa
Francesca DE LUCA 1,∗, Alberto TROCCOLI 1, Larry W. DUNCAN 2, Sergei A. SUBBOTIN 3,4,Lieven WAEYENBERGE 5, Daniel L. COYNE 6, Francis C. BRENTU 7 and Renato N. INSERRA 8
1 CNR, Istituto per la Protezione delle Piante, Via G. Amendola 165/A, Bari 70126, Italy2 University of Florida, IFAS, Citrus Research and Education Center, Department of Entomology,
Lake Alfred, FL 33850, USA3 Plant Pest Diagnostic Center, California Department of Food and Agriculture, 3294 Meadowview Road,
Sacramento, CA 95832, USA4 Center of Parasitology of A.N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences,
Leninskii Prospect 33, Moscow 117071, Russia5 ILVO, Institute for Agricultural and Fisheries Research, Plant, Crop Protection, Burg. Van Gansberghelaan 96,
9820 Merelbeke, Ghent, Belgium6 International Institute of Tropical Agriculture (IITA), Carolyn House, 26 Dingwall Road, Croydon CR9 3EE, UK
7 Forest and Horticultural Crops Research Centre, Kade, Institute of Agricultural Research,College of Agriculture and Consumer Sciences, University of Ghana, Legon, Accra, Ghana
8 Florida Department of Agriculture and Consumer Services, DPI, Nematology Section, P.O. Box 147100,Gainesville, FL 32614-7100, USA
Received: 24 January 2012; revised: 15 March 2012Accepted for publication: 20 March 2012
Summary – A new root-lesion nematode, particularly pathogenic to Musa spp. and causing important plantain losses in Ghana, isdescribed and named Pratylenchus speijeri n. sp. The cryptic status of this species within the P. coffeae species complex has beenassessed and confirmed in this study. An extensive comparison of the morphological and molecular characteristics of this new specieswith those of P. coffeae and other related amphimictic species did not result in an unambiguous separation of this species from P.coffeae because only a few morphological features of diagnostic value were found. Sequence and phylogenetic analyses of the D2-D3expansion segments of the 28S rRNA gene, the ITS rRNA gene and a portion of the hsp90 gene of P. speijeri n. sp. and P. coffeae speciescomplex populations from different sources generated majority consensus BI trees with three major clades: P. speijeri n. sp. from Musaspp. roots in Ghana; unidentified or putative new Pratylenchus sp. C1 from Colocasia esculenta roots in Japan and P. coffeae withnon-homogeneous relationships from different hosts and distant geographical areas. These results confirmed the validity of P. speijerin. sp. as a new taxon and indicated that P. coffeae populations from Colocasia in Japan also need to be considered as a new species.Sequence differences in the ITS were used to design group- and species-specific primers to detect P. speijeri n. sp. and other species ofP. coffeae species complex. The use of these species-specific primers for the separation of P. speijeri n. sp., Pratylenchus sp. C1 and P.coffeae has important practical application in breeding programmes for agriculture in West Africa.
Keywords – Ghana, molecular, morphology, morphometrics, Musa spp., new species, phylogeny, Pratylenchus coffeae, SEM, speciescomplex, taxonomy.
The coffee root-lesion nematode, Pratylenchus coffeae(Zimmermann, 1898) Filipjev & Schuurmans Stekhoven,1941, is a common nematode pest of Musa spp. (ba-nana and plantain) in the tropics (Gowen et al., 2005).However, while particularly damaging in some instances,
the level of damage caused to Musa spp. by P. cof-feae varies in different geographical areas (Dubois &Coyne, 2011). Such variability in the yield reduction byP. coffeae has been attributed to the presence of nema-tode populations with different host preference, patho-
types or strains of P. coffeae, or even to different Praty-lenchus spp. that are particularly pathogenic to Musa spp.(Coyne, 2009). One such aggressive P. coffeae populationinflicted severe damage to plantain growing in microplotsin Ghana (Brentu et al., 2004). The study by Brentu etal. (2004) followed the consistent association of heavyplantain losses with this nematode in Ghana (Schill etal., 1997; Udzu, 1997). The higher reproductive poten-tial of the Ghana population compared to that of otherP. coffeae isolates from Vietnam was shown in popula-tion dynamics tests conducted on carrot discs (Nguyen,2010). The morphology and molecular characterisation ofa root-lesion nematode identified as P. coffeae and origi-nating from plantain in Ghana were reported by Duncanet al. (1999). In spite of the morphological similarity be-tween the Ghana P. coffeae and other P. coffeae popula-tions, phylogenetic analyses using the D2-D3 expansionregions of the 28S rRNA gene indicated that the Ghanaisolate was different from P. coffeae (Duncan et al., 1999).Analysis of published data (Uehara et al., 1998; Duncanet al., 1999; Andrés et al., 2000; Mizukubo et al., 2003),field observations (Brentu et al., 2004) and our detailedstudy showed that P. coffeae from Ghana should be con-sidered as a cryptic species, almost morphologically in-distinguishable, but biologically and molecularly distinctfrom P. coffeae.
Considering the economic significance of root-lesionnematodes on plantain in West Africa, and the need todistinguish these damaging species, we provide here themorphological and molecular characterisation of a newroot-lesion nematode, Pratylenchus speijeri n. sp.
The specific objectives of this paper were to: i) present acomprehensive description of P. speijeri n. sp. with differ-ential diagnosis to similar and related species, includinga topotype population (K6) of P. coffeae from Indonesia;ii) provide molecular characterisation of P. speijeri n. sp.and reconstruct its relationship with other representativesof the P. coffeae species complex using three molecularmarkers (sequences of the D2-D3 expansion segments ofthe 28S rDNA, the ITS rDNA and the portion of the hsp90gene); iii) develop a PCR-RFLP diagnostic tool for differ-entiation of P. speijeri n. sp. from other species of P. cof-feae species complex; and iv) design PCR with a species-specific primer for detection of P. speijeri n. sp. and PCRwith group-specific primers for detection of all species ofthe P. coffeae species complex.
Materials and methods
ROOT-LESION NEMATODE POPULATIONS USED
Root-lesion nematode isolates were imported into Flor-ida with special permits. Carrot cultures of the plan-tain population of P. coffeae B1 were provided by DrJ. Pinochet. This B1 population was originally collectedfrom a major plantain production area in Kade, Ghana,and maintained in culture on the false horn plantain cv.Apantu-pa (Musa spp., AAB-group) at the same locality,Forest and Horticultural Crops Research Centre, Kade,of the University of Ghana, located 120 km north-westof Accra (6°09′N, 0°55′W), Ghana, where it is avail-able for further studies. This population also was trans-ported in carrot cultures by the late Paul Speijer to Bel-gium and given to Dr J. Pinochet and other colleagues.The topotype P. coffeae population K6 (Duncan et al.,1999) was collected from coffee in Kaliwining, easternJava, Indonesia. Nematodes (B1 and K6) were recov-ered from carrot discs and coffee roots and transferredto carrot discs and maintained at 23°C at the Univer-sity of Florida CREC, Lake Alfred, FL, USA, for almost8 years (Huettel, 1985). The carrot disc-cultured nema-todes used by Duncan et al. (1999) for preliminary mor-phological and molecular analyses of these two popu-lations (B1 and K6) were re-examined for the currentstudy for morphological, sequence and phylogenetic anal-yses to verify their purity. Additional populations char-acterised in this comparative molecular study included asecond B1 population from the same locality in Ghanaand six geographically distant P. coffeae populations (Ta-ble 1).
MORPHOMETRIC AND MORPHOLOGICAL ANALYSIS
Only adult nematodes cultured on carrot discs wereused during the study. Live specimens were immobilisedby gently heating and then mounted in water agar on aslide for measurements and photographs (Esser, 1986).Additional measurements and drawings were made usingspecimens killed and fixed in hot aqueous 2% formalde-hyde + 1% propionic acid, dehydrated in ethanol vapourand mounted in dehydrated glycerin (Hooper, 1970).Measurements of specimens were made with an ocularmicrometer and drawings with a camera lucida. Pho-tographs were taken using two Leica (Wild MPS 46/52and Leica DFC 320) cameras mounted on Nikon (Op-tiphot) and Leica DM 2500 compound microscopes.
2 Nematology
Pratylenchus speijeri n. sp. from Musa in West Africa
Table 1. Samples from the Pratylenchus coffeae species complex used in the present molecular study.
Species Locality Host Collection codesfor DNA ornematode cultures
GenBank accession number Source ofmaterials and/orreferenceD2-D3 of
28S rRNAITS rRNA hsp90
P. speijeri n. sp. Kade,Ghana
Musa sp. Ghana AF170433,JN809841-JN809845
JN809828-JN809831
HE601547 J. Pinochet; F.C.Brentu; Duncanet al. (1999)
P. coffeae Kaliwining,Java,Indonesia
Coffea arabica K6, JAVA AF170443,JN809860
JN809840 HE601548 L.W. Duncan;Duncan et al.(1999)
P. coffeae Arcahaie,Haiti
Musa sp. PcHT JN809847-JN809849,JN809852,JN809853
JN809834 – J.-L. Sarah
P. coffeae Kumamoto,Japan
Ipomoea batatus CA201 EU130847-EU130849
JN809835,JN809836
– T. Mizukubo;Subbotin et al.(2008)
P. coffeae Viet Nam Musa sp. PcLW JN809858,JN809859
JN809837-JN809839
– T.T. Nguyen
P. coffeae Martinique Coffea sp. PcMT JN809846,JN809854-JN809857
– – J.-L. Sarah
P. coffeae FL, USA Aglaonema sp. CD820 JN809850,JN809851
Specimens for scanning electron microscope (SEM) ob-servations were cold-fixed in glutaraldehyde buffered with0.1 M phosphate buffer (pH 7.2), post-fixed for 1 h in2% osmium tetroxide, dehydrated in a graded series ofethanol, critical point-dried with CO2 and sputter-coatedwith gold palladium (Eisenback, 1985). Nematodes wereobserved with a Hitachi S530 microscope at 15-20 kV ac-celerating voltage.
DNA EXTRACTION, PCR, CLONING AND
SEQUENCING
DNA was extracted from both female and male root-lesion nematode specimens. Specimens were handpickedand placed singly on a glass slide in 3 μl of the lysisbuffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 15 mMMgCl2, 0.1% Triton X-100, 0.01% gelatine with 90 μgml−1 proteinase K) and then cut into small pieces by usinga sterilised syringe needle under a dissecting microscope.The samples were incubated at 65°C for 1 h and thenat 95°C for 15 min to deactivate the proteinase K. PCR,
cloning and sequencing were done in three laboratories:Istituto per la Protezione delle Piante, Italy; ILVO, Bel-gium; PPDC, CDFA, USA. The protocols were describedin detail by De Luca et al. (2004), Waeyenberge et al.(2000), Subbotin and Moens (2006) and Subbotin et al.(2008), respectively. The following sets of primers wereused for amplification of gene fragments in the presentstudy: i) D2-D3 expansion segments of 28S rRNA geneusing forward D2A and reverse D3B primers; ii) ITS1-5.8-ITS2-rRNA using forward F194 and reverse F195primers or forward TW81 and reverse AB28 primers; andiii) hsp90 using forward U831 and reverse L1110 primers(Table 2). New sequences have been submitted to the Gen-Bank database under the numbers indicated in Table 1 andFigures 1-3.
PCR-RFLP
The PCR product of D2-D3 of 28S rRNA gene waspurified using the QIAquick Gel Extraction Kit (Qia-gen, Valencia, CA, USA). A 3 μl volume of purified
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Table 2. Primer sets used in the present study.
Primer code Sequence (5′ → 3′) Amplified gene References
TW81 GTTTCCGTAGGTGAACCTGC ITS-rRNA Tanha Maafi et al. (2003)AB28 ATATGCTTAAGTTCAGCGGGTF194 CGTAACAAGGTAGCTGTAGG ITS-rRNA Ferris et al. (1993)F195 TCCTCCGCTAAATGATATD2A ACAAGTACCGTGAGGGAAAGTTG D2-D3 of 28S rRNA Subbotin et al. (2006)D3B TCGGAAGGAACCAGCTACTAU831 AAYAARACMAAGCCNTYTGGAC hsp90 Skantar & Carta (2005)L1110 TCRCARTTVTCCATGATRAAVACTW81 GTTTCCGTAGGTGAACCTGC ITS-rRNA This studycoffeae group-specific CTTAAGCCATGTGCCAACTCTW81 GTTTCCGTAGGTGAACCTGC ITS-rRNA This studyspeijeri species-specific GTGCACTGATGTTATTATGTATGG
product was digested by either Cfr42I or Bpu1102I re-striction enzyme in the buffer stipulated by the man-ufacturer. The digested DNA was run on a 1% TAEbuffered agarose gel, stained with ethidium bromide, vi-sualised on a UV transilluminator and photographed.The exact lengths of each restriction fragments fromthe PCR products were obtained by a virtual diges-tion of the sequences using WebCutter 2.0 (available atwww.firstmarket.com/cutter/cut2.html).
PCR WITH GROUP- AND SPECIES-SPECIFIC PRIMERS
The ITS sequences of P. coffeae species complexwere aligned with other available Pratylenchus sequencesin the GenBank and then the sequences specific to P.coffeae species complex and to P. speijeri n. sp. wereidentified. This information was used to design group-specific primers amplifying rRNA gene fragment from allspecies of the P. coffeae species complex and a species-specific primer amplifying rRNA gene fragment fromP. speijeri n. sp. (Table 2). The primers were tested incombination with the universal primer TW81 with severalPratylenchus samples in PCR separately and together in aduplex PCR condition. A 2 μl volume of extracted DNAwas transferred into a 0.2 ml Eppendorf tube containing:2.5 μl 10× Taq incubation buffer, 5 μl Q solution, 0.5 μldNTPs mixture (Taq PCR Core Kit, Qiagen), 0.15 μl ofeach primer (1.0 μg μl−1), 0.1 μl Taq polymerase anddouble distilled water to a final volume of 25 μl. The PCRamplification profile consisted of 4 min at 94°C; 30 cyclesof 1 min at 94°C, 45 s at 55°C and 45 s at 72°C, followedby a final step of 10 min at 72°C. A 4 μl volume of the
PCR product was run on a 1.4% TAE buffered agarosegel, stained and photographed.
PHYLOGENETIC ANALYSES
The newly obtained sequences for each gene werealigned using ClustalX 1.83 (Thompson et al., 1997) withdefault parameters with corresponding published gene se-quences, respectively (Al-Banna et al., 1997; Uehara etal., 1998; Duncan et al., 1999; Skantar & Carta, 2004;Subbotin et al., 2008; Cheng et al., 2009; Nguyen, 2010;Zhao, unpubl.; Wang & Peng, unpubl.; Wang et al., un-publ.; Yu et al., unpubl.; Waeyenberge et al., unpubl.;Lee et al., unpubl.). Outgroup taxa for each dataset werechosen according to the results of previously publisheddata (Subbotin et al., 2006). Sequence alignment of eachgene was manually edited using GenDoc 2.5.0. Sequencedatasets were analysed with Bayesian inference (BI) us-ing MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001). BIanalysis under the GTR + I + G model for each gene wasinitiated with a random starting tree and was run with fourchains for 1.0 × 106 generations. The Markov chains weresampled at intervals of 100 generations. Two runs wereperformed for each analysis. The log-likelihood values ofthe sample points stabilised after approximately 1000 gen-erations. After discarding burn-in samples and evaluatingconvergence the remaining samples were retained for fur-ther analysis. The topologies were used to generate a 50%majority rule consensus tree. Posterior probabilities (PP)are given on appropriate clades. Sequence differences be-tween samples were calculated with PAUP* 4b10 (Swof-ford, 2003) as an absolute distance matrix and the percent-age was adjusted for missing data.
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Pratylenchus speijeri n. sp. from Musa in West Africa
Results
MOLECULAR CHARACTERISATION OF Pratylenchusspeijeri N. SP. AND ITS PHYLOGENETIC
RELATIONSHIPS WITH OTHER SPECIES FROM THE P.coffeae SPECIES COMPLEX
PCR with D2A and D3B primers yielded an ampliconranging from 780 to 795 bp in length depending on samplestudied. Twenty new D2-D3 sequences of Pratylenchus,five of them belonging to the new species, were obtainedin this study. Alignment of the D2-D3 of the 28S rDNAincluded 60 sequences of P. coffeae species complexand was 758 bp in length. The phylogenetic analysisgenerated the majority consensus BI tree with threemajor clades within the P. coffeae species complex:clade 1) P. coffeae (PP = 95); clade 2) Pratylenchussp. C1 from Colocasia esculenta in Japan (PP = 100);and clade 3) P. speijeri n. sp. (PP = 94) (Fig. 1).Intraspecific variations for P. speijeri n. sp. were 0-0.3%(0-2 bp); P. coffeae for majority samples 0-1.1% (0-9 bp)and maximum 1.5% (15 bp) including a sample fromMartinique. No variation was observed for Pratylenchussp. C1. Interspecific variation between P. speijeri n. sp.and other species ranged from 2.4 to 3.4% (17-24 bp).
PCR with F194 and F195 and TW81 and AB28 primersfor the ITS sequences yielded a fragment ranging from1025 to 1057 bp and from 1002 to 1039 bp, respectively,for different samples of the P. coffeae species complex.The di-nucleotide repeats or degenerate version of the re-peat (GT)n and (CA)n, which were detected, contributedto length variation between species and also within indi-vidual nematodes (Troccoli et al., 2008; Palomares-Riuset al., 2010). Thirteen new sequences, four of them be-longing to the new species, were obtained in this study.The ITS sequence alignment included 44 sequences of theP. coffeae species complex and was 1099 bp in length. TheBI tree included several clades, comprising clade 1, P. cof-feae isolates with uncertain relationships and two highlysupported subclades with PP = 100; clade 2, Pratylenchussp. C1; and clade 3, P. speijeri n. sp. (Fig. 2). The P. spei-jeri n. sp. clade included four sequences of populationsfrom Ghana and the sequence of a geographically distantpopulation collected from Musa sp. in China. Intraspecificvariations for P. speijeri n. sp. were 0.2-1.1% (2-11 bp)and for P. coffeae 0-5.2% (0-51 bp). Interspecific sequencevariation between P. speijeri n. sp. and other species was4.2-8.6% (36-82 bp).
PCR amplification of the hsp90 gene by U831 andL1110 primers yielded a fragment that varied by approx-
imately 301-321 bp depending on sample. Three new se-quences, one each for P. speijeri n. sp., P. coffeae andP. hippeastri, were obtained in the present study. Thehsp90 sequence alignment included ten sequences andwas 361 bp in length. In the BI tree P. speijeri n. sp. clus-tered with P. coffeae (Fig. 3), and differed from it by 1.7%(5 bp).
MOLECULAR DIAGNOSTICS OF Pratylenchusspeijeri N. SP.
Enzyme Cfr42I (Fig. 4A) cut PCR product of D2-D3 of the 28S rRNA gene of P. speijeri n. sp. intotwo fragments (574 and 221 bp), whereas PCR productsof Pratylenchus sp. C1 and P. coffeae were unrestricted(795 bp and 781 bp, respectively). Enzyme Bpu1102I(Fig. 4B) generated three fragments for P. speijeri n. sp.(395, 232, 168 bp), two for Pratylenchus sp. C1 (561,232 bp), and did not cut P. coffeae (781 bp).
The RFLP profiles of the ITS amplified products forP. speijeri n. sp. (= P. coffeae from Ghana) generated byHinf I were obtained by Pourjame et al. (1999) and thoseby CfoI, DdeI, HindIII, HpaII and PstI were given byWaeyenberge et al. (2000). PCR-ITS-RFLP profiles of P.speijeri n. sp. were clearly different from those of otherP. coffeae isolates. However, the high levels of polymor-phism for RFLP patterns of some species of the P. coffeaespecies complex do not allow a reliable use of the ITSmarker for RFLP identification of this nematode group.
MOLECULAR DIAGNOSTIC USING PCR WITH SPECIFIC
PRIMERS
Uehara et al. (1998) were the first to propose PCR as-say with specific primers, designed on the ITS sequences,for the diagnosis of P. coffeae. According to our phylo-genetic results, the isolate used in the Uehara et al. studycorresponds to a new or unidentified species indicated inour paper as Pratylenchus sp. C1. Our in silico analysisrevealed that the specific primers designed by Uehara etal. (1998) can amplify Pratylenchus sp. C1 and P. cof-feae, but do not amplify P. speijeri n. sp. This finding isin agreement with the PCR results obtained by Duncan etal. (1999), who used Uehara’s primers for a large range ofP. coffeae populations, including P. speijeri n. sp. (isolateB1) and did not observe a specific band with this isolate.
In our study, we designed a specific primer for P.speijeri n. sp. and a group of specific primers for theP. coffeae species complex (P. coffeae, P. speijeri n.sp. and Pratylenchus sp. C1). Results of duplex PCR
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Fig. 1. Phylogenetic relationships within populations and species of the Pratylenchus coffeae species complex as inferred from Bayesiananalysis using the D2-D3 of 28S rRNA gene sequence dataset with the GTR + I + G model. Posterior probability more than 70% isgiven for appropriate clades. Newly obtained sequences are indicated in bold. *Sequences from Nguyen (2010); **only D3 segment ofa sample originally identified as P. musicola.
6 Nematology
Pratylenchus speijeri n. sp. from Musa in West Africa
Fig. 2. Phylogenetic relationships within populations and species of the Pratylenchus coffeae species complex as inferred from Bayesiananalysis using the ITS rRNA gene sequence dataset with the GTR + I + G model. Posterior probability more than 70% is given forappropriate clades. Newly obtained sequences are indicated in bold. *Sequence from Cheng et al. (2009) incorrectly annotated asCaenorhabditis elegans; **sequence from Uehara et al. (1998).
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Fig. 3. Phylogenetic relationships within populations andspecies of the Pratylenchus coffeae species complex as inferredfrom Bayesian analysis using the hsp90 gene sequence datasetwith the GTR + I + G model. Posterior probability more than70% is given for appropriate clades. Newly obtained sequencesare indicated in bold.
Fig. 4. PCR-D2-D3-RFLP for the Pratylenchus coffeae speciescomplex. A: Cfr42I; B: Bpu1102I; Lanes: M: 100 bp DNAladder (Promega); 1, 2, 3, 4: Pratylenchus sp. C1 (Japan,Colocasia esculenta, CA80 and CA96); 5: P. coffeae (USA, FL,Aglaonema sp., CD820); 6: P. coffeae (Viet Nam, Musa sp.); 7:P. coffeae (Haiti, Musa sp.).
Fig. 5. Duplex PCR with the Pratylenchus coffeae group-specificand P. speijeri n. sp. species-specific primers. Lanes: M: 100 bpDNA marker (Promega); 1, 2, 3, 4: Pratylenchus sp. C1 (Japan,Colocasia esculenta, CA80 and CA96); 5: P. coffeae (USA, FL,Aglaonema sp., CD820); 6: P. neglectus; 7: P. brachyurus; 8:control without DNA. Arrows indicate a group-specific bandfor Pratylenchus coffeae species complex (417-425 bp) and aspecies-specific band for P. speijeri n. sp. (102 bp).
with species-specific primers are given in Figure 5. Thecombination of universal primer TW81 with a species-specific primer yielded a single PCR product of 102 bpfor P. speijeri n. sp. and group-specific primers yieldedan amplicon of 417-425 bp for samples belonging to P.coffeae species complex. These primer combinations weresuccessfully tested with all samples studied.
MORPHOLOGICAL CHARACTERISATION AND
DESCRIPTION OF Pratylenchus speijeri N. SP.
The results obtained by the sequence and phylogeneticanalyses conducted in this study support the conclusionthat the lesion nematode parasitising plantain in Ghana isa new species and this is described herein.
Pratylenchus speijeri* n. sp.= P. coffeae B1 apud Duncan et al., 1999
= P. coffeae apud Brentu et al., 2004(Figs 6-10)
MEASUREMENTS
See Table 2 in Duncan et al. (1999; population origi-nally identified as P. coffeae B1) and Table 3 (P. speijerin. sp. and P. coffeae K6 populations from Indonesia).
* Specific epithet in honour of Paul Speijer who dedicated hislife to studying nematode diseases of Musa in Africa and wastragically killed in a plane crash in West Africa on 30 January2000.
8 Nematology
Pratylenchus speijeri n. sp. from Musa in West Africa
Fig. 6. Pratylenchus speijeri n. sp. A, B: Female and male entire body; C: Female pharyngeal region; D: Male pharyngeal region; E:Female anterior region; F: Schematic view of undivided face pattern of female; G: Female vulval region with spermatheca; H: Femaleposterior region; I, J: Male tail; K-O: Female tail.
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Fig. 7. Light micrographs of Pratylenchus speijeri n. sp. A, B: Female pharyngeal region showing hemizonid and excretory pore; C:Male pharyngeal region; D: Female anterior end; E: Female vulval region; F, H: Female tail; G: Female lateral field; I: Femaleposterior body portion; J: Male tail. (Scale bars = 20 μm.)
10 Nematology
Pratylenchus speijeri n. sp. from Musa in West Africa
Fig. 8. Common tail shapes observed in specimens of Pratylenchus speijeri n. sp. Note tail terminus indentation (F, J), a commonfeature of this root-lesion nematode.
DESCRIPTION
Female
Body habitus almost straight to open C-shaped. Lip re-gion slightly offset from body contour, with two annulidistinctly higher than adjoining body annuli. First lip an-nulus with angular edge in profile, narrower and thinnerthan second annulus. In a single specimen, three distinctannuli on a side of lip region and two on other side were
observed. In en face view with SEM, lip region appearingplain, smooth, with all labial sectors fused together andwith an oral disc. Stylet rather long, robust, conus 8.5 ±0.4 (8.0-9.5) μm long, forming on average 50% of entirestylet length. Stylet shaft tubular and slender, basal knobsprominent, rounded, slightly anteriorly flattened. Pha-ryngeal procorpus narrowing just anterior to small, ovalmetacorpus. Valve of median bulb conspicuous. Isthmuselongate, very slender, ending in an almost cylindrical
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Fig. 9. SEM morphology of Pratylenchus speijeri n. sp. Left and right: Undivided face pattern of female with all labial sectors fusedtogether and with oral disc; Centre: Female face lateral view showing second lip annulus higher than first.
gland lobe overlapping intestine for ca 40 μm. Secretory-excretory pore located just posterior to hemizonid. Hemi-zonion usually highly visible, 8-10 body annuli posteriorto hemizonid. Body annulation clear, prominent, lateralfield with four smooth incisures. Outline of outer bandsbecoming indented towards tail end, between phasmid andtail tip. Ovary mono-prodelphic, rather short, oocytes ar-ranged in a single row. Spermatheca large, round to oval,occasionally rectangular in shape, full of sperm, 18.4 ±4.0 (11.0-25.5) μm long, posterior margin 41.6 ± 6.2(30-54) μm from vagina. Vulva slightly sunken in body,located between two prominent lips. Post-uterine sac ca1.5 vulval body diam. long, usually undifferentiated. Ina few specimens, a short post-rectal extension of intes-tine observed. Phasmids located just anterior to mid-tail,17.1 ± 2.2 (13.5-21.0) μm from tail tip. Tail subcylindri-cal, tapering towards tip. Tail terminus variable in shape,in most specimens (85%) truncate, with indented, striated(or irregularly annulated) or less frequently smooth mar-gin. In more than half of specimens observed, a more orless pronounced indentation present. Specimens with con-ically pointed or subhemispherical, smooth tail terminirarely observed (see Frederick & Tarjan, 1989 for termi-nology).
Male
Similar to female except in posterior end of body, witha slightly smaller body length. Anterior part of body more
slender than in female. Lip region usually higher thanin female. Stylet slightly smaller than that of female,with narrower knobs in transverse view. Pharyngeal bulbsmall, ovate; isthmus slender, elongate, ending in along, narrow glandular lobe. Testis outstretched, short.Spicules paired, weakly cephalated, ventrally arcuate.Gubernaculum slightly curved. Tail conical, elongate,characteristically bent on ventral side, enveloped by apoorly protruding, crenate bursa.
TYPE HOST AND LOCALITY
Roots of the false horn plantain cv. Apantu-pa (Musaspp., AAB-group) at the Forest and Horticultural CropsResearch Centre, Kade, University of Ghana, Accra(6°09′N, 0°55′W), Ghana. The soil is a deep and welldrained Acrisol, the annual precipitation 1660 mm and theclimate is humid-tropical.
OTHER HOST AND LOCALITIES
Other samples identified by us as P. speijeri n. sp.,based on published sequences (Cheng et al., 2009), camefrom banana roots collected in Guangzhou, P.R. China.
TYPE MATERIAL
Holotype, 80 female and 36 male paratypes, mountedon glass slides deposited in the nematode collection at
12 Nematology
Pratylenchus speijeri n. sp. from Musa in West Africa
Fig. 10. Comparative line drawings of morphological features of the closely related Pratylenchus auracensis (P.a.), P. coffeae (P.c.), P.floridensis (P.f.), P. jaehni (P.j.), P. loosi (P.l.), P. parafloridensis (P.p.) and P. speijeri n. sp. (P.s.). (5, 18, 25 after Seinhorst, 1977, and7, 20, 27 after Múnera et al., 2009, modified.)
the Istituto per la Protezione delle Piante, CNR, Bari,Italy (collection nos IPP-K-1034 to -36 and K-1039 toK-1055). Additional paratypes were distributed to theUnited States Department of Agriculture Nematode Col-lection, Beltsville, MD, USA (five females and threemales, collection no. IPP-K-1032), University of Cal-
ifornia Riverside Nematode Collection, Riverside, CA,
USA (four females and two males, collection no. IPP-K-
The Netherlands (four females and two males, collection
no. IPP-K-1036) and Instituto de Agricultura Sostenible,
Vol. 00(0), 2012 13
F. De Luca et al.
Tabl
e3.
Mor
phom
etri
csof
Prat
ylen
chus
spei
jeri
n.sp
.and
P.co
ffea
eK
6fr
omco
ffee
inJa
va,I
ndon
esia
.Spe
cim
ens
wer
em
easu
red
aliv
e,th
enfix
edan
dre
-mea
sure
d.A
llm
easu
rem
ents
are
inμ
man
din
the
form
:m
ean
±s.
d.(r
ange
).
Cha
ract
erP.
spei
jeri
n.sp
.P.
coffe
aeK
6
Fem
ale
Mal
eFe
mal
e
Hol
otyp
ePa
raty
pes
Para
type
sn
–20
(fixe
d)10
(fixe
d)20
(liv
e)20
(fixe
d)L
521
533
±21
.9(4
73-5
63)
489
±27
.9(4
46-5
50)
670
±59
.2(5
29-7
99)
602
±51
.4(5
20-7
15)
a26
.027
.7±
2.6
(23.
4-32
.7)
29.7
±1.
8(2
7.1-
33.0
)28
.0±
2.3
(21.
6-31
.7)
28.7
±3.
1(2
3.4-
34.0
)b
6.2
6.6
±0.
6(5
.8-8
.0)
6.3
±0.
2(5
.8-6
.5)
7.1
±0.
4(6
.5-8
.0)
6.7
±0.
4(5
.6-7
.2)
b′4.
24.
4±
0.3
(3.8
-5.0
)4.
3±
0.2
(4.0
-4.6
)–
–c
18.2
18.8
±1.
2(1
7.1-
21.6
)16
.2±
0.9
(14.
2-17
.6)
21.2
±2.
2(1
7.1-
25.8
)20
.9±
2.8
(17.
0-31
.0)
c′2.
22.
3±
0.2
(1.8
-2.6
)3.
2±
0.2
(2.9
-3.5
)–
–V
orT
79.5
80.0
±1.
2(7
7.6-
82.0
)35
.0±
5.3
(28-
45)
80.8
±1.
3(7
6.6-
82.5
)80
.5±
1.5
(76.
0-82
.5)
Styl
etle
ngth
17.5
17.0
±0.
4(1
6.5-
18.0
)15
.2±
0.6
(14.
5-16
.5)
17.2
±0.
5(1
6.5-
18.0
)16
.9±
0.2
(16.
5-17
.0)
Styl
etkn
obw
idth
4.0
4.0
±0.
4(2
.5-4
.5)
3.0
±0.
5(2
.5-3
.5)
4.5
±0.
3(4
.0-5
.0)
4.2
±0.
2(4
.0-4
.5)
Styl
etkn
obhe
ight
2.5
2.5
±0.
3(2
.0-2
.5)
1.9
±0.
2(1
.5-2
.0)
3.0
±0.
2(2
.9-3
.2)
2.7
±0.
2(2
.0-3
.5)
DG
Ofr
omst
ylet
base
2.5
3.0
±0.
5(2
.0-4
.0)
3.5
±0.
0(3
.5)
3.1
±0.
3(2
.5-3
.5)
2.8
±0.
5(2
.5-4
.0)
Ant
erio
ren
dto
:ce
ntre
ofm
etac
orpu
s52
53±
3.5
(46.
0-62
)52
±2.
6(4
7-56
)–
–ca
rdia
83.5
81±
5.4
(69-
93)
79±
5.2
(72-
89)
94±
5.7
(78-
105)
90±
6.2
(78-
102)
end
ofph
aryn
geal
glan
dlo
be12
512
2±
7.3
(107
-135
)11
4±
5.6
(110
-125
)–
–se
cret
ory/
excr
etor
ypo
re80
81±
3.6
(73-
87)
74±
4.9
(66-
82)
91±
6.8
(76-
102)
84±
6.0
(76-
100)
Phar
ynge
alov
erla
p42
40.0
±4.
7(3
0.0-
46.5
)35
.5±
4.7
(28.
5-43
.5)
53.4
±9.
6(3
0.0-
84.0
)49
.8±
10.3
(34.
0-72
.5)
Max
.bod
ydi
am.
2019
.5±
2.3
(16.
0-23
.5)
16.5
±0.
8(1
5.5-
18.5
)24
.0±
2.5
(20.
5-30
.5)
21.2
±2.
3(1
7.5-
25.5
)V
ulva
lbod
ydi
am.
17.5
17.5
±2.
0(1
4.5-
21.5
)–
22.5
±2.
0(1
7.0-
26.0
)19
.5±
1.6
(17.
0-22
.0)
Ana
lbod
ydi
am.
1312
.5±
1.1
(11.
0-14
.5)
9.5
±0.
8(8
.5-1
1.0)
15.0
±1.
1(1
3.0-
17.0
)13
.2±
1.1
(11.
5-15
.0)
Ant
erio
rge
nita
ltra
ctle
ngth
208
168
±34
.0(9
9-21
1)17
1±
28.0
(123
-222
)–
–Sp
erm
athe
ca-v
agin
a35
.541
.5±
6.2
(30-
54)
–50
.2±
11.7
(28.
0-77
.0)
47.1
±11
.6(2
7.0-
70.5
)Ta
ille
ngth
28.5
28±
2.1
(23-
32)
30±
1.9
(27-
34)
32±
2.6
(26-
37)
29±
3.3
(21-
36)
No.
ofta
ilan
nuli
2023
±2.
5(1
8-26
)–
––
Spic
ule
leng
th–
–17
.5±
0.4
(16.
5-18
.0)
––
Gub
erna
culu
mle
ngth
––
5.5
±0.
1(5
.0-5
.5)
––
Vul
vato
anus
dist
ance
7877
±7.
5(6
6-98
)–
96±
13.1
(70-
138)
87±
12.8
(70-
135)
Post
-ute
rine
sac
26.5
24.5
±3.
3(2
0.5-
31.5
)–
33.2
±6.
5(2
4.5-
49.5
)29
.5±
6.5
(19.
5-49
.5)
Lat
eral
field
wid
th5.
56.
5±
1.3
(5.5
-10.
0)6.
0±
0.2
(5.5
-6.0
)7.
2±
0.7
(5.9
-8.5
)–
14 Nematology
Pratylenchus speijeri n. sp. from Musa in West Africa
CSIC, Córdoba, Spain (four females and three males, col-lection no. IPP-K-1037).
DIAGNOSIS AND RELATIONSHIPS
Pratylenchus speijeri n. sp. is characterised by a slightlyoffset lip region bearing two annuli which are distinctlyhigher than the adjoining body annuli, second lip annulushigher than the first, SEM face smooth, undivided, stylet17 (16.5-18) μm long, with massive rounded knobs, pha-ryngeal overlap rather long, lateral field non-areolated andwith four incisures, body annulation prominent, spermath-eca large, mostly round to oval, full of sperm, vulva usu-ally with prominent lips, tail sub-cylindrical with truncate,usually indented and striated or smooth terminus, malescommon and with a characteristic ventrally curved, elon-gate tail. The matrix code of the new species, according toCastillo and Vovlas (2007) is: A1, B2, C3, D3, E2, 3, F3,G2, H2, I2, 3, J1, K1.
Pratylenchus speijeri n. sp. has an undivided andplain face, as seen with SEM (Group 1 according toCorbett & Clark, 1983) in combination with two lipannuli (Fig. 9), a functional spermatheca and numerousmales. These characters separate P. speijeri n. sp. fromall other Pratylenchus species having a divided face,in combination with two or more lip annuli, a non-functional spermatheca and absence of males. Relatedspecies sharing with P. speijeri n. sp. two lip annuli,undivided and plain face, as seen with SEM, a functionalspermatheca and numerous males include P. araucensisMúnera, Bert & Decraemer, 2009, P. coffeae (sensuDuncan et al., 1999 and Inserra et al., 2001), the twocryptic species P. floridensis De Luca, Troccoli, Duncan,Subbotin, Waeyenberge, Moens & Inserra, 2010 and P.parafloridensis De Luca, Troccoli, Duncan, Subbotin,Waeyenberge, Moens & Inserra, 2010, P. jaehni Inserra,Duncan, Troccoli, Dunn, Maia dos Santos, Kaplan &Vovlas, 2001 and P. loosi Loof, 1960. Pratylenchusspeijeri n. sp. differs from P. araucensis mainly bythe longer stylet (16.5-18.0 vs 14.7-15.9 μm). Othercharacters such as body and pharynx length, pharyngealoverlap and PUS are greater in P. speijeri n. sp. than in P.araucensis (mean values: 533, 122, 39.8, 24.6 vs 462, 99,24.2, 18.1 μm, respectively), although the range valuesof these characters overlap. Thus the separation of thesetwo species, which share the same host (Musa spp.), reliesmainly on the length of the stylet.
From P. coffeae, which also parasitises Musa spp., thenew species differs in having shorter body length, pharynxlength, pharyngeal overlap and vulva-anus distance (mean
values: 533 vs 602, 84 vs 132, 40 vs 49.8, and 77 vs87.2 μm, respectively). Pratylenchus speijeri n. sp. alsohas wider and slightly shorter stylet knobs (transverse andlongitudinal axis 8.5 vs 4.2, 2.5 vs 2.7 μm, respectively)and a tail terminus that is more often indented than inP. coffeae. However, overlap of the range values of thesecharacters makes the morphological separation of thesetwo species difficult and unreliable.
Pratylenchus speijeri n. sp. differs from the crypticspecies P. floridensis and P. parafloridensis by a longerstylet (16.5-18.0 vs 14-15.8 and 14.5-16 μm, respec-tively), a more posterior vulva (80 vs 77% in both species),and the truncate tail with indented terminus vs bluntlypointed or sub-hemispherical with smooth terminus in thetwo cryptic species. From P. jaehni and P. loosi, P. speijerin. sp. differs mainly by the slightly longer stylet (16.5-18vs 14.5-16.0 and 15-16 μm, respectively) and the trun-cate and indented tail terminus, which is commonly hemi-spherical or sub-hemispherical and smooth in P. jaehniand bluntly pointed and smooth in P. loosi.
Other Pratylenchus species sharing with P. speijeri n.sp. the combination of two lip annuli, functional sper-matheca and presence of males have been described with-out examining their lip patterns. From these incompletelydescribed species, P. speijeri n. sp. differs from P. alleniFerris, 1961 and P. artemisiae Zheng & Chen, 1994 by thelonger stylet (16.5-18 vs 13.5-15 and 11.5-14.5 μm, re-spectively) and tail shape (truncate indented vs rounded);from P. brzeskii Karssen, Waeyenberge & Moens, 2000by the slightly shorter stylet (16.5-18 vs 18-19 μm) andtail shape (truncate indented vs narrowly rounded andsmooth); from P. flakkensis Seinhorst, 1968 by the moreposterior vulva position (77.6-82 vs 73-77%); from P. gib-bicaudatus Minagawa, 1982 by the longer stylet (16.5-18vs 14-16.5 μm) and a functional spermatheca (with spermvs without sperm); from P. kumamotoensis Mizukubo,Sugimura & Uesugi, 2007 by the shorter post-uterine sac,longer stylet (20.5-31.5 and 16.5-18 vs 37-45 and 14-15 μm, respectively) and tail shape (conically pointed,with smooth terminus in P. kumamotoensis); from P. neo-brachyurus Siddiqi, 1994 by the longer body and stylet(473-563 and 16.5-18 vs 310-410 and 14.5-17.0 μm, re-spectively); and from P. panamaensis Siddiqi, Dabur &Bajaj, 1991, P. roseus Zarina & Maqbool, 1998 and P. sil-vaticus Brzeski, 1998 mainly by the shape of the tail ter-minus (truncate indented vs hemispherical and annulated,rounded and coarsely annulated and clavate and striated,respectively).
Vol. 00(0), 2012 15
F. De Luca et al.
The Pratylenchus coffeae species complex and theusefulness of molecular data
During the last few years the use of SEM, LM andmolecular approaches to study P. coffeae, or representa-tives of the P. coffeae species complex, has resulted inthe descriptions of several new cryptic species, namelyP. jaehni, P. floridensis, P. parafloridensis and P. speijerin. sp. The morphological similarity makes separating P.speijeri n. sp. from P. coffeae based only on morphometricand morphological characters unreliable, whereas molec-ular analysis can clearly distinguish these root lesion ne-matode species.
Sequences and phylogenetic analyses of the D2-D3expansion segments of 28S rRNA gene and the ITS rRNAgene well supported the distinction between P. speijerin. sp. and P. coffeae. The phylogenetic studies clearlydemonstrated that P. speijeri n. sp. formed a separateclade from P. coffeae and thus represents a distinctspecies. A PCR-RAPD study of 15 isolates of root-lesionnematodes, including P. speijeri n. sp. (= P. coffeae fromGhana), with 18 decameric primers generated 227 bands(Duncan et al., 1999). In the phenogram presented bythose authors the Ghanaian population clustered withP. coffeae topotype and genetically related samples, butoccupied a basal position in this group. Isozyme patternanalysis of P. speijeri n. sp. (= Ghanaian population of P.coffeae) was conducted by Andrés et al. (2000). Among40 studied populations comprising nine Pratylenchusspecies, the greatest intraspecific diversity was foundwithin P. coffeae populations and P. speijeri n. sp. differedin esterase and phosphoglucose isomerase phenotypesfrom Guatemalan and Honduran populations of P. coffeae.
Our phylogenetic and sequence analysis revealed thata Japanese population of P. coffeae from taro (C. escu-lenta), named here as Pratylenchus sp. C1, differed fromP. speijeri n. sp. and P. coffeae and may represent a sep-arate species. Mizukubo et al. (2003) conducted detailedanalysis of PCR-RFLP types, compatibility on host plantsand crossing experiments of 20 isolates of the P. cof-feae species complex. Their study revealed three RFLPtypes for the P. coffeae species complex. The JapanesePratylenchus sp. C1 likely corresponds to RFLP type B,whereas P. coffeae represents RFLP type A, according toMizukubo’s classification. Laboratory hybridisation testsbetween these RFLP types generated abundant F1 hy-brids and inbreeding of F1 progeny produced a few F2
progeny, suggesting an incomplete reproductive isolation(Mizukubo et al., 2003). Differences in host compatibil-
ity to sweet potato and taro were also observed for theseRFLP phenotypes. Mizukubo et al. (2003) concluded thatthese phenotypes might represent different subspecies.
In the present work, we used two fragments of riboso-mal RNA genes and one fragment of a protein coding geneto reconstruct relationships within the P. coffeae speciescomplex and to design molecular diagnostic tools. Recentprogress made in the study of the genomics of P. coffeae(Haegeman et al., 2011) will allow the selection and ap-plication of additional genetic markers for deeper under-standing of relationships within this species complex.
Conclusion
Although the morphological characterisation of P. spei-jeri n. sp. did not result in an unambiguous separationfrom the closest related species, P. coffeae, important fea-tures of diagnostic value, such as larger stylet knobs anda more often indented tail terminus, were identified. Inorder to overcome the inadequacy of the morphologicalanalysis for a reliable differentiation of this new root-lesion nematode, a major objective of this study was thedevelopment of species-specific molecular tools for thereliable identification of P. speijeri n. sp. We were able toelucidate the useful diagnostic properties of the enzymeCfr421 specific for P. speijeri n. sp. and Bpu1102I, whichis specific for the separation of P. speijeri n. sp., Praty-lenchus sp. C1 and other P. coffeae populations with un-certain relationships in PCR-RFLP analysis of the D2-D3expansion segments of 28S rRNA genes. Finally, we de-signed species- and group-specific primers that amplifiedspecific fragments from DNA of P. speijeri n. sp. and allspecies presently known in the P. coffeae species complex(P. coffeae, P. speijeri n. sp. and Pratylenchus sp. C1).The use of these molecular tools have important practi-cal applications, not only for routine diagnosis, but to re-evaluate the worldwide distribution of these economicallyimportant species and to develop Musa germplasm resis-tant to these damaging root-lesion nematodes for the ulti-mate benefit of banana and plantain producers globally.
Acknowledgements
F.D.L. and A.T. contributed equally to the present work.F.D.L. acknowledges support from the Italian Ministryof Research (MIUR)-Fondo FAR- Legge 297/1999 Art.12/lab-Project Grant DM 19410. Funds provided by theItalian Ministry of Economy and Finance to the CNR for
16 Nematology
Pratylenchus speijeri n. sp. from Musa in West Africa
the project ‘Innovazione e Sviluppo del Mezzogiorno –Conoscenze Integrate per Sostenibilità ed Innovazione delMade in Italy Agroalimentare’ – Legge n. 191/2009 werealso utilised for this study.
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